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Sample records for interleukin-1beta-induced beta-cell apoptosis

  1. Angiotensin II modulates interleukin-1{beta}-induced inflammatory gene expression in vascular smooth muscle cells via interfering with ERK-NF-{kappa}B crosstalk

    SciTech Connect

    Xu, Shanqin; Zhi, Hui; Hou, Xiuyun; Jiang, Bingbing

    2011-07-08

    Highlights: {yields} We examine how angiotensin II modulates ERK-NF-{kappa}B crosstalk and gene expression. {yields} Angiotensin II suppresses IL-1{beta}-induced prolonged ERK and NF-{kappa}B activation. {yields} ERK-RSK1 signaling is required for IL-1{beta}-induced prolonged NF-{kappa}B activation. {yields} Angiotensin II modulates NF-{kappa}B responsive genes via regulating ERK-NF-{kappa}B crosstalk. {yields} ERK-NF-{kappa}B crosstalk is a novel mechanism regulating inflammatory gene expression. -- Abstract: Angiotensin II is implicated in cardiovascular diseases, which is associated with a role in increasing vascular inflammation. The present study investigated how angiotensin II modulates vascular inflammatory signaling and expression of inducible nitric oxide synthase (iNOS) and vascular cell adhesion molecule (VCAM)-1. In cultured rat aortic vascular smooth muscle cells (VSMCs), angiotensin II suppressed interleukin-1{beta}-induced prolonged phosphorylation of extracellular signal-regulated kinase (ERK) and ribosomal S6 kinase (RSK)-1, and nuclear translocation of nuclear factor (NF)-{kappa}B, leading to decreased iNOS but enhanced VCAM-1 expression, associated with an up-regulation of mitogen-activated protein kinase phosphatase-1 expression. Knock-down of RSK1 selectively down regulated interleukin-1{beta}-induced iNOS expression without influencing VCAM-1 expression. In vivo experiments showed that interleukin-1{beta}, iNOS, and VCAM-1 expression were detectable in the aortic arches of both wild-type and apolipoprotein E-deficient (ApoE{sup -/-}) mice. VCAM-1 and iNOS expression were higher in ApoE{sup -/-} than in wild type mouse aortic arches. Angiotensin II infusion (3.2 mg/kg/day, for 6 days, via subcutaneous osmotic pump) in ApoE{sup -/-} mice enhanced endothelial and adventitial VCAM-1 and iNOS expression, but reduced medial smooth muscle iNOS expression associated with reduced phosphorylation of ERK and RSK-1. These results indicate that angiotensin

  2. Baculovirus p35 increases pancreatic {beta}-cell resistance to apoptosis

    SciTech Connect

    Hollander, Kenneth; Bar-Chen, Michal; Efrat, Shimon . E-mail: sefrat@post.tau.ac.il

    2005-07-01

    {beta}-cells die by apoptosis in type 1 diabetes as a result of autoimmune attack mediated by cytokines, and in type 2 diabetes by various perpetrators including human islet amyloid polypeptide (hIAPP). The cascade of apoptotic events induced by cytokines and hIAPP is mediated through caspases and reactive oxygen species. The baculovirus p35 protein is a potent anti-apoptotic agent shown to be effective in a variety of species and able to inhibit a number of apoptotic pathways. Here, we aimed at determining the protective potential of p35 in {beta}-cells exposed to cytokines and hIAPP, as well as the effects of p35 on {beta}-cell function. The p35 gene was introduced into {beta}TC-tet cells, a differentiated murine {beta}-cell line capable of undergoing inducible growth-arrest. Both proliferating and growth-arrested cells expressing p35 manifested increased resistance to cytokines and hIAPP, compared with control cells, as judged by cell viability, DNA fragmentation, and caspase-3 activity assays. p35 was significantly more protective in growth-arrested, compared with proliferating, cells. No significant differences were observed in proliferation and insulin content between cells expressing p35 and control cells. In contrast, p35 manifested a perturbing effect on glucose-induced insulin secretion. These findings suggest that p35 could be incorporated as part of a multi-pronged approach of immunoprotective strategies to provide protection from recurring autoimmunity for transplanted {beta}-cells, as well as in preventive gene therapy in type 1 diabetes. p35 may also be protective from {beta}-cell damage caused by hIAPP in type 2 diabetes.

  3. Interleukin-1beta induces death in chondrocyte-like ATDC5 cells through mitochondrial dysfunction and energy depletion in a reactive nitrogen and oxygen species-dependent manner.

    PubMed

    Yasuhara, Rika; Miyamoto, Yoichi; Akaike, Takaaki; Akuta, Teruo; Nakamura, Masanori; Takami, Masamichi; Morimura, Naoko; Yasu, Kayoko; Kamijo, Ryutaro

    2005-07-15

    IL-1 (interleukin-1) acts as a key mediator of the degeneration of articular cartilage in RA (rheumatoid arthritis) and OA (osteoarthritis),where chondrocyte death is observed. It is still controversial, however, whether IL-1 induces chondrocyte death. In the present study, the viability of mouse chondrocyte-like ATDC5 cells was reduced by the treatment with IL-1beta for 48 h or longer. IL-1beta augmented the expression of the catalytic gp91 subunit of NADPH oxidase, gp91phox, as well as inducible NO synthase in ATDC5 cells. Generation of nitrated guanosine and tyrosine suggested the formation of reactive nitrogen species including ONOO- (peroxynitrite), a reaction product of NO and O2-, in ATDC5 cells and rat primary chondrocytes treated with IL-1beta. Death of ATDC5 cells after IL-1beta treatment was prevented by an NADPH-oxidase inhibitor, AEBSF[4-(2-aminoethyl)benzene-sulphonyl fluoride], an NO synthase inhibitor, L-NAME (NG-nitro-L-arginine methyl ester), and a ONOO- scavenger, uric acid. The viability of ATDC5 cells was reduced by the ONOO(-)-generator 3-(4-morpholinyl)sydnonimine hydrochloride, but not by either the NO-donor 1-hydroxy-2-oxo-3-(N-methyl-2-aminopropyl)-3-methyl-1-triazene or S-nitrosoglutathione. Disruption of mitochondrial membrane potential and ATP deprivation were observed in IL-1beta-treated ATDC5 cells, both of which were restored by L-NAME, AEBSF or uric acid. On the other hand, no morphological or biochemical signs indicating apoptosis were observed in these cells. These results suggest that the death of chondrocyte-like ATDC5 cells was mediated at least in part by mitochondrial dysfunction and energy depletion through ONOO- formation after IL-1beta treatment. PMID:15784009

  4. Knockdown of prolactin receptors in a pancreatic beta cell line: effects on DNA synthesis, apoptosis, and gene expression.

    PubMed

    Arumugam, Ramamani; Fleenor, Don; Freemark, Michael

    2014-08-01

    Prolactin (PRL) and placental lactogen stimulate beta cell replication and insulin production in vitro and in vivo. The molecular mechanisms by which lactogens promote beta cell expansion are unclear. We treated rat insulinoma cells with a PRL receptor (PRLR) siRNA to determine if PRLR signaling is required for beta cell DNA synthesis and cell survival and to identify beta cell cycle genes whose expression depends upon lactogen action. Effects of PRLR knockdown were compared with those of PRL treatment. PRLR knockdown (-80 %) reduced DNA synthesis, increased apoptosis, and inhibited expression of cyclins D2 and B2, IRS-2, Tph1, and the anti-apoptotic protein PTTG1; p21 and BCL6 mRNAs increased. Conversely, PRL treatment increased DNA synthesis, reduced apoptosis, and enhanced expression of A, B and D2 cyclins, CDK1, IRS-2, FoxM1, BCLxL, and PTTG1; BCL6 declined. PRLR signaling is required for DNA synthesis and survival of rat insulinoma cells. The effects of lactogens are mediated by down-regulation of cell cycle inhibitors (BCL6, p21) and induction of A, B, and D2 cyclins, IRS-2, Tph1, FoxM1, and the anti-apoptotic proteins BCLxL and PTTG1. PMID:24114406

  5. Effects of low intensity laser acupoint irradiation on inhibiting islet beta-cell apoptosis in rats with type 2 diabetes

    NASA Astrophysics Data System (ADS)

    Xiong, Guoxin; Xiong, Leilei; Li, Xinzhong

    2016-09-01

    To investigate the effects of low intensity semiconductor laser acupoint irradiation on inhibiting islet beta-cell apoptosis in rats with type 2 diabetes, a method using a high-fat diet and low-dose intraperitoneal injections of streptozotocin established a type 2 diabetes mellitus rat model. Model rats were randomly divided into a laser acupoint irradiation group, rosiglitazone control group, and placebo group; each group had 10 rats. In addition, 10 normal male rats were selected for the normal control group. The Housanli, Neiting and Yishu acupoints of the rats in the laser acupoint irradiation group were irradiated with a 10 mW semiconductor laser; each point was irradiated for 15 min, once every 2 d over 28 d, for a total of 14 episodes of irradiation. The rosiglitazone group rats were given rosiglitazone (0.2 mg kg‑1) intragastrically; the placebo group rats were given 0.9% brine (0.2 mg kg‑1) intragastrically, once daily, for four consecutive weeks. The change of fasting blood glucose was determined before and after each treatment. The islet beta-cell apoptosis was determined. The islet beta-cell apoptosis rates of the laser acupoint irradiation group and the rosiglitazone group were significantly lower than the rate of the placebo group. Even though the rate was lower in the laser acupoint irradiation group than in the rosiglitazone group, there was no significant difference between them. It is shown that acupoint irradiation with a semiconductor laser can effectively inhibit islet beta-cell apoptosis in rats with type 2 diabetes.

  6. Calcium-activated Calpain-2 Is a Mediator of Beta Cell Dysfunction and Apoptosis in Type 2 Diabetes*

    PubMed Central

    Huang, Chang-jiang; Gurlo, Tatyana; Haataja, Leena; Costes, Safia; Daval, Marie; Ryazantsev, Sergey; Wu, Xiuji; Butler, Alexandra E.; Butler, Peter C.

    2010-01-01

    The islet in type 2 diabetes (T2DM) and the brain in neurodegenerative diseases share progressive cell dysfunction, increased apoptosis, and accumulation of locally expressed amyloidogenic proteins (islet amyloid polypeptide (IAPP) in T2DM). Excessive activation of the Ca2+-sensitive protease calpain-2 has been implicated as a mediator of oligomer-induced cell death and dysfunction in neurodegenerative diseases. To establish if human IAPP toxicity is mediated by a comparable mechanism, we overexpressed human IAPP in rat insulinoma cells and freshly isolated human islets. Pancreas was also obtained at autopsy from humans with T2DM and nondiabetic controls. We report that overexpression of human IAPP leads to the formation of toxic oligomers and increases beta cell apoptosis mediated by increased cytosolic Ca2+ and hyperactivation of calpain-2. Cleavage of α-spectrin, a marker of calpain hyperactivation, is increased in beta cells in T2DM. We conclude that overactivation of Ca2+-calpain pathways contributes to beta cell dysfunction and apoptosis in T2DM. PMID:19861418

  7. Maternal antioxidants prevent beta cell apoptosis and promote formation of dual hormone-expressing endocrine cells in male offspring following fetal and neonatal nicotine exposure

    PubMed Central

    BRUIN, Jennifer E; WOYNILLOWICZ, Amanda K; HETTINGA, Bart P; TARNOPOLSKY, Mark A; MORRISON, Katherine M; GERSTEIN, Hertzel C; HOLLOWAY, Alison C

    2013-01-01

    Aim Fetal and neonatal nicotine exposure causes beta cell oxidative stress and apoptosis in neonates, leading to adult-onset dysglycemia. The goal of this study was to determine whether an antioxidant intervention could prevent nicotine-induced beta cell loss. Methods Nulliparous female Wistar rats received daily subcutaneous injections of either saline or nicotine bitartrate (1.0 mg/kg/d) for 2 weeks prior to mating until weaning. Nicotine-exposed dams received either normal chow or diet containing antioxidants (1000 IU/kg vitamin E, 0.25% w/w coenzyme Q10 and 0.1% w/w alpha-lipoic acid) during mating, pregnancy and lactation; saline-exposed dams received normal chow. Pancreas tissue was collected from male offspring at 3 weeks of age to measure beta cell fraction, apoptosis, proliferation and the presence of cells co-expressing insulin and glucagon. Results The birth weight of the offspring born to nicotine-exposed dams receiving dietary antioxidants was significantly reduced. Most interestingly, the antioxidant intervention to nicotine-exposed dams prevented the beta cell loss and apoptosis observed in nicotine exposed male offspring whose mothers did not receive antioxidants. Male pups born to nicotine-treated mothers receiving antioxidants also had a trend towards increased beta cell proliferation and a significant increase in islets containing insulin/glucagon bi-hormonal cells relative to the other two treatment groups. Conclusion This study demonstrates that exposure to maternal antioxidants protects beta cells from the damaging effects of nicotine thus preserving beta cell mass. PMID:22385833

  8. Ubiquitin D Regulates IRE1α/c-Jun N-terminal Kinase (JNK) Protein-dependent Apoptosis in Pancreatic Beta Cells.

    PubMed

    Brozzi, Flora; Gerlo, Sarah; Grieco, Fabio Arturo; Juusola, Matilda; Balhuizen, Alexander; Lievens, Sam; Gysemans, Conny; Bugliani, Marco; Mathieu, Chantal; Marchetti, Piero; Tavernier, Jan; Eizirik, Décio L

    2016-06-01

    Pro-inflammatory cytokines contribute to pancreatic beta cell apoptosis in type 1 diabetes at least in part by inducing endoplasmic reticulum (ER) stress and the consequent unfolded protein response (UPR). It remains to be determined what causes the transition from "physiological" to "apoptotic" UPR, but accumulating evidence indicates that signaling by the ER transmembrane protein IRE1α is critical for this transition. IRE1α activation is regulated by both intra-ER and cytosolic cues. We evaluated the role for the presently discovered cytokine-induced and IRE1α-interacting protein ubiquitin D (UBD) on the regulation of IRE1α and its downstream targets. UBD was identified by use of a MAPPIT (mammalian protein-protein interaction trap)-based IRE1α interactome screen followed by comparison against functional genomic analysis of human and rodent beta cells exposed to pro-inflammatory cytokines. Knockdown of UBD in human and rodent beta cells and detailed signal transduction studies indicated that UBD modulates cytokine-induced UPR/IRE1α activation and apoptosis. UBD expression is induced by the pro-inflammatory cytokines interleukin (IL)-1β and interferon (IFN)-γ in rat and human pancreatic beta cells, and it is also up-regulated in beta cells of inflamed islets from non-obese diabetic mice. UBD interacts with IRE1α in human and rodent beta cells, modulating IRE1α-dependent activation of JNK and cytokine-induced apoptosis. Our data suggest that UBD provides a negative feedback on cytokine-induced activation of the IRE1α/JNK pro-apoptotic pathway in cytokine-exposed beta cells. PMID:27044747

  9. Exendin-4 Protects Mitochondria from Reactive Oxygen Species Induced Apoptosis in Pancreatic Beta Cells

    PubMed Central

    Li, Zhen; Zhou, Zhiguang; Huang, Gan; Hu, Fang; Xiang, Yufei; He, Lining

    2013-01-01

    Objective Mitochondrial oxidative stress is the basis for pancreatic β-cell apoptosis and a common pathway for numerous types of damage, including glucotoxicity and lipotoxicity. We cultivated mice pancreatic β-cell tumor Min6 cell lines in vitro and observed pancreatic β-cell apoptosis and changes in mitochondrial function before and after the addition of Exendin-4. Based on these observations, we discuss the protective role of Exendin-4 against mitochondrial oxidative damage and its relationship with Ca2+-independent phospholipase A2. Methods We established a pancreatic β-cell oxidative stress damage model using Min6 cell lines cultured in vitro with tert-buty1 hydroperoxide and hydrogen peroxide. We then added Exendin-4 to observe changes in the rate of cell apoptosis (Annexin-V-FITC-PI staining flow cytometry and DNA ladder). We detected the activity of the caspase 3 and 8 apoptotic factors, measured the mitochondrial membrane potential losses and reactive oxygen species production levels, and detected the expression of cytochrome c and Smac/DLAMO in the cytosol and mitochondria, mitochondrial Ca2-independent phospholipase A2 and Ca2+-independent phospholipase A2 mRNA. Results The time-concentration curve showed that different percentages of apoptosis occurred at different time-concentrations in tert-buty1 hydroperoxide- and hydrogen peroxide-induced Min6 cells. Incubation with 100 µmol/l of Exendin-4 for 48 hours reduced the Min6 cell apoptosis rate (p<0.05). The mitochondrial membrane potential loss and total reactive oxygen species levels decreased (p<0.05), and the release of cytochrome c and Smac/DLAMO from the mitochondria was reduced. The study also showed that Ca2+-independent phospholipase A2 activity was positively related to Exendin-4 activity. Conclusion Exendin-4 reduces Min6 cell oxidative damage and the cell apoptosis rate, which may be related to Ca2-independent phospholipase A2. PMID:24204601

  10. Activation of PPAR{delta} up-regulates fatty acid oxidation and energy uncoupling genes of mitochondria and reduces palmitate-induced apoptosis in pancreatic {beta}-cells

    SciTech Connect

    Wan, Jun; Jiang, Li; Lue, Qingguo; Ke, Linqiu; Li, Xiaoyu; Tong, Nanwei

    2010-01-15

    Recent evidence indicates that decreased oxidative capacity, lipotoxicity, and mitochondrial aberrations contribute to the development of insulin resistance and type 2 diabetes. The goal of this study was to investigate the effects of peroxisome proliferator-activated receptor {delta} (PPAR{delta}) activation on lipid oxidation, mitochondrial function, and insulin secretion in pancreatic {beta}-cells. After HIT-T15 cells (a {beta}-cell line) were exposed to high concentrations of palmitate and GW501516 (GW; a selective agonist of PPAR{delta}), we found that administration of GW increased the expression of PPAR{delta} mRNA. GW-induced activation of PPAR{delta} up-regulated carnitine palmitoyltransferase 1 (CPT1), long-chain acyl-CoA dehydrogenase (LCAD), pyruvate dehydrogenase kinase 4 (PDK4), and uncoupling protein 2 (UCP2); alleviated mitochondrial swelling; attenuated apoptosis; and reduced basal insulin secretion induced by increased palmitate in HIT cells. These results suggest that activation of PPAR{delta} plays an important role in protecting pancreatic {beta}-cells against aberrations caused by lipotoxicity in metabolic syndrome and diabetes.

  11. GLIS3, a susceptibility gene for type 1 and type 2 diabetes, modulates pancreatic beta cell apoptosis via regulation of a splice variant of the BH3-only protein Bim.

    PubMed

    Nogueira, Tatiane C; Paula, Flavia M; Villate, Olatz; Colli, Maikel L; Moura, Rodrigo F; Cunha, Daniel A; Marselli, Lorella; Marchetti, Piero; Cnop, Miriam; Julier, Cécile; Eizirik, Decio L

    2013-05-01

    Mutations in human Gli-similar (GLIS) 3 protein cause neonatal diabetes. The GLIS3 gene region has also been identified as a susceptibility risk locus for both type 1 and type 2 diabetes. GLIS3 plays a role in the generation of pancreatic beta cells and in insulin gene expression, but there is no information on the role of this gene on beta cell viability and/or susceptibility to immune- and metabolic-induced stress. GLIS3 knockdown (KD) in INS-1E cells, primary FACS-purified rat beta cells, and human islet cells decreased expression of MafA, Ins2, and Glut2 and inhibited glucose oxidation and insulin secretion, confirming the role of this transcription factor for the beta cell differentiated phenotype. GLIS3 KD increased beta cell apoptosis basally and sensitized the cells to death induced by pro-inflammatory cytokines (interleukin 1β + interferon-γ) or palmitate, agents that may contribute to beta cell loss in respectively type 1 and 2 diabetes. The increased cell death was due to activation of the intrinsic (mitochondrial) pathway of apoptosis, as indicated by cytochrome c release to the cytosol, Bax translocation to the mitochondria and activation of caspases 9 and 3. Analysis of the pathways implicated in beta cell apoptosis following GLIS3 KD indicated modulation of alternative splicing of the pro-apoptotic BH3-only protein Bim, favouring expression of the pro-death variant BimS via inhibition of the splicing factor SRp55. KD of Bim abrogated the pro-apoptotic effect of GLIS3 loss of function alone or in combination with cytokines or palmitate. The present data suggest that altered expression of the candidate gene GLIS3 may contribute to both type 1 and 2 type diabetes by favouring beta cell apoptosis. This is mediated by alternative splicing of the pro-apoptotic protein Bim and exacerbated formation of the most pro-apoptotic variant BimS. PMID:23737756

  12. GLIS3, a Susceptibility Gene for Type 1 and Type 2 Diabetes, Modulates Pancreatic Beta Cell Apoptosis via Regulation of a Splice Variant of the BH3-Only Protein Bim

    PubMed Central

    Colli, Maikel L.; Moura, Rodrigo F.; Cunha, Daniel A.; Marselli, Lorella; Marchetti, Piero; Cnop, Miriam; Julier, Cécile; Eizirik, Decio L.

    2013-01-01

    Mutations in human Gli-similar (GLIS) 3 protein cause neonatal diabetes. The GLIS3 gene region has also been identified as a susceptibility risk locus for both type 1 and type 2 diabetes. GLIS3 plays a role in the generation of pancreatic beta cells and in insulin gene expression, but there is no information on the role of this gene on beta cell viability and/or susceptibility to immune- and metabolic-induced stress. GLIS3 knockdown (KD) in INS-1E cells, primary FACS-purified rat beta cells, and human islet cells decreased expression of MafA, Ins2, and Glut2 and inhibited glucose oxidation and insulin secretion, confirming the role of this transcription factor for the beta cell differentiated phenotype. GLIS3 KD increased beta cell apoptosis basally and sensitized the cells to death induced by pro-inflammatory cytokines (interleukin 1β + interferon-γ) or palmitate, agents that may contribute to beta cell loss in respectively type 1 and 2 diabetes. The increased cell death was due to activation of the intrinsic (mitochondrial) pathway of apoptosis, as indicated by cytochrome c release to the cytosol, Bax translocation to the mitochondria and activation of caspases 9 and 3. Analysis of the pathways implicated in beta cell apoptosis following GLIS3 KD indicated modulation of alternative splicing of the pro-apoptotic BH3-only protein Bim, favouring expression of the pro-death variant BimS via inhibition of the splicing factor SRp55. KD of Bim abrogated the pro-apoptotic effect of GLIS3 loss of function alone or in combination with cytokines or palmitate. The present data suggest that altered expression of the candidate gene GLIS3 may contribute to both type 1 and 2 type diabetes by favouring beta cell apoptosis. This is mediated by alternative splicing of the pro-apoptotic protein Bim and exacerbated formation of the most pro-apoptotic variant BimS. PMID:23737756

  13. PED/PEA-15 Inhibits Hydrogen Peroxide-Induced Apoptosis in Ins-1E Pancreatic Beta-Cells via PLD-1

    PubMed Central

    Raciti, Gregory Alexander; Zatterale, Federica; Nigro, Cecilia; Mirra, Paola; Falco, Roberta; Ulianich, Luca; Di Jeso, Bruno; Formisano, Pietro; Miele, Claudia; Beguinot, Francesco

    2014-01-01

    The small scaffold protein PED/PEA-15 is involved in several different physiologic and pathologic processes, such as cell proliferation and survival, diabetes and cancer. PED/PEA-15 exerts an anti-apoptotic function due to its ability to interfere with both extrinsic and intrinsic apoptotic pathways in different cell types. Recent evidence shows that mice overexpressing PED/PEA-15 present larger pancreatic islets and increased beta-cells mass. In the present work we investigated PED/PEA-15 role in hydrogen peroxide-induced apoptosis in Ins-1E beta-cells. In pancreatic islets isolated from TgPED/PEA-15 mice hydrogen peroxide-induced DNA fragmentation was lower compared to WT islets. TUNEL analysis showed that PED/PEA-15 overexpression increases the viability of Ins-1E beta-cells and enhances their resistance to apoptosis induced by hydrogen peroxide exposure. The activity of caspase-3 and the cleavage of PARP-1 were markedly reduced in Ins-1E cells overexpressing PED/PEA-15 (Ins-1EPED/PEA-15). In parallel, we observed a decrease of the mRNA levels of pro-apoptotic genes Bcl-xS and Bad. In contrast, the expression of the anti-apoptotic gene Bcl-xL was enhanced. Accordingly, DNA fragmentation was higher in control cells compared to Ins-1EPED/PEA-15 cells. Interestingly, the preincubation with propranolol, an inhibitor of the pathway of PLD-1, a known interactor of PED/PEA-15, responsible for its deleterious effects on glucose tolerance, abolishes the antiapoptotic effects of PED/PEA-15 overexpression in Ins-1E beta-cells. The same results have been obtained by inhibiting PED/PEA-15 interaction with PLD-1 in Ins-1EPED/PEA-15. These results show that PED/PEA-15 overexpression is sufficient to block hydrogen peroxide-induced apoptosis in Ins-1E cells through a PLD-1 mediated mechanism. PMID:25489735

  14. Serum levels of pancreatic stone protein (PSP)/reg1A as an indicator of beta-cell apoptosis suggest an increased apoptosis rate in hepatocyte nuclear factor 1 alpha (HNF1A-MODY) carriers from the third decade of life onward

    PubMed Central

    2012-01-01

    Background Mutations in the transcription factor hepatocyte nuclear factor-1-alpha (HNF1A) result in the commonest type of maturity onset diabetes of the young (MODY). HNF1A-MODY carriers have reduced pancreatic beta cell mass, partially due to an increased rate of apoptosis. To date, it has not been possible to determine when apoptosis is occurring in HNF1A-MODY.We have recently demonstrated that beta cell apoptosis stimulates the expression of the pancreatic stone protein/regenerating (PSP/reg) gene in surviving neighbour cells, and that PSP/reg1A protein is subsequently secreted from these cells. The objective of this study was to determine whether serum levels of PSP/reg1A are elevated during disease progression in HNF1A-MODY carriers, and whether it may provide information regarding the onset of beta-cell apoptosis. Methods We analysed serum PSP/reg1A levels and correlated with clinical and biochemical parameters in subjects with HNF1A-MODY, glucokinase (GCK-MODY), and type 1 diabetes mellitus. A control group of normoglycaemic subjects was also analysed. Results PSP/reg1A serum levels were significantly elevated in HNF1A-MODY (n = 37) subjects compared to controls (n = 60) (median = 12.50 ng/ml, IQR = 10.61-17.87 ng/ml versus median = 10.72 ng/ml, IQR = 8.94-12.54 ng/ml, p = 0.0008). PSP/reg1A correlated negatively with insulin levels during OGTT, (rho = −0.40, p = 0.02). Interestingly we noted a significant positive correlation of PSP/reg1A with age of the HNF1A-MODY carriers (rho = 0.40 p = 0.02) with an age of 25 years separating carriers with low and high PSP/reg1A levels. Patients with type 1 diabetes mellitus also had elevated serum levels of PSP/reg1A compared to controls, however this was independent of the duration of diabetes. Conclusion Our data suggest that beta cell apoptosis contributes increasingly to the pathophysiology of HNF1A-MODY in patients 25 years and over. PSP/reg1A may be

  15. D-saccharic acid-1,4-lactone ameliorates alloxan-induced diabetes mellitus and oxidative stress in rats through inhibiting pancreatic beta-cells from apoptosis via mitochondrial dependent pathway

    SciTech Connect

    Bhattacharya, Semantee; Manna, Prasenjit; Sil, Parames C.

    2011-12-15

    Oxidative stress plays a vital role in diabetic complications. To suppress the oxidative stress mediated damage in diabetic pathophysiology, a special focus has been given on naturally occurring antioxidants present in normal diet. D-saccharic acid 1,4-lactone (DSL), a derivative of D-glucaric acid, is present in many dietary plants and is known for its detoxifying and antioxidant properties. The aim of the present study was to evaluate the beneficial role of DSL against alloxan (ALX) induced diabetes in the pancreas tissue of Swiss albino rats. A dose-dependent study for DSL (20-120 mg/kg body weight) was carried out to find the effective dose of the compound in ALX-induced diabetic rats. ALX exposure elevated the blood glucose, glycosylated Hb, decreased the plasma insulin and disturbed the intra-cellular antioxidant machineries whereas oral administration of DSL at a dose of 80 mg/kg body weight restored these alterations close to normal. Investigating the mechanism of the protective activity of DSL we observed that it prevented the pancreatic {beta}-cell apoptosis via mitochondria-dependent pathway. Results showed decreased mitochondrial membrane potential, enhanced cytochrome c release in the cytosol and reciprocal regulation of Bcl-2 family proteins in the diabetic rats. These events were also found to be associated with increased level of Apaf-1, caspase 9, and caspase 3 that ultimately led to pancreatic {beta}-cell apoptosis. DSL treatment, however, counteracted these changes. In conclusion, DSL possesses the capability of ameliorating the oxidative stress in ALX-induced diabetes and thus could be a promising approach in lessening diabetic complications. Highlights: Black-Right-Pointing-Pointer Oxidative stress is suggested as a key event in the pathogenesis of diabetes. Black-Right-Pointing-Pointer D-saccharic acid 1,4-lactone (DSL) reduces the alloxan-induced diabetes mellitus. Black-Right-Pointing-Pointer DSL normalizes cellular antioxidant machineries

  16. Lithospermic acid B protects beta-cells from cytokine-induced apoptosis by alleviating apoptotic pathways and activating anti-apoptotic pathways of Nrf2-HO-1 and Sirt1

    SciTech Connect

    Lee, Byung-Wan; Chun, Sung Wan; Kim, Soo Hyun; Lee, Yongho; Kang, Eun Seok; Cha, Bong-Soo; Lee, Hyun Chul

    2011-04-01

    Lithospermic acid B (LAB) has been reported to protect OLETF rats, an established type 2 diabetic animal model, from the development of diabetes-related vascular complications. We investigated whether magnesium lithospermate B (LAB) has a protective role under cytokine-induced apoptosis in INS-1 cells in vitro and whether it slows the development of diabetes in OLETF rats in vivo. Pretreatment with 50 {mu}M LAB significantly reduced the 1000 U/mL INF-{gamma} and 100 U/mL IL-1{beta}-induced INS-1 cell death. LAB significantly alleviated cytokine-induced phosphorylations of p38 and JNK in accordance with a decrease in cleaved caspase-3 activity in beta-cells. LAB also protected against the cytokine-induced caspase-3 apoptotic pathway via significant activation of Nrf2-HO (heme-oxigenase)-1 and Sirt1 expression. OLETF rats treated with 40 mg/kg/day LAB showed a significant improvement in glucose tolerance compared to untreated OLETF control rats in vivo. Our results suggest that the cytoprotective effects of LAB on pancreatic {beta}-cells are related with both alleviating apoptotic pathways and activating anti-apoptotic pathways of Nrf2-HO-1 and Sirt1.

  17. Mechanisms of beta-cell death in type 2 diabetes.

    PubMed

    Donath, Marc Y; Ehses, Jan A; Maedler, Kathrin; Schumann, Desiree M; Ellingsgaard, Helga; Eppler, Elisabeth; Reinecke, Manfred

    2005-12-01

    A decrease in the number of functional insulin-producing beta-cells contributes to the pathophysiology of type 2 diabetes. Opinions diverge regarding the relative contribution of a decrease in beta-cell mass versus an intrinsic defect in the secretory machinery. Here we review the evidence that glucose, dyslipidemia, cytokines, leptin, autoimmunity, and some sulfonylureas may contribute to the maladaptation of beta-cells. With respect to these causal factors, we focus on Fas, the ATP-sensitive K+ channel, insulin receptor substrate 2, oxidative stress, nuclear factor-kappaB, endoplasmic reticulum stress, and mitochondrial dysfunction as their respective mechanisms of action. Interestingly, most of these factors are involved in inflammatory processes in addition to playing a role in both the regulation of beta-cell secretory function and cell turnover. Thus, the mechanisms regulating beta-cell proliferation, apoptosis, and function are inseparable processes. PMID:16306327

  18. Interleukin-1beta induces hyperpolarization and modulates synaptic inhibition in preoptic and anterior hypothalamic neurons.

    PubMed

    Tabarean, I V; Korn, H; Bartfai, T

    2006-09-15

    Most of the inflammatory effects of the cytokine interleukin 1beta (IL-1beta) are mediated by induction of cyclooxygenase (COX)2 and the subsequent synthesis and release of prostaglandin E2. This transcription-dependent process takes 45-60 min, but IL-1beta, a well-characterized endogenous pyrogen also exerts faster neuronal actions in the preoptic area/anterior hypothalamus. Here, we have studied the fast (1-3 min) signaling by IL-1beta using whole-cell patch clamp recordings in preoptic area/anterior hypothalamus neurons. Exposure to IL-1beta (0.1-1 nM) hyperpolarized a subset ( approximately 20%) of preoptic area/anterior hypothalamus neurons, decreased their input resistance and reduced their firing rate. These effects were associated with an increased frequency of bicuculline-sensitive spontaneous inhibitory postsynaptic currents and putative miniature inhibitory postsynaptic currents, strongly suggesting a presynaptic mechanism of action. These effects require the type 1 interleukin 1 receptor (IL-1R1), and the adapter protein myeloid differentiation primary response protein (MyD88), since they were not observed in cultures obtained from IL-1R1 (-/-) or from MyD88 (-/-) mice. Ceramide, a second messenger of the IL-1R1-dependent fast signaling cascade, is produced by IL-1R1-MyD88-mediated activation of the neutral sphingomyelinase. C2-ceramide, its cell penetrating analog, also increased the frequency of miniature inhibitory postsynaptic currents in a subset of cells. Both IL-1beta and ceramide reduced the delayed rectifier and the A-type K(+) currents in preoptic area/anterior hypothalamus neurons. The latter effect may account in part for the increased spontaneous inhibitory postsynaptic current frequency as suggested by experiments with the A-type K(+) channel blockers 4-aminopyridine. Taken together our data suggest that IL-1beta inhibits the activity of preoptic area/anterior hypothalamus neurons by increasing the presynaptic release of GABA. PMID:16777343

  19. Interleukin-1 beta induces synthesis and secretion of interleukin-6 in human chondrocytes.

    PubMed

    Bender, S; Haubeck, H D; Van de Leur, E; Dufhues, G; Schiel, X; Lauwerijns, J; Greiling, H; Heinrich, P C

    1990-04-24

    Increased concentrations of interleukin-6 (IL-6) have been found in the synovial fluid of patients with osteoarthritis, rheumatoid arthritis and crystal-related joint diseases. It is therefore of great interest to identify the cells responsible for the production of IL-6, and to investigate whether IL-6 plays a role in the pathogenesis of degenerative or inflammatory joint diseases. Here we show that human interleukin-1 beta (IL-1 beta) induces IL-6 synthesis and secretion in differentiated human chondrocytes. In organ cultures resembling closely the in vivo system 10(6) chondrocytes incubated with 100 units of interleukin-1 beta per ml of medium led to the release of 6 X 10(3) units of IL-6 within 24 h. Chondrocytes cultured in agarose or as monolayers similarly incubated with IL-1 beta produced even higher amounts of IL-6: 70 X 10(3) units per 10(6) cells within 24 h. The induction of IL-6 synthesis by IL-1 beta was also shown at the mRNA level. IL-6 secreted by stimulated chondrocytes showed heterogeneity upon Western blot analysis. PMID:2335234

  20. Glucolipotoxicity of the Pancreatic Beta Cell

    PubMed Central

    Poitout, Vincent; Amyot, Julie; Semache, Meriem; Zarrouki, Bader; Hagman, Derek; Fontés, Ghislaine

    2009-01-01

    Summary The concept of glucolipotoxicity refers to the combined, deleterious effects of elevated glucose and fatty acid levels on pancreatic beta-cell function and survival. Significant progress has been made in recent years towards a better understanding of the cellular and molecular basis of glucolipotoxicity in the beta cell. The permissive effect of elevated glucose on the detrimental actions of fatty acids stems from the influence of glucose on intracellular fatty-acid metabolism, promoting the synthesis of cellular lipids. The combination of excessive levels of fatty acids and glucose therefore leads to decreased insulin secretion, impaired insulin gene expression, and beta-cell death by apoptosis, all of which probably have distinct underlying mechanisms. Recent studies from our laboratory have identified several pathways implicated in fatty-acid inhibition of insulin gene expression, including the extracellular-regulated kinase (ERK1/2) pathway; the metabolic sensor Per-Arnt-Sim kinase (PASK); and the ATF6 branch of the unfolded protein response. We have also confirmed in vivo in rats that the decrease in insulin gene expression is an early defect which precedes any detectable abnormality in insulin secretion. While the role of glucolipotoxicity in humans is still debated, the inhibitory effects of chronically elevated fatty acid levels has been clearly demonstrated in several studies, at least in individuals genetically predisposed to developing type 2 diabetes. It is therefore likely that glucolipotoxicity contributes to beta-cell failure in type 2 diabetes as well as to the decline in beta-cell function observed after the onset of the disease. PMID:19715772

  1. Unraveling the contribution of pancreatic beta-cell suicide in autoimmune type 1 diabetes✩

    PubMed Central

    Jaberi-Douraki, Majid; Schnell, Santiago; Pietropaolo, Massimo; Khadra, Anmar

    2014-01-01

    In type 1 diabetes, an autoimmune disease mediated by autoreactive T-cells that attack insulin-secreting pancreatic beta-cells, it has been suggested that disease progression may additionally require protective mechanisms in the target tissue to impede such auto-destructive mechanisms. We hypothesize that the autoimmune attack against beta-cells causes endoplasmic reticulum stress by forcing the remaining beta-cells to synthesize and secrete defective insulin. To rescue beta-cell from the endoplasmic reticulum stress, beta-cells activate the unfolded protein response to restore protein homeostasis and normal insulin synthesis. Here we investigate the compensatory role of unfolded protein response by developing a multi-state model of type 1 diabetes that takes into account beta-cell destruction caused by pathogenic autoreactive T-cells and apoptosis triggered by endoplasmic reticulum stress. We discuss the mechanism of unfolded protein response activation and how it counters beta-cell extinction caused by an autoimmune attack and/or irreversible damage by endoplasmic reticulum stress. Our results reveal important insights about the balance between beta-cell destruction by autoimmune attack (beta-cell homicide) and beta-cell apoptosis by endoplasmic reticulum stress (beta-cell suicide). It also provides an explanation as to why the unfolded protein response may not be a successful therapeutic target to treat type 1 diabetes. PMID:24831415

  2. Characterization of FKGK18 as inhibitor of group VIA Ca2+-independent phospholipase A2 (iPLA2β): candidate drug for preventing beta-cell apoptosis and diabetes.

    PubMed

    Ali, Tomader; Kokotos, George; Magrioti, Victoria; Bone, Robert N; Mobley, James A; Hancock, William; Ramanadham, Sasanka

    2013-01-01

    Ongoing studies suggest an important role for iPLA2β in a multitude of biological processes and it has been implicated in neurodegenerative, skeletal and vascular smooth muscle disorders, bone formation, and cardiac arrhythmias. Thus, identifying an iPLA2βinhibitor that can be reliably and safely used in vivo is warranted. Currently, the mechanism-based inhibitor bromoenol lactone (BEL) is the most widely used to discern the role of iPLA2β in biological processes. While BEL is recognized as a more potent inhibitor of iPLA2 than of cPLA2 or sPLA2, leading to its designation as a "specific" inhibitor of iPLA2, it has been shown to also inhibit non-PLA2 enzymes. A potential complication of its use is that while the S and R enantiomers of BEL exhibit preference for cytosol-associated iPLA2β and membrane-associated iPLA2γ, respectively, the selectivity is only 10-fold for both. In addition, BEL is unstable in solution, promotes irreversible inhibition, and may be cytotoxic, making BEL not amenable for in vivo use. Recently, a fluoroketone (FK)-based compound (FKGK18) was described as a potent inhibitor of iPLA2β. Here we characterized its inhibitory profile in beta-cells and find that FKGK18: (a) inhibits iPLA2β with a greater potency (100-fold) than iPLA2γ, (b) inhibition of iPLA2β is reversible, (c) is an ineffective inhibitor of α-chymotrypsin, and (d) inhibits previously described outcomes of iPLA2β activation including (i) glucose-stimulated insulin secretion, (ii) arachidonic acid hydrolysis; as reflected by PGE2 release from human islets, (iii) ER stress-induced neutral sphingomyelinase 2 expression, and (iv) ER stress-induced beta-cell apoptosis. These findings suggest that FKGK18 is similar to BEL in its ability to inhibit iPLA2β. Because, in contrast to BEL, it is reversible and not a non-specific inhibitor of proteases, it is suggested that FKGK18 is more ideal for ex vivo and in vivo assessments of iPLA2β role in biological functions. PMID

  3. WS6 induces both alpha and beta cell proliferation without affecting differentiation or viability

    PubMed Central

    Boerner, Brian P.; George, Nicholas M.; Mir, Shakeel U.R.; Sarvetnick, Nora E.

    2016-01-01

    Agents that stimulate human pancreatic beta cell proliferation are needed to improve diabetes mellitus treatment. Recently, a small molecule, WS6, was observed to stimulate human beta cell proliferation. However, little is known about its other effects on human islets. To better understand the role of WS6 as a possible beta cell regenerative therapy, we carried out in-depth phenotypic analysis of WS6-treated human islets, exploring its effects on non-beta cell proliferation, beta cell differentiation, and islet cell viability. WS6 not only stimulated beta cell proliferation in cultured human islets (in agreement with previous reports), but also human alpha cell proliferation, indicating that WS6 is not a beta cell-specific mitogen. WS6 did not change the proportion of insulin-positive beta cells or the expression of beta cell-specific transcription factors, suggesting that WS6 does not alter beta cell differentiation, and WS6 had no effect on human islet cell apoptosis or viability. In conclusion, WS6 stimulates proliferation of both human beta and alpha cells while maintaining cellular viability and the beta cell differentiated phenotype. These findings expand the literature on WS6 and support the suggestion that WS6 may help increase human islet mass needed for successful treatment of diabetes. PMID:25739404

  4. Phenylpropenoic Acid Glucoside from Rooibos Protects Pancreatic Beta Cells against Cell Death Induced by Acute Injury

    PubMed Central

    Himpe, Eddy; Cunha, Daniel A.; Song, Imane; Bugliani, Marco; Marchetti, Piero; Cnop, Miriam; Bouwens, Luc

    2016-01-01

    Objective Previous studies demonstrated that a phenylpropenoic acid glucoside (PPAG) from rooibos (Aspalathus linearis) extract had anti-hyperglycemic activity and significant protective effects on the pancreatic beta cell mass in a chronic diet-induced diabetes model. The present study evaluated the cytoprotective effect of the phytochemical on beta cells exposed to acute cell stress. Methods Synthetically prepared PPAG was administered orally in mice treated with a single dose of streptozotocin to acutely induce beta cell death and hyperglycemia. Its effect was assessed on beta cell mass, proliferation and apoptotic cell death. Its cytoprotective effect was also studied in vitro on INS-1E beta cells and on human pancreatic islet cells. Results Treatment with the phytochemical PPAG protected beta cells during the first days after the insult against apoptotic cell death, as evidenced by TUNEL staining, and prevented loss of expression of anti-apoptotic protein BCL2 in vivo. In vitro, PPAG protected INS-1E beta cells from streptozotocin-induced apoptosis and necrosis in a BCL2-dependent and independent way, respectively, depending on glucose concentration. PPAG also protected human pancreatic islet cells against the cytotoxic action of the fatty acid palmitate. Conclusions These findings show the potential use of PPAG as phytomedicine which protects the beta cell mass exposed to acute diabetogenic stress. PMID:27299564

  5. Regulation of pancreatic beta-cell mass.

    PubMed

    Bouwens, Luc; Rooman, Ilse

    2005-10-01

    Beta-cell mass regulation represents a critical issue for understanding diabetes, a disease characterized by a near-absolute (type 1) or relative (type 2) deficiency in the number of pancreatic beta cells. The number of islet beta cells present at birth is mainly generated by the proliferation and differentiation of pancreatic progenitor cells, a process called neogenesis. Shortly after birth, beta-cell neogenesis stops and a small proportion of cycling beta cells can still expand the cell number to compensate for increased insulin demands, albeit at a slow rate. The low capacity for self-replication in the adult is too limited to result in a significant regeneration following extensive tissue injury. Likewise, chronically increased metabolic demands can lead to beta-cell failure to compensate. Neogenesis from progenitor cells inside or outside islets represents a more potent mechanism leading to robust expansion of the beta-cell mass, but it may require external stimuli. For therapeutic purposes, advantage could be taken from the surprising differentiation plasticity of adult pancreatic cells and possibly also from stem cells. Recent studies have demonstrated that it is feasible to regenerate and expand the beta-cell mass by the application of hormones and growth factors like glucagon-like peptide-1, gastrin, epidermal growth factor, and others. Treatment with these external stimuli can restore a functional beta-cell mass in diabetic animals, but further studies are required before it can be applied to humans. PMID:16183912

  6. The class I histone deacetylase inhibitor MS-275 prevents pancreatic beta cell death induced by palmitate.

    PubMed

    Plaisance, Valérie; Rolland, Laure; Gmyr, Valéry; Annicotte, Jean-Sébastien; Kerr-Conte, Julie; Pattou, François; Abderrahmani, Amar

    2014-01-01

    Elevation of the dietary saturated fatty acid palmitate contributes to the reduction of functional beta cell mass in the pathogenesis of type 2 diabetes. The diabetogenic effect of palmitate is achieved by increasing beta cell death through induction of the endoplasmic reticulum (ER) stress markers including activating transcription factor 3 (Atf3) and CAAT/enhancer-binding protein homologous protein-10 (Chop). In this study, we investigated whether treatment of beta cells with the MS-275, a HDAC1 and HDAC3 activity inhibitor which prevents beta cell death elicited by cytokines, is beneficial for combating beta cell dysfunction caused by palmitate. We show that culture of isolated human islets and MIN6 cells with MS-275 reduced apoptosis evoked by palmitate. The protective effect of MS-275 was associated with the attenuation of the expression of Atf3 and Chop. Silencing of HDAC3, but not of HDAC1, mimicked the effects of MS-275 on the expression of the two ER stress markers and apoptosis. These data point to HDAC3 as a potential drug target for preserving beta cells against lipotoxicity in diabetes. PMID:25610877

  7. The Class I Histone Deacetylase Inhibitor MS-275 Prevents Pancreatic Beta Cell Death Induced by Palmitate

    PubMed Central

    Plaisance, Valérie; Rolland, Laure; Gmyr, Valéry; Annicotte, Jean-Sébastien; Kerr-Conte, Julie; Pattou, François; Abderrahmani, Amar

    2014-01-01

    Elevation of the dietary saturated fatty acid palmitate contributes to the reduction of functional beta cell mass in the pathogenesis of type 2 diabetes. The diabetogenic effect of palmitate is achieved by increasing beta cell death through induction of the endoplasmic reticulum (ER) stress markers including activating transcription factor 3 (Atf3) and CAAT/enhancer-binding protein homologous protein-10 (Chop). In this study, we investigated whether treatment of beta cells with the MS-275, a HDAC1 and HDAC3 activity inhibitor which prevents beta cell death elicited by cytokines, is beneficial for combating beta cell dysfunction caused by palmitate. We show that culture of isolated human islets and MIN6 cells with MS-275 reduced apoptosis evoked by palmitate. The protective effect of MS-275 was associated with the attenuation of the expression of Atf3 and Chop. Silencing of HDAC3, but not of HDAC1, mimicked the effects of MS-275 on the expression of the two ER stress markers and apoptosis. These data point to HDAC3 as a potential drug target for preserving beta cells against lipotoxicity in diabetes. PMID:25610877

  8. MST1: a promising therapeutic target to restore functional beta cell mass in diabetes.

    PubMed

    Ardestani, Amin; Maedler, Kathrin

    2016-09-01

    The loss of insulin-producing beta cells by apoptosis is a hallmark of all forms of diabetes mellitus. Strategies to prevent beta cell apoptosis and dysfunction are urgently needed to restore the insulin-producing cells and to prevent severe diabetes progression. We recently identified the serine/threonine kinase known as mammalian sterile 20-like kinase 1 (MST1) as a critical regulator of apoptotic beta cell death and dysfunction. MST1 activates several apoptotic signalling pathways, which further stimulate its own cleavage, leading to a vicious cycle of cell death. This led us to hypothesise that MST1 signalling is central to the initiation of beta cell death in diabetes. We found that MST1 is strongly activated in a diabetic beta cell and induces not only its death but also directly impairs insulin secretion through promoting proteasomal degradation of key beta cell transcription factor, pancreatic and duodenal homeobox 1 (PDX1), which is critical for insulin production.Pre-clinical studies in various animal models of diabetes have reported that MST1 deficiency remarkably restores normoglycaemia and beta cell function and prevents the development of diabetes. Importantly, MST1 deficiency can revert fully diabetic beta cells to a non-diabetic state. MST1 may serve as a target for the development of novel therapies for diabetes that trigger the cause of the disease, namely, the destruction of the beta cells. The major current focus of our investigation is to identify and test the efficacy of potent inhibitors of this death signalling pathway to protect beta cells against the effects of autoimmune attack in type 1 diabetes and to preserve beta cell mass and function in type 2 diabetes. This review summarises a presentation given at the 'Can we make a better beta cell?' symposium at the 2015 annual meeting of the EASD. It is accompanied by two other reviews on topics from this symposium (by Heiko Lickert and colleagues, DOI: 10.1007/s00125-016-3949-9 , and by Harry

  9. Pituitary tumor transforming gene-null male mice exhibit impaired pancreatic beta cell proliferation and diabetes

    PubMed Central

    Wang, Zhiyong; Moro, Enrico; Kovacs, Kalman; Yu, Run; Melmed, Shlomo

    2003-01-01

    The mammalian securin, pituitary tumor transforming gene (PTTG), regulates sister chromatid separation during mitosis. Mice or cell lines deficient in PTTG expression, however, are surprisingly viable. Here we show that PTTG disruption in mice (PTTG−/−) severely impairs glucose homeostasis leading to diabetes during late adulthood, especially in males associated with nonautoimmune insulinopenia and reversed alpha/beta cell ratio. Islet beta cell mass in PTTG−/− mice was already diminished before development of frank diabetes and only increased minimally during growth. BrdUrd incorporation of islet cells in PTTG-null mice was ≈65% lower (P < 0.005) than in the WT pancreas, whereas apoptosis rates were similar. PTTG−/− beta cells had pleiotropic nuclei, suggesting defects in cell division. The results indicated that securin is indispensable for normal pancreatic beta cell proliferation. PMID:12626748

  10. Osteocalcin protects pancreatic beta cell function and survival under high glucose conditions

    SciTech Connect

    Kover, Karen; Yan, Yun; Tong, Pei Ying; Watkins, Dara; Li, Xiaoyu; Tasch, James; Hager, Melissa; Clements, Mark; Moore, Wayne V.

    2015-06-19

    Diabetes is characterized by progressive beta cell dysfunction and loss due in part to oxidative stress that occurs from gluco/lipotoxicity. Treatments that directly protect beta cell function and survival in the diabetic milieu are of particular interest. A growing body of evidence suggests that osteocalcin, an abundant non-collagenous protein of bone, supports beta cell function and proliferation. Based on previous gene expression data by microarray, we hypothesized that osteocalcin protects beta cells from glucose-induced oxidative stress. To test our hypothesis we cultured isolated rat islets and INS-1E cells in the presence of normal, high, or high glucose ± osteocalcin for up to 72 h. Oxidative stress and viability/mitochondrial function were measured by H{sub 2}O{sub 2} assay and Alamar Blue assay, respectively. Caspase 3/7 activity was also measured as a marker of apoptosis. A functional test, glucose stimulated insulin release, was conducted and expression of genes/protein was measured by qRT-PCR/western blot/ELISA. Osteocalcin treatment significantly reduced high glucose-induced H{sub 2}O{sub 2} levels while maintaining viability/mitochondrial function. Osteocalcin also significantly improved glucose stimulated insulin secretion and insulin content in rat islets after 48 h of high glucose exposure compared to untreated islets. As expected sustained high glucose down-regulated gene/protein expression of INS1 and BCL2 while increasing TXNIP expression. Interestingly, osteocalcin treatment reversed the effects of high glucose on gene/protein expression. We conclude that osteocalcin can protect beta cells from the negative effects of glucose-induced oxidative stress, in part, by reducing TXNIP expression, thereby preserving beta cell function and survival. - Highlights: • Osteocalcin reduces glucose-induced oxidative stress in beta cells. • Osteocalcin preserves beta cell function and survival under stress conditions. • Osteocalcin reduces glucose

  11. A synopsis of factors regulating beta cell development and beta cell mass.

    PubMed

    Prasadan, Krishna; Shiota, Chiyo; Xiangwei, Xiao; Ricks, David; Fusco, Joseph; Gittes, George

    2016-10-01

    The insulin-secreting beta cells in the endocrine pancreas regulate blood glucose levels, and loss of functional beta cells leads to insulin deficiency, hyperglycemia (high blood glucose) and diabetes mellitus. Current treatment strategies for type-1 (autoimmune) diabetes are islet transplantation, which has significant risks and limitations, or normalization of blood glucose with insulin injections, which is clearly not ideal. The type-1 patients can lack insulin counter-regulatory mechanism; therefore, hypoglycemia is a potential risk. Hence, a cell-based therapy offers a better alternative for the treatment of diabetes. Past research was focused on attempting to generate replacement beta cells from stem cells; however, recently there has been an increasing interest in identifying mechanisms that will lead to the conversion of pre-existing differentiated endocrine cells into beta cells. The goal of this review is to provide an overview of several of the key factors that regulate new beta cell formation (neogenesis) and beta cell proliferation. PMID:27105622

  12. NADPH oxidase mediates glucolipotoxicity-induced beta cell dysfunction--clinical implications.

    PubMed

    McCarty, Mark F; Barroso-Aranda, Jorge; Contreras, Francisco

    2010-03-01

    An impairment of glucose-stimulated insulin secretion--reflecting decreased glucokinase expression--and a moderate decrease in beta cell mass attributable to increased apoptosis, constitute the key features of beta cell failure in type 2 diabetes. Oxidative stress, provoked by prolonged exposure to excessive levels of glucose and/or fatty acids (glucolipotoxicity), appears to be a key mediator of these defects. Oxidant-provoked JNK activation induces nuclear export of the PDX-1 transcription factor, required for expression of glucokinase and other beta cell proteins. Conversely, increases in cAMP induced by incretin hormones promote the nuclear importation of PDX-1, counteracting the diabetogenic impact of oxidant stress; this may explain the utility of measures that slow dietary carbohydrate absorption for diabetes prevention. The ability of oxidative stress to boost apoptosis in beta cells is poorly understood, but may also entail JNK activation. Recent work establishes a phagocyte-type NADPH oxidase as the chief source of glucotoxicity-mediated oxidative stress in beta cells. Since bilirubin is now known to function physiologically as an inhibitor of NADPH oxidase, and phycocyanobilin (PCB) derived from spirulina likewise can inhibit this enzyme complex, supplemental PCB may have utility in the prevention and control of diabetes, and Gilbert syndrome, associated with chronically elevated free bilirubin, may be associated with decreased diabetes risk. PMID:19576699

  13. Translation attenuation through eIF2α phosphorylation prevents oxidative stress and maintains the differentiated state in beta cells

    PubMed Central

    Back, Sung Hoon; Scheuner, Donalyn; Han, JaeSeok; Song, Benbo; Ribick, Mark; Wang, Junying; Gildersleeve, Robert D.; Pennathur, Subramaniam; Kaufman, Randal J.

    2009-01-01

    SUMMARY Accumulation of unfolded protein within the endoplasmic reticulum (ER) lumen attenuates mRNA translation through activation of the protein kinase PERK and subsequent phosphorylation of eukaryotic initiation factor 2 on Ser51 of the alpha subunit (eIF2α). Genetic disruption of the PERK/eIF2α pathway in humans and mice produces severe pancreatic beta cell deficiency and post-natal lethality. To elucidate the role of eIF2α phosphorylation in beta cells, we have rescued the lethality of homozygous eIF2α Ser51Ala mice by expression of a loxP-flanked wild-type eIF2α transgene. Beta cell-specific transgene deletion to prevent eIF2α phosphorylation caused a severe diabetic phenotype due to heightened, unregulated proinsulin translation, defective intracellular trafficking of secretory and plasma membrane proteins, increased oxidative damage, reduced expression of stress response and beta cell-specific genes, and apoptosis. However, glucose intolerance and beta cell death in these mice were attenuated by antioxidant treatment. We conclude that phosphorylation of eIF2α coordinately attenuates mRNA translation, prevents oxidative stress, and optimizes ER protein folding to support insulin production in the beta cell. These findings that show increased proinsulin synthesis causes oxidative stress leading to beta cell failure may reflect events in the beta cell loss associated with insulin resistance in type 2 diabetes. PMID:19583950

  14. Toward beta cell replacement for diabetes

    PubMed Central

    Johannesson, Bjarki; Sui, Lina; Freytes, Donald O; Creusot, Remi J; Egli, Dieter

    2015-01-01

    The discovery of insulin more than 90 years ago introduced a life-saving treatment for patients with type 1 diabetes, and since then, significant progress has been made in clinical care for all forms of diabetes. However, no method of insulin delivery matches the ability of the human pancreas to reliably and automatically maintain glucose levels within a tight range. Transplantation of human islets or of an intact pancreas can in principle cure diabetes, but this approach is generally reserved for cases with simultaneous transplantation of a kidney, where immunosuppression is already a requirement. Recent advances in cell reprogramming and beta cell differentiation now allow the generation of personalized stem cells, providing an unlimited source of beta cells for research and for developing autologous cell therapies. In this review, we will discuss the utility of stem cell-derived beta cells to investigate the mechanisms of beta cell failure in diabetes, and the challenges to develop beta cell replacement therapies. These challenges include appropriate quality controls of the cells being used, the ability to generate beta cell grafts of stable cellular composition, and in the case of type 1 diabetes, protecting implanted cells from autoimmune destruction without compromising other aspects of the immune system or the functionality of the graft. Such novel treatments will need to match or exceed the relative safety and efficacy of available care for diabetes. PMID:25733347

  15. Toward beta cell replacement for diabetes.

    PubMed

    Johannesson, Bjarki; Sui, Lina; Freytes, Donald O; Creusot, Remi J; Egli, Dieter

    2015-04-01

    The discovery of insulin more than 90 years ago introduced a life-saving treatment for patients with type 1 diabetes, and since then, significant progress has been made in clinical care for all forms of diabetes. However, no method of insulin delivery matches the ability of the human pancreas to reliably and automatically maintain glucose levels within a tight range. Transplantation of human islets or of an intact pancreas can in principle cure diabetes, but this approach is generally reserved for cases with simultaneous transplantation of a kidney, where immunosuppression is already a requirement. Recent advances in cell reprogramming and beta cell differentiation now allow the generation of personalized stem cells, providing an unlimited source of beta cells for research and for developing autologous cell therapies. In this review, we will discuss the utility of stem cell-derived beta cells to investigate the mechanisms of beta cell failure in diabetes, and the challenges to develop beta cell replacement therapies. These challenges include appropriate quality controls of the cells being used, the ability to generate beta cell grafts of stable cellular composition, and in the case of type 1 diabetes, protecting implanted cells from autoimmune destruction without compromising other aspects of the immune system or the functionality of the graft. Such novel treatments will need to match or exceed the relative safety and efficacy of available care for diabetes. PMID:25733347

  16. Transcription factors involved in glucose-stimulated insulin secretion of pancreatic beta cells

    SciTech Connect

    Shao, Shiying; Fang, Zhong; Yu, Xuefeng; Zhang, Muxun

    2009-07-10

    GSIS, the most important function of pancreatic beta cell, is essential for maintaining the glucose homeostasis. Transcription factors are known to control different biological processes such as differentiation, proliferation and apoptosis. In pancreas, some transcription factors are involved in regulating the function of beta cells. In this review, the role of these transcription factors including Pdx-1, FoxO1, SREBP-1c, and MafA in GSIS is highlighted. The related molecular mechanisms are analyzed as well. Furthermore, the association between the role of transcription factors in GSIS and the development of T2DM is discussed.

  17. On the origin of the beta cell.

    PubMed

    Oliver-Krasinski, Jennifer M; Stoffers, Doris A

    2008-08-01

    The major forms of diabetes are characterized by pancreatic islet beta-cell dysfunction and decreased beta-cell numbers, raising hope for cell replacement therapy. Although human islet transplantation is a cell-based therapy under clinical investigation for the treatment of type 1 diabetes, the limited availability of human cadaveric islets for transplantation will preclude its widespread therapeutic application. The result has been an intense focus on the development of alternate sources of beta cells, such as through the guided differentiation of stem or precursor cell populations or the transdifferentiation of more plentiful mature cell populations. Realizing the potential for cell-based therapies, however, requires a thorough understanding of pancreas development and beta-cell formation. Pancreas development is coordinated by a complex interplay of signaling pathways and transcription factors that determine early pancreatic specification as well as the later differentiation of exocrine and endocrine lineages. This review describes the current knowledge of these factors as they relate specifically to the emergence of endocrine beta cells from pancreatic endoderm. Current therapeutic efforts to generate insulin-producing beta-like cells from embryonic stem cells have already capitalized on recent advances in our understanding of the embryonic signals and transcription factors that dictate lineage specification and will most certainly be further enhanced by a continuing emphasis on the identification of novel factors and regulatory relationships. PMID:18676806

  18. Proliferating pancreatic beta-cells upregulate ALDH.

    PubMed

    Liu, Yinglan; Jiang, Xiaoxin; Zeng, Yong; Zhou, Hui; Yang, Jing; Cao, Renxian

    2014-12-01

    High levels of aldehyde dehydrogenase (ALDH) activity have been regarded as a specific feature of progenitor cells and stem cells. Hence, as an indicator of ALDH activity, aldefluor fluorescence has been widely used for the identification and isolation of stem and progenitor cells. ALDH activity was recently detected in embryonic mouse pancreas, and specifically and exclusively in adult centroacinar and terminal duct cells, suggesting that these duct cells may harbor cells of endocrine and exocrine differentiation potential in the adult pancreas. Here, we report the presence of aldefluor+ beta-cells in a beta-cell proliferation model, partial pancreatectomy. The aldefluor+ beta-cells are essentially all positive for Ki-67 and expressed high levels of cell-cycle activators such as CyclinD1, CyclinD2, and CDK4, suggesting that they are mitotic cells. Our data thus reveal a potential change in ALDH activity of proliferating beta-cells, which provides a novel method for the isolation and analysis of proliferating beta-cells. Moreover, our data also suggest that aldefluor lineage-tracing is not a proper method for analyzing progenitor or stem activity in the adult pancreas. PMID:25028343

  19. Dopamine Modulates Insulin Release and Is Involved in the Survival of Rat Pancreatic Beta Cells

    PubMed Central

    Iglesias Osma, Maria Carmen; Blanco, Enrique J.; Carretero Hernández, Marta; Sánchez Robledo, Virginia; Catalano Iniesta, Leonardo; Carrero, Sixto

    2015-01-01

    The local synthesis of dopamine and its effects on insulin release have been described in isolated islets. Thus, it may be accepted that dopamine exerts an auto-paracrine regulation of insulin secretion from pancreatic beta cells. The aim of the present study is to analyze whether dopamine is a regulator of the proliferation and apoptosis of rat pancreatic beta cells after glucose-stimulated insulin secretion. Glucose stimulated pancreatic islets obtained from male Wistar rats were cultured with 1 or 10 μM dopamine from 1 to 12 h. Insulin secretion was analyzed by RIA. The cellular proliferation rate of pancreatic islets and beta cells was studied with immunocytochemical double labelling for both insulin and PCNA (proliferating cell nuclear antigen), and active caspase-3 was detected to evaluate apoptosis. The secretion of insulin from isolated islets was significantly inhibited (p<0.01), by treatment with 1 and 10 μM dopamine, with no differences between either dose as early as 1 h after treatment. The percentage of insulin-positive cells in the islets decreased significantly (p<0.01) after 1 h of treatment up to 12 h. The proliferation rate of insulin-positive cells in the islets decreased significantly (p<0.01) following treatment with dopamine. Apoptosis in pancreatic islets and beta cells was increased by treatment with 1 and 10 μM dopamine along 12 h. In conclusion, these results suggest that dopamine could modulate the proliferation and apoptosis of pancreatic beta cells and that dopamine may be involved in the maintenance of pancreatic islets. PMID:25886074

  20. Role of Bioactive Food Components in Diabetes Prevention: Effects on Beta-Cell Function and Preservation

    PubMed Central

    Oh, Yoon Sin; Jun, Hee-Sook

    2014-01-01

    Bioactive compounds found in fruits and vegetables can have anti-oxidant, anti-inflammatory, and anti-carcinogenic effects and can be protective against various diseases and metabolic disorders. These beneficial effects make them good candidates for the development of new functional foods with potential protective and preventive properties for type 1 and type 2 diabetes. This review summarizes the most relevant results concerning the effects of various bioactive compounds such as flavonoids, vitamins, and carotenoids on several aspects of beta-cell functionality. Studies using animal models with induced diabetes and diabetic patients support the hypothesis that bioactive compounds could ameliorate diabetic phenotypes. Published data suggest that there might be direct effects of bioactive compounds on enhancing insulin secretion and preventing beta-cell apoptosis, and some compounds might modulate beta-cell proliferation. Further research is needed to establish any clinical effects of these compounds. PMID:25092987

  1. Plant-Derived Compounds Targeting Pancreatic Beta Cells for the Treatment of Diabetes

    PubMed Central

    Oh, Yoon Sin

    2015-01-01

    Diabetes is a global health problem and a national economic burden. Although several antidiabetic drugs are available, the need for novel therapeutic agents with improved efficacy and few side effects remains. Drugs derived from natural compounds are more attractive than synthetic drugs because of their diversity and minimal side effects. This review summarizes the most relevant effects of various plant-derived natural compounds on the functionality of pancreatic beta cells. Published data suggest that natural compounds directly enhance insulin secretion, prevent pancreatic beta cell apoptosis, and modulate pancreatic beta cell differentiation and proliferation. It is essential to continuously investigate natural compounds as sources of novel pharmaceuticals. Therefore, more studies into these compounds' mechanisms of action are warranted for their development as potential anti-diabetics. PMID:26587047

  2. ER stress and the decline and fall of pancreatic beta cells in type 1 diabetes

    PubMed Central

    Brozzi, Flora

    2016-01-01

    Components of the unfolded protein response (UPR) modulate beta cell inflammation and death in early type 1 diabetes (T1D). The UPR is a mechanism by which cells react to the accumulation of misfolded proteins in the endoplasmic reticulum (ER). It aims to restore cellular homeostasis, but in case of chronic or overwhelming ER stress the persistent activation of the UPR triggers apoptosis, contributing to the loss of beta cells in both T1D and type 2 diabetes. It remains to be determined how and why the transition from ‘physiological’ to ‘pathological’ UPR takes place. A key component of the UPR is the ER transmembrane protein IRE1α (inositol-requiring enzyme 1α). IRE1α activity is modulated by both intra-ER signals and by the formation of protein complexes at its cytosolic domain. The amplitude and duration of IRE1α signaling is critical for the transition between the adaptive and cell death programs, with particular relevance for the activation of the pro-apoptotic c-Jun N-terminal kinase (JNK) in beta cells. In the present review we discuss the available information on IRE1α-regulating proteins in beta cells and their downstream targets, and the important differences observed between cytokine-induced UPR in human and rodent beta cells. PMID:26899404

  3. Human Fucci Pancreatic Beta Cell Lines: New Tools to Study Beta Cell Cycle and Terminal Differentiation

    PubMed Central

    Carlier, Géraldine; Maugein, Alicia; Cordier, Corinne; Pechberty, Séverine; Garfa-Traoré, Meriem; Martin, Patrick; Scharfmann, Raphaël; Albagli, Olivier

    2014-01-01

    Regulation of cell cycle in beta cells is poorly understood, especially in humans. We exploited here the recently described human pancreatic beta cell line EndoC-βH2 to set up experimental systems for cell cycle studies. We derived 2 populations from EndoC-βH2 cells that stably harbor the 2 genes encoding the Fucci fluorescent indicators of cell cycle, either from two vectors, or from a unique bicistronic vector. In proliferating non-synchronized cells, the 2 Fucci indicators revealed cells in the expected phases of cell cycle, with orange and green cells being in G1 and S/G2/M cells, respectively, and allowed the sorting of cells in different substeps of G1. The Fucci indicators also faithfully red out alterations in human beta cell proliferative activity since a mitogen-rich medium decreased the proportion of orange cells and inflated the green population, while reciprocal changes were observed when cells were induced to cease proliferation and increased expression of some beta cell genes. In the last situation, acquisition of a more differentiated beta cell phenotype correlates with an increased intensity in orange fluorescence. Hence Fucci beta cell lines provide new tools to address important questions regarding human beta cell cycle and differentiation. PMID:25259951

  4. Specific Silencing of the REST Target Genes in Insulin-Secreting Cells Uncovers Their Participation in Beta Cell Survival

    PubMed Central

    Gesina, Emilie; Caille, Dorothee; Gjinovci, Asllan; Waeber, Gerard; Meda, Paolo; Haefliger, Jacques-Antoine

    2012-01-01

    The absence of the transcriptional repressor RE-1 Silencing Transcription Factor (REST) in insulin-secreting beta cells is a major cue for the specific expression of a large number of genes. These REST target genes were largely ascribed to a function of neurotransmission in a neuronal context, whereas their role in pancreatic beta cells has been poorly explored. To identify their functional significance, we have generated transgenic mice expressing REST in beta cells (RIP-REST mice), and previously discovered that REST target genes are essential to insulin exocytosis. Herein we characterized a novel line of RIP-REST mice featuring diabetes. In diabetic RIP-REST mice, high levels of REST were associated with postnatal beta cell apoptosis, which resulted in gradual beta cell loss and sustained hyperglycemia in adults. Moreover, adenoviral REST transduction in INS-1E cells led to increased cell death under control conditions, and sensitized cells to death induced by cytokines. Screening for REST target genes identified several anti-apoptotic genes bearing the binding motif RE-1 that were downregulated upon REST expression in INS-1E cells, including Gjd2, Mapk8ip1, Irs2, Ptprn, and Cdk5r2. Decreased levels of Cdk5r2 in beta cells of RIP-REST mice further confirmed that it is controlled by REST, in vivo. Using siRNA-mediated knock-down in INS-1E cells, we showed that Cdk5r2 protects beta cells against cytokines and palmitate-induced apoptosis. Together, these data document that a set of REST target genes, including Cdk5r2, is important for beta cell survival. PMID:23029270

  5. Specific silencing of the REST target genes in insulin-secreting cells uncovers their participation in beta cell survival.

    PubMed

    Martin, David; Allagnat, Florent; Gesina, Emilie; Caille, Dorothee; Gjinovci, Asllan; Waeber, Gerard; Meda, Paolo; Haefliger, Jacques-Antoine

    2012-01-01

    The absence of the transcriptional repressor RE-1 Silencing Transcription Factor (REST) in insulin-secreting beta cells is a major cue for the specific expression of a large number of genes. These REST target genes were largely ascribed to a function of neurotransmission in a neuronal context, whereas their role in pancreatic beta cells has been poorly explored. To identify their functional significance, we have generated transgenic mice expressing REST in beta cells (RIP-REST mice), and previously discovered that REST target genes are essential to insulin exocytosis. Herein we characterized a novel line of RIP-REST mice featuring diabetes. In diabetic RIP-REST mice, high levels of REST were associated with postnatal beta cell apoptosis, which resulted in gradual beta cell loss and sustained hyperglycemia in adults. Moreover, adenoviral REST transduction in INS-1E cells led to increased cell death under control conditions, and sensitized cells to death induced by cytokines. Screening for REST target genes identified several anti-apoptotic genes bearing the binding motif RE-1 that were downregulated upon REST expression in INS-1E cells, including Gjd2, Mapk8ip1, Irs2, Ptprn, and Cdk5r2. Decreased levels of Cdk5r2 in beta cells of RIP-REST mice further confirmed that it is controlled by REST, in vivo. Using siRNA-mediated knock-down in INS-1E cells, we showed that Cdk5r2 protects beta cells against cytokines and palmitate-induced apoptosis. Together, these data document that a set of REST target genes, including Cdk5r2, is important for beta cell survival. PMID:23029270

  6. Effects of exendin-4 on glucose tolerance, insulin secretion, and beta-cell proliferation depend on treatment dose, treatment duration and meal contents

    SciTech Connect

    Arakawa, Masayuki; Ebato, Chie; Mita, Tomoya; Hirose, Takahisa; Kawamori, Ryuzo; Fujitani, Yoshio; Watada, Hirotaka

    2009-12-18

    Beta-cell proliferation is regulated by various metabolic demands including peripheral insulin resistance, obesity, and hyperglycemia. In addition to enhancement of glucose-induced insulin secretion, agonists for glucagon-like peptide-1 receptor (GLP-1R) stimulate proliferation and inhibit apoptosis of beta-cells, thereby probably preserve beta-cell mass. To evaluate the beta-cell preserving actions of GLP-1R agonists, we assessed the acute and chronic effects of exendin-4 on beta-cell proliferation, mass and glucose tolerance in C57BL/6J mice under various conditions. Short-term administration of high-dose exendin-4 transiently stimulated beta-cell proliferation. Comparative transcriptomic analysis showed upregulation of IGF-1 receptor and its downstream effectors in islets. Treatment of mice with exendin-4 daily for 4 weeks (long-term administration) and feeding high-fat diet resulted in significant inhibition of weight gain and improvement of glucose tolerance with reduced insulin secretion and beta-cell mass. These findings suggest that long-term GLP-1 treatment results in insulin sensitization of peripheral organs, rather than enhancement of beta-cell proliferation and function, particularly when animals are fed high-fat diet. Thus, the effects of exendin-4 on glucose tolerance, insulin secretion, and beta-cell proliferation largely depend on treatment dose, duration of treatment and meal contents. While GLP-1 enhances proliferation of beta-cells in some diabetic mice models, our results suggest that GLP-1 stimulates beta-cell growth only when expansion of beta-cell mass is required to meet metabolic demands.

  7. Monitoring of beta cell replacement outcomes.

    PubMed

    Chang, Charles A; Haque, Waqas Z; Yoshimatsu, Gumpei; Balajii, Prathab S; Lawrence, Michael C; Naziruddin, Bashoo

    2016-03-01

    Pancreatic islet transplantation is a promising beta cell replacement treatment for patients with "brittle" type 1 diabetes (T1D) or intractable chronic pancreatitis to restore or preserve pancreatic endocrine function. Early after transplant, a significant islet mass is lost due to an innate inflammatory response, and further loss of the islet graft occurs over time due to immune response, drug toxicity, or metabolic exhaustion. Thus, clinically feasible techniques are essential to monitor islet graft function and survival to maintain appropriate therapy. Currently, islet graft function is monitored using blood glucose levels, insulin and C-peptide levels, and islet imaging. However, these tests are influenced by physiological changes, including beta cell stimulation. Biomarkers that are independent of metabolic stimuli would be more accurate and reliable in detecting islet damage. Antibodies against islet autoantigens are useful but not reliable markers of islet injury due to their presence during the pretransplant period. Several islet-specific proteins such as Glutamate decarboxylase-65, doublecortin, protein phosphatase 1, regulatory (inhibitor) subunit 1A, ubiquitin C-terminal hydrolase-L1, and the high-mobility group box-1 protein have been proposed as candidates to monitor islet damage, but these biomarkers have short half-lives and unreliable detection. Unmethylated insulin DNA has been studied in T1D patients and has been documented as a highly correlative and selective biomarker for beta cell death. More recently, microRNAs (miRNAs) that are selectively expressed in islets have been shown to provide sensitive and accurate quantification of islet damage. Analysis of plasma samples from autologous and allogeneic islet transplant patients has demonstrated the value of miRNA-375 as a specific biomarker to accurately assess islet damage. Use of selective, sensitive, and measurably reproducible biomarkers of islets will lead to effective monitoring of beta

  8. MicroRNAs as regulators of beta-cell function and dysfunction.

    PubMed

    Osmai, Mirwais; Osmai, Yama; Bang-Berthelsen, Claus H; Pallesen, Emil M H; Vestergaard, Anna L; Novotny, Guy W; Pociot, Flemming; Mandrup-Poulsen, Thomas

    2016-05-01

    In the last decade, there has been an explosion in both the number of and knowledge about miRNAs associated with both type 1 and type 2 diabetes. Even though we are presently in the initial stages of understanding how this novel class of posttranscriptional regulators are involved in diabetes, recent studies have demonstrated that miRNAs are important regulators of the islet transcriptome, controlling apoptosis, differentiation and proliferation, as well as regulating unique islet and beta-cell functions and pathways such as insulin expression, processing and secretion. Furthermore, a large number of miRNAs have been linked to diabetogenic processes induced by elevated levels of glucose, free fatty acids and inflammatory cytokines. Thus, miRNAs are novel therapeutic targets with the potential of protecting the beta-cell, and there is proof of principle that miRNA antagonists, so-called antagomirs, are effective in vivo for other disorders. miRNAs are exported out of cells in exosomes, raising the intriguing possibility of cell-to-cell communication between distant tissues via miRNAs and that miRNAs can be used as biomarkers of beta-cell function, mass and survival. The purpose of this review is to provide a status on how miRNAs control beta-cell function and viability in health and disease. Copyright © 2015 John Wiley & Sons, Ltd. PMID:26418758

  9. Cocaine- and Amphetamine-regulated Transcript (CART) Protects Beta Cells against Glucotoxicity and Increases Cell Proliferation*

    PubMed Central

    Sathanoori, Ramasri; Olde, Björn; Erlinge, David; Göransson, Olga; Wierup, Nils

    2013-01-01

    Cocaine- and amphetamine-regulated transcript (CART) is an islet peptide that promotes glucose-stimulated insulin secretion in beta cells via cAMP/PKA-dependent pathways. In addition, CART is a regulator of neuronal survival. In this study, we examined the effect of exogenous CART 55–102 on beta cell viability and dissected its signaling mechanisms. Evaluation of DNA fragmentation and chromatin condensation revealed that CART 55–102 reduced glucotoxicity-induced apoptosis in both INS-1 (832/13) cells and isolated rat islets. Glucotoxicity in INS-1 (832/13) cells also caused a 50% reduction of endogenous CART protein. We show that CART increased proliferation in INS-1 (832/13) cells, an effect that was blocked by PKA, PKB, and MEK1 inhibitors. In addition, CART induced phosphorylation of CREB, IRS, PKB, FoxO1, p44/42 MAPK, and p90RSK in INS-1 (832/13) cells and isolated rat islets, all key mediators of cell survival and proliferation. Thus, we demonstrate that CART 55-102 protects beta cells against glucotoxicity and promotes proliferation. Taken together our data point to the potential use of CART in therapeutic interventions targeted at enhancing functional beta cell mass and long-term insulin secretion in T2D. PMID:23250745

  10. Cocaine- and amphetamine-regulated transcript (CART) protects beta cells against glucotoxicity and increases cell proliferation.

    PubMed

    Sathanoori, Ramasri; Olde, Björn; Erlinge, David; Göransson, Olga; Wierup, Nils

    2013-02-01

    Cocaine- and amphetamine-regulated transcript (CART) is an islet peptide that promotes glucose-stimulated insulin secretion in beta cells via cAMP/PKA-dependent pathways. In addition, CART is a regulator of neuronal survival. In this study, we examined the effect of exogenous CART 55-102 on beta cell viability and dissected its signaling mechanisms. Evaluation of DNA fragmentation and chromatin condensation revealed that CART 55-102 reduced glucotoxicity-induced apoptosis in both INS-1 (832/13) cells and isolated rat islets. Glucotoxicity in INS-1 (832/13) cells also caused a 50% reduction of endogenous CART protein. We show that CART increased proliferation in INS-1 (832/13) cells, an effect that was blocked by PKA, PKB, and MEK1 inhibitors. In addition, CART induced phosphorylation of CREB, IRS, PKB, FoxO1, p44/42 MAPK, and p90RSK in INS-1 (832/13) cells and isolated rat islets, all key mediators of cell survival and proliferation. Thus, we demonstrate that CART 55-102 protects beta cells against glucotoxicity and promotes proliferation. Taken together our data point to the potential use of CART in therapeutic interventions targeted at enhancing functional beta cell mass and long-term insulin secretion in T2D. PMID:23250745

  11. Insulin receptor alternative splicing is regulated by insulin signaling and modulates beta cell survival

    PubMed Central

    Malakar, Pushkar; Chartarifsky, Lital; Hija, Ayat; Leibowitz, Gil; Glaser, Benjamin; Dor, Yuval; Karni, Rotem

    2016-01-01

    Type 2 Diabetes (T2DM) affects more than 300 million people worldwide. One of the hallmarks of T2DM is peripheral insulin resistance, in part due to unproductive insulin signaling through the insulin receptor. The insulin receptor (INSR) exists as two isoforms, INSR-A and INSR-B, which results from skipping or inclusion of exon 11 respectively. What determines the relative abundance of the different insulin receptor splice variants is unknown. Moreover, it is not yet clear what the physiological roles of each of the isoforms are in normal and diseased beta cells. In this study, we show that insulin induces INSR exon 11 inclusion in pancreatic beta cells in both human and mouse. This occurs through activation of the Ras-MAPK/ERK signaling pathway and up-regulation of the splicing factor SRSF1. Induction of exon 11 skipping by a splice-site competitive antisense oligonucleotide inhibited the MAPK-ERK signaling pathway downstream of the insulin receptor, sensitizing the pancreatic β-cell line MIN6 to stress-induced apoptosis and lipotoxicity. These results assign to insulin a regulatory role in INSR alternative splicing through the Ras-MAPK/ERK signaling pathway. We suggest that in beta cells, INSR-B has a protective role, while INSR-A expression sensitizes beta cells to programmed cell death. PMID:27526875

  12. Conversion of adult pancreatic alpha-cells to beta-cells after extreme beta-cell loss.

    PubMed

    Thorel, Fabrizio; Népote, Virginie; Avril, Isabelle; Kohno, Kenji; Desgraz, Renaud; Chera, Simona; Herrera, Pedro L

    2010-04-22

    Pancreatic insulin-producing beta-cells have a long lifespan, such that in healthy conditions they replicate little during a lifetime. Nevertheless, they show increased self-duplication after increased metabolic demand or after injury (that is, beta-cell loss). It is not known whether adult mammals can differentiate (regenerate) new beta-cells after extreme, total beta-cell loss, as in diabetes. This would indicate differentiation from precursors or another heterologous (non-beta-cell) source. Here we show beta-cell regeneration in a transgenic model of diphtheria-toxin-induced acute selective near-total beta-cell ablation. If given insulin, the mice survived and showed beta-cell mass augmentation with time. Lineage-tracing to label the glucagon-producing alpha-cells before beta-cell ablation tracked large fractions of regenerated beta-cells as deriving from alpha-cells, revealing a previously disregarded degree of pancreatic cell plasticity. Such inter-endocrine spontaneous adult cell conversion could be harnessed towards methods of producing beta-cells for diabetes therapies, either in differentiation settings in vitro or in induced regeneration. PMID:20364121

  13. Rat neonatal beta cells lack the specialised metabolic phenotype of mature beta cells

    PubMed Central

    Jermendy, A.; Toschi, E.; Aye, T.; Koh, A.; Aguayo-Mazzucato, C.; Sharma, A.; Weir, G. C.; Sgroi, D.

    2011-01-01

    Aims/hypothesis Fetal and neonatal beta cells have poor glucose-induced insulin secretion and only gain robust glucose responsiveness several weeks after birth. We hypothesise that this unresponsiveness is due to a generalised immaturity of the metabolic pathways normally found in beta cells rather than to a specific defect. Methods Using laser-capture microdissection we excised beta cell-enriched cores of pancreatic islets from day 1 (P1) neonatal and young adult Sprague–Dawley rats in order to compare their gene-expression profiles using Affymetrix U34A microarrays (neonatal, n=4; adult, n=3). Results Using dChip software for analysis, 217 probe sets for genes/38 expressed sequence tags (ESTs) were significantly higher and 345 probe sets for genes/33 ESTs significantly lower in beta cell-enriched cores of neonatal islets compared with those of adult islets. Among the genes lower in the neonatal beta cells were key metabolic genes including mitochondrial shuttles (malate dehydrogenase, glycerol-3-phosphate dehydrogenase and glutamate oxalacetate transaminase), pyruvate carboxylase and carnitine palmitoyl transferase 2. Differential expression of these enzyme genes was confirmed by quantitative PCR on RNA from isolated neonatal (P2 until P28) and adult islets and with immunostaining of pancreas. Even by 28 days of age some of these genes were still expressed at lower levels than in adults. Conclusions/interpretation The lack of glucose responsiveness in neonatal islets is likely to be due to a generalised immaturity of the metabolic specialisation of pancreatic beta cells. PMID:21240476

  14. In vivo regeneration of insulin-producing beta-cells.

    PubMed

    Jun, Hee-Sook

    2010-01-01

    Type 1 and type 2 diabetes mellitus are considered to be caused by defective control of blood glucose resulting from a reduced beta-cell mass. Thus, the restoration of a functional beta-cell mass by replacing the damaged beta-cells or stimulating beta-cell regeneration is a logical approach for the treatment of diabetes. Strategies for increasing the beta-cell mass include stimulating beta-cell replication and differentiation and inhibiting beta-cell death. Treatment with various growth factors such as GLP-1, BTC, HGF, and EGF and forced expression of beta-cell transcription factors such as Pdx-1, NeuroD, and MafA resulted in the regeneration of beta-cells in vivo. Another approach is the administration of stem/progenitor cells, which can differentiate into insulin-producing cells. However, there are no satisfactory methods yet for clinical application. Understanding the mechanisms of the regenerative process of pancreatic beta-cells will pave the way for the development of regenerative medicine for treatment of diabetes. PMID:20217517

  15. Pancreatic beta-cell hyperactivity in morbidly obese adolescents.

    PubMed

    Mercado, Arlene B; Castells, Salvador

    2006-12-01

    beta-cell hyperactivity, with increased beta-cell mass in the pancreas, contributes to insulin oversecretion in response to insulin resistance. beta-cell mass expansion, also known as "endocrine pancreas plasticity", is an adaptation to variations in insulin demand, is generally observed in obese persons and in women during late pregnancy. In obese persons, increased free fatty acids contribute to beta-cell growth. It is believed that type 2 diabetes develops in those persons unable to respond to an increased insulin demand with a high rate of beta-cell proliferation. Impairment of insulin secretion may originate from a genetic predisposition as well as aggravated by high lipid and glucose levels. Better understanding of endocrine pancreas plasticity and its regeneration mechanisms could lead to new treatment modalities for type 2 diabetes. Review of literature of pancreatic beta-cell hyperactivity in obesity and its existence in morbidly obese adolescents is hereby presented. PMID:17237743

  16. ADVANCES IN MOLECULAR IMAGING OF PANCREATIC BETA CELLS

    PubMed Central

    Lin, Mai; Lubag, Angelo; McGuire, Michael J.; Seliounine, Serguei Y.; Tsyganov, Edward N.; Antich, Peter P.; Sherry, A. Dean; Brown, Kathlynn C.; Sun, Xiankai

    2009-01-01

    The development of non-invasive imaging methods for early diagnosis of the beta cell associated metabolic diseases, including type 1 and type 2 diabetes (T1D and T2D), has recently drawn considerable interest from the molecular imaging community as well as clinical investigators. Due to the challenges imposed by the location of the pancreas, the sparsely dispersed beta cell population within the pancreas, and the poor understanding of the pathogenesis of the diseases, clinical diagnosis of beta cell abnormalities is still limited. Current diagnostic methods are invasive, often inaccurate, and usually performed post-onset of the disease. Advances in imaging techniques for probing beta cell mass and function are needed to address this critical health care problem. A variety of currently available imaging techniques have been tested for the assessment of the pancreatic beta cell islets. Here we discuss the current advances in magnetic resonance imaging (MRI), bioluminescence imaging (BLI), and nuclear imaging for the study of beta cell diseases. Spurred by early successes in nuclear imaging techniques for beta cells, especially positron emission tomography (PET), the need for beta cell specific ligands has expanded. Progress in the field for obtaining such ligands is presented. Additionally, we report our preliminary efforts of developing such a peptidic ligand for PET imaging of the pancreatic beta cells. PMID:18508529

  17. Group VIA Ca2+-independent phospholipase A2 (iPLA2beta) and its role in beta-cell programmed cell death.

    PubMed

    Lei, Xiaoyong; Barbour, Suzanne E; Ramanadham, Sasanka

    2010-06-01

    Activation of phospholipases A(2) (PLA(2)s) leads to the generation of biologically active lipid mediators that can affect numerous cellular events. The Group VIA Ca(2+)-independent PLA(2), designated iPLA(2)beta, is active in the absence of Ca(2+), activated by ATP, and inhibited by the bromoenol lactone suicide inhibitor (BEL). Over the past 10-15 years, studies using BEL have demonstrated that iPLA(2)beta participates in various biological processes and the recent availability of mice in which iPLA(2)beta expression levels have been genetically-modified are extending these findings. Work in our laboratory suggests that iPLA(2)beta activates a unique signaling cascade that promotes beta-cell apoptosis. This pathway involves iPLA(2)beta dependent induction of neutral sphingomyelinase, production of ceramide, and activation of the intrinsic pathway of apoptosis. There is a growing body of literature supporting beta-cell apoptosis as a major contributor to the loss of beta-cell mass associated with the onset and progression of Type 1 and Type 2 diabetes mellitus. This underscores a need to gain a better understanding of the molecular mechanisms underlying beta-cell apoptosis so that improved treatments can be developed to prevent or delay the onset and progression of diabetes mellitus. Herein, we offer a general review of Group VIA Ca(2+)-independent PLA(2) (iPLA(2)beta) followed by a more focused discussion of its participation in beta-cell apoptosis. We suggest that iPLA(2)beta-derived products trigger pathways which can lead to beta-cell apoptosis during the development of diabetes. PMID:20083151

  18. Cordycepin modulates inflammatory and catabolic gene expression in interleukin-1beta-induced human chondrocytes from advanced-stage osteoarthritis: an in vitro study

    PubMed Central

    Hu, Pengfei; Chen, Weiping; Bao, Jiapeng; Jiang, Lifeng; Wu, Lidong

    2014-01-01

    Cordycepin is widely used as for its various pharmacological activities, such as anti-inflammation, anti-angiogenesis, anti-aging, anti-tumor and anti-proliferation. However, the precise role of cordycepin on chondrocytes is not clear. In the present study, we examined the inhibitory effects of cordycepin on interleukin-1 beta (IL-1β)-induced glycosaminoglycan (GAG) release, nitric oxide production as well as gene expressions of inflammatory and catabolic mediators in human cartilage and chondrocytes. Cartilage explants and human chondrocytes were cultured in the absence or in the presence of IL-1β (10 ng/ml) and with or without cordycepin (5-100 μM). GAG content in the cartilage explants was measured by using the dimethylmethylene blue method and Safranin O staining. Nitric oxide level was determined by Griess reaction. Expressions of MMP-1, MMP-13, cathepsin K, cathepsin S, ADAMTS-4 (a disintegrin and metalloproteinase with thrombospondin motifs-4) and ADAMTS-5, inducible nitric oxide synthase (iNOS) and cyclooxgenase-2 (COX-2) were evaluated by real-time quantitative PCR. We found that cordycepin suppressed IL-1β-stimulated GAG release. Gene expressions of catabolic enzymes, including MMP-1, MMP-13, cathepsin K, cathepsin S, ADAMTS-4 and ADAMTS-5, were decreased by cordycepin in a dose-dependent manner. In addition, cordycepin inhibited IL-1β-induced COX-2 and iNOS expression at the transcript level as well as blocked NO production. Our results suggest that cordycepin may possess chondroprotective effect by preventing cartilage denegation and interfering inflammatory response in the pathogenesis of OA. PMID:25400736

  19. Interleukin-1beta-induced extracellular matrix degradation and glycosaminoglycan release is inhibited by curcumin in an explant model of cartilage inflammation.

    PubMed

    Clutterbuck, Abigail L; Mobasheri, Ali; Shakibaei, Mehdi; Allaway, David; Harris, Pat

    2009-08-01

    Osteoarthritis (OA) is a degenerative and inflammatory disease of synovial joints that is characterized by the loss of articular cartilage, for which there is increasing interest in natural remedies. Curcumin (diferuloylmethane) is the main polyphenol in the spice turmeric, derived from rhizomes of the plant Curcuma longa. Curcumin has potent chemopreventive properties and has been shown to inhibit nuclear factor kappaB-mediated inflammatory signaling in many cell types, including chondrocytes. In this study, normal articular cartilage was harvested from metacarpophalangeal and metatarsophalangeal joints of eight horses, euthanized for reasons other than research purposes, to establish an explant model mimicking the inflammatory events that occur in OA. Initially, cartilage explants (N= 8) were stimulated with increasing concentrations of the proinflammatory cytokine IL-1beta to select effective doses for inducing cartilage degeneration in the explant model. Separate cartilage explants were then cotreated with IL-1beta at either 10 ng/mL (n= 3) or 25 ng/mL (n= 3) and curcumin (0.1 micromol/L, 0.5 micromol/L, 1 micromol/L, 10 micromol/L, and 100 micromol/L). After 5 days, the percentage of glycosaminoglycan (GAG) release from the explants was assessed using a dimethylmethylene blue colorimetric assay. Curcumin (100 micromol/L) significantly reduced IL-1beta-stimulated GAG release in the explants by an average of 20% at 10 ng/mL and 27% at 25 ng/mL back to unstimulated control levels (P < 0.001). Our results suggest that this explant model effectively simulates the proinflammatory cytokine-mediated release of articular cartilage components seen in OA. Furthermore, the evidence suggests that the inflammatory cartilage explant model is useful for studying the effects of curcumin on inflammatory pathways and gene expression in IL-1beta-stimulated chondrocytes. PMID:19723086

  20. Aucubin prevents interleukin-1 beta induced inflammation and cartilage matrix degradation via inhibition of NF-κB signaling pathway in rat articular chondrocytes.

    PubMed

    Wang, Sheng-Nan; Xie, Guo-Ping; Qin, Cheng-He; Chen, Yi-Rong; Zhang, Kai-Rui; Li, Xue; Wu, Qian; Dong, Wei-Qiang; Yang, Jun; Yu, Bin

    2015-02-01

    Proinflammatory cytokine interleukin-1β (IL-1β) plays a crucial role in the pathogenesis of Osteoarthritis (OA) by stimulating several mediators contributed to cartilage degradation. Aucubin, a natural compound derived from plants which has been shown to possess diverse biological activities including anti-inflammatory property, may benefit the IL-1β stimulated chondrocytes. The present study was aimed to investigate the effects of Aucubin on IL-1β stimulated rat chondrocytes. Rat chondrocytes were cultured and pretreated with Aucubin (1, 10, 20, 50μM), and then stimulated with or without IL-1β (10ng/ml). Gene and protein expression of MMP-3, MMP-9, MMP-13, cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) was determined by real-time PCR and Western blotting respectively. Nitric oxide (NO) production was quantified by Griess reagent. Phosphorylation and nuclear translocation of p65 were detected by western blotting and immunofluorescence, respectively. We found that Aucubin significantly reversed the elevated gene and protein expression of MMP-3, MMP-9, MMP-13, iNOS, COX-2 and the production of NO induced by IL-1β challenge in rat chondrocytes. Furthermore, Aucubin was able to suppress the IL-1β-mediated phosphorylation and nuclear translocation of p65, indicating Aucubin may possibly act via the NF-κB signaling pathway. The present study proposes that Aucubin may be a potential therapeutic choice in the treatment of OA due to its anti-inflammatory and chondroprotective features. PMID:25576403

  1. Interleukin-1beta-induced type IIA secreted phospholipase A2 gene expression and extracellular activity in rat vascular endothelial cells.

    PubMed

    Schwemmer, M; Aho, H; Michel, J B

    2001-06-01

    Two phospholipase A2 (PLA2) isoforms, secretory and cytosolic, have been implicated in inflammation. Secretory type IIA PLA2 (sPLA2-IIA), which hydrolyzes fatty acids bound at the sn-2 position of glycerophospholipids, has been detected universally in a variety of mammalian tissues and cells. The expression of the sPLA2-IIA gene and its extracellular activity were shown to be regulated by different factors such as hypoxia, cytokines and phorbol esters. In the present study, we examined the effects of interleukin-1beta (IL-1beta) on the expression of the 14kDa sPLA2-IIA, determined using reverse transcription polymerase chain reaction and radiometric Escherichia coli enzyme assay in primary cultures of rat endothelial cells and in two different rat endothelial cell lines (SVAREC and RBE4). These experiments revealed that IL-1beta induces sPLA2-IIa gene expression and secretion of the enzyme in endothelial cells in a dose- and time-dependent manner. The cAMP-elevator forskolin did not augment the cytokine-induced elevation of sPLA2-IIa enzyme activity but significantly increased the IL-1beta-stimulated sPLA2-IIa mRNA contents in endothelial cells. PMID:11469536

  2. Closing in on Mass Production of Mature Human Beta Cells.

    PubMed

    Kieffer, Timothy J

    2016-06-01

    Human pluripotent stem cell differentiation protocols based on mimicking developmental pathways are getting close to generating fully fledged pancreatic endocrine cells, including insulin-producing beta cells. However, challenges remain in identifying pathways to trigger the attainment of robust glucose responsiveness that occurs postnatally in beta cells. PMID:27257758

  3. Effect of Exendin-4 on Autophagy Clearance in Beta Cell of Rats with Tacrolimus-induced Diabetes Mellitus

    PubMed Central

    Lim, Sun Woo; Jin, Long; Jin, Jian; Yang, Chul Woo

    2016-01-01

    Growing evidence suggests that GLP-1 protects beta cells against various cellular injuries by modulating autophagy. In this study, we examined whether exendin-4 (Ex-4), a GLP-1 analog, had preventive effects on tacrolimus (Tac)-induced beta cell injury by improving autophagy clearance. Rats with Tac-induced diabetes mellitus exhibited increased autophagy-associated protein expression, light chain 3B levels, and autophagic vacuole numbers in pancreatic beta cells. Additionally, Tac increased autophagy in a dose- and time-dependent manner in vitro, and inhibition of autophagosome using 3-methyladenine reduced Tac-induced islet cell injury by decreasing reactive oxygen species production and apoptosis. Ex-4 treatment decreased Tac-induced hyperglycaemia, oxidative stress, and apoptosis, accompanied by decreased autophagy-associated protein expression and autophagosome numbers. In vivo and in vitro studies showed that Tac treatment impaired lysosomal function and autophagosome-lysosome fusion; these processes were improve by Ex-4 treatment. Moreover, addition of bafilomycin A1, an inhibitor of lysosomal function, abolished the protective effects of Ex-4. Our findings reveal that Tac-induced diabetes mellitus was a state of excessive burden of autophagosomes and impairment of autophagy clearance and that Ex-4 protected against Tac-induced pancreatic islet injury by reducing the burden of autophagosomes via activation of autophagosome clearance. Thus, Ex-4 had therapeutic effects on Tac-induced pancreatic beta cell injury. PMID:27436514

  4. Adenoviral infection or deferoxamine? Two approaches to overexpress VEGF in beta-cell lines.

    PubMed

    Langlois, Allan; Bietiger, William; Sencier, Marie-Christine; Maillard, Elisa; Pinget, Michel; Kessler, Laurence; Sigrist, Severine

    2009-07-01

    Rapid and adequate revascularization of transplanted islets is important for their survival and function during transplantation. Vascular endothelial growth factor (VEGF) could play a critical role with respect to islet revascularization. The aim of this study was to compare two strategies that are used to overexpress VEGF in beta-cells: (1) gene therapy through adenoviral infection and (2) a pharmacological approach using deferoxamine (DFO). beta-Cell lines from rat insulinoma (RINm5F) were either infected using an adenovirus encoding the gene of human VEGF 165 or incubated with DFO. One day after treatment, the viability of RINm5F cells was preserved with 10 micromol/L of DFO (103.95 +/- 5.66% toward control; n = 4). In addition, adenoviral infection maintained the viability of cells for all the concentrations used. In both treatments, overexpression of VEGF was in a comparable level. Finally, the ratio of Bax/Bcl-2 indicated that the apoptosis increased in infected beta-cells whereas treatment with DFO seems to be antiapoptotic. Our results suggest that the use of DFO could be a realistic approach to improve the vascularization of islets during transplantation. PMID:19527112

  5. Uncovering Factors Related to Pancreatic Beta-Cell Function

    PubMed Central

    Curran, Aoife M.; Ryan, Miriam F.; Drummond, Elaine; Gibney, Eileen R.; Gibney, Michael J.; Roche, Helen M.; Brennan, Lorraine

    2016-01-01

    Aim The incidence of type 2 diabetes has increased rapidly on a global scale. Beta-cell dysfunction contributes to the overall pathogenesis of type 2 diabetes. However, factors contributing to beta-cell function are not clear. The aims of this study were (i) to identify factors related to pancreatic beta-cell function and (ii) to perform mechanistic studies in vitro. Methods Three specific measures of beta-cell function were assessed for 110 participants who completed an oral glucose tolerance test as part of the Metabolic Challenge Study. Anthropometric and biochemical parameters were assessed as potential modulators of beta-cell function. Subsequent in vitro experiments were performed using the BRIN-BD11 pancreatic beta-cell line. Validation of findings were performed in a second human cohort. Results Waist-to-hip ratio was the strongest anthropometric modulator of beta-cell function, with beta-coefficients of -0.33 (p = 0.001) and -0.30 (p = 0.002) for beta-cell function/homeostatic model assessment of insulin resistance (HOMA-IR), and disposition index respectively. Additionally, the resistin-to-adiponectin ratio (RA index) emerged as being strongly associated with beta-cell function, with beta-coefficients of -0.24 (p = 0.038) and -0.25 (p = 0.028) for beta-cell function/HOMA-IR, and disposition index respectively. Similar results were obtained using a third measure for beta-cell function. In vitro experiments revealed that the RA index was a potent regulator of acute insulin secretion where a high RA index (20ng ml-1 resistin, 5nmol l-1 g-adiponectin) significantly decreased insulin secretion whereas a low RA index (10ng ml-1 resistin, 10nmol l-1 g-adiponectin) significantly increased insulin secretion. The RA index was successfully validated in a second human cohort with beta-coefficients of -0.40 (p = 0.006) and -0.38 (p = 0.008) for beta-cell function/ HOMA-IR, and disposition index respectively. Conclusions Waist-to-hip ratio and RA index were identified

  6. Targeting insulin-producing beta cells for regenerative therapy.

    PubMed

    Migliorini, Adriana; Roscioni, Sara S; Lickert, Heiko

    2016-09-01

    Pancreatic beta cells differ in terms of glucose responsiveness, insulin secretion and proliferative capacity; however, the molecular pathways that regulate this cellular heterogeneity are unknown. We have identified the Wnt-planar cell polarity (PCP) effector Flattop (FLTP) as a biomarker that identifies mature beta cells in the islets of Langerhans. Interestingly, three-dimensional architecture and Wnt-PCP ligands are sufficient to trigger mouse and human beta cell maturation. These results highlight the fact that novel biomarkers shed light on the long-standing mystery of beta cell heterogeneity and identify the Wnt-PCP pathway as triggering beta cell maturation. Understanding heterogeneity in the islets of Langerhans might allow targeting of beta cell subpopulations for regenerative therapy and provide building principles for stem cell-derived islets. This review summarises a presentation given at the 'Can we make a better beta cell?' symposium at the 2015 annual meeting of the EASD. It is accompanied by two other reviews on topics from this symposium (by Amin Ardestani and Kathrin Maedler, DOI: 10.1007/s00125-016-3892-9 , and by Harry Heimberg and colleagues, DOI: 10.1007/s00125-016-3879-6 ) and a commentary by the Session Chair, Shanta Persaud (DOI: 10.1007/s00125-016-3870-2 ). PMID:27412250

  7. Sources of beta cells inside the pancreas.

    PubMed

    De Groef, Sofie; Staels, Willem; Van Gassen, Naomi; Lemper, Marie; Yuchi, Yixing; Sojoodi, Mozhdeh; Bussche, Leen; Heremans, Yves; Leuckx, Gunter; De Leu, Nico; Van de Casteele, Mark; Baeyens, Luc; Heimberg, Harry

    2016-09-01

    The generation of beta(-like) cells to compensate for their absolute or relative shortage in type 1 and type 2 diabetes is an obvious therapeutic strategy. Patients first received grafts of donor islet cells over 25 years ago, but this procedure has not become routine in clinical practice because of a donor cell shortage and (auto)immune problems. Transplantation of differentiated embryonic and induced pluripotent stem cells may overcome some but not all the current limitations. Reprogramming exocrine cells towards functional beta(-like) cells would offer an alternative abundant and autologous source of beta(-like) cells. This review focuses on work by our research group towards achieving such a source of cells. It summarises a presentation given at the 'Can we make a better beta cell?' symposium at the 2015 annual meeting of the EASD. It is accompanied by two other reviews on topics from this symposium (by Amin Ardestani and Kathrin Maedler, DOI: 10.1007/s00125-016-3892-9 , and by Heiko Lickert and colleagues, DOI: 10.1007/s00125-016-3949-9 ) and a commentary by the Session Chair, Shanta Persaud (DOI: 10.1007/s00125-016-3870-2 ). PMID:27053238

  8. Serotonin competence of mouse beta cells during pregnancy.

    PubMed

    Goyvaerts, Lotte; Schraenen, Anica; Schuit, Frans

    2016-07-01

    Pregnancy is a key mammalian reproductive event in which growth and differentiation of the fetus imposes extra metabolic and hormonal demands on the mother. Its successful outcome depends on major changes in maternal blood circulation, metabolism and endocrine function. One example is the endocrine pancreas, where beta cells undergo a number of changes in pregnancy that result in enhanced functional beta cell mass in order to compensate for the rising metabolic needs for maternal insulin. During the last 5 years, a series of studies have increased our understanding of the molecular events involved in this functional adaptation. In the mouse, a prominent functional change during pregnancy is the capacity of some beta cells to produce serotonin. In this review we will discuss the mechanism and potential effects of pregnancy-related serotonin production in beta cells, considering functional consequences at the local intra-islet and systemic level. PMID:27056372

  9. Pancreatic beta cells express a diverse set of homeobox genes.

    PubMed Central

    Rudnick, A; Ling, T Y; Odagiri, H; Rutter, W J; German, M S

    1994-01-01

    Homeobox genes, which are found in all eukaryotic organisms, encode transcriptional regulators involved in cell-type differentiation and development. Several homeobox genes encoding homeodomain proteins that bind and activate the insulin gene promoter have been described. In an attempt to identify additional beta-cell homeodomain proteins, we designed primers based on the sequences of beta-cell homeobox genes cdx3 and lmx1 and the Drosophila homeodomain protein Antennapedia and used these primers to amplify inserts by PCR from an insulinoma cDNA library. The resulting amplification products include sequences encoding 10 distinct homeodomain proteins; 3 of these proteins have not been described previously. In addition, an insert was obtained encoding a splice variant of engrailed-2, a homeodomain protein previously identified in the central nervous system. Northern analysis revealed a distinct pattern of expression for each homeobox gene. Interestingly, the PCR-derived clones do not represent a complete sampling of the beta-cell library because no inserts encoding cdx3 or lmx1 protein were obtained. Beta cells probably express additional homeobox genes. The abundance and diversity of homeodomain proteins found in beta cells illustrate the remarkable complexity and redundancy of the machinery controlling beta-cell development and differentiation. Images PMID:7991607

  10. IL-13 improves beta-cell survival and protects against IL-1beta-induced beta-cell death

    PubMed Central

    Rütti, Sabine; Howald, Cédric; Arous, Caroline; Dermitzakis, Emmanouil; Halban, Philippe A.; Bouzakri, Karim

    2015-01-01

    Objectives IL-13 is a cytokine classically produced by anti-inflammatory T-helper-2 lymphocytes; it is decreased in the circulation of type 2 diabetic patients and impacts positively on liver and skeletal muscle. Although IL-13 can exert positive effects on beta-cell lines, its impact and mode of action on primary beta-cell function and survival remain largely unexplored. Methods Beta-cells were cultured for 48 h in the presence of IL-13 alone or in combination with IL-1β or cytokine cocktail (IL-1β, IFNγ, TNFα). Results IL-13 protected human and rat beta-cells against cytokine induced death. However, IL-13 was unable to protect from IL-1β impaired glucose stimulated insulin secretion and did not influence NFκB nuclear relocalization induced by IL-1β. IL-13 induced phosphorylation of Akt, increased IRS2 protein expression and counteracted the IL-1β induced regulation of several beta-cell stress response genes. Conclusions The prosurvival effects of IL-13 thus appear to be mediated through IRS2/Akt signaling with NFκB independent regulation of gene expression. In addition to previously documented beneficial effects on insulin target tissues, these data suggest that IL-13 may be useful for treatment of type 2 diabetes by preserving beta-cell mass or slowing its rate of decline. PMID:26909320

  11. Detailed transcriptome atlas of the pancreatic beta cell

    PubMed Central

    Kutlu, Burak; Burdick, David; Baxter, David; Rasschaert, Joanne; Flamez, Daisy; Eizirik, Decio L; Welsh, Nils; Goodman, Nathan; Hood, Leroy

    2009-01-01

    Background Gene expression patterns provide a detailed view of cellular functions. Comparison of profiles in disease vs normal conditions provides insights into the processes underlying disease progression. However, availability and integration of public gene expression datasets remains a major challenge. The aim of the present study was to explore the transcriptome of pancreatic islets and, based on this information, to prepare a comprehensive and open access inventory of insulin-producing beta cell gene expression, the Beta Cell Gene Atlas (BCGA). Methods We performed Massively Parallel Signature Sequencing (MPSS) analysis of human pancreatic islet samples and microarray analyses of purified rat beta cells, alpha cells and INS-1 cells, and compared the information with available array data in the literature. Results MPSS analysis detected around 7600 mRNA transcripts, of which around a third were of low abundance. We identified 2000 and 1400 transcripts that are enriched/depleted in beta cells compared to alpha cells and INS-1 cells, respectively. Microarray analysis identified around 200 transcription factors that are differentially expressed in either beta or alpha cells. We reanalyzed publicly available gene expression data and integrated these results with the new data from this study to build the BCGA. The BCGA contains basal (untreated conditions) gene expression level estimates in beta cells as well as in different cell types in human, rat and mouse pancreas. Hierarchical clustering of expression profile estimates classify cell types based on species while beta cells were clustered together. Conclusion Our gene atlas is a valuable source for detailed information on the gene expression distribution in beta cells and pancreatic islets along with insulin producing cell lines. The BCGA tool, as well as the data and code used to generate the Atlas are available at the T1Dbase website (T1DBase.org). PMID:19146692

  12. Islet Stellate Cells Isolated from Fibrotic Islet of Goto-Kakizaki Rats Affect Biological Behavior of Beta-Cell.

    PubMed

    Li, Feng-Fei; Chen, Bi-Jun; Li, Wei; Li, Ling; Zha, Min; Zhou, S; Bachem, M G; Sun, Zi-Lin

    2016-01-01

    We previously isolated islet stellate cells (ISCs) from healthy Wistar rat islets. In the present study, we isolated "already primed by diabetic environment" ISCs from islets of Goto-Kakizaki rats, determined the gene profile of these cells, and assessed the effects of these ISCs on beta-cell function and survival. We detected gene expression of ISCs by digital gene expression. INS-1 cell proliferation, apoptosis, and insulin production were measured after being treated with ISCs supernatant (SN). We observed the similar expression pattern of ISCs and PSCs, but 1067 differentially expressed genes. Insulin production in INS-1 cells cultured with ISC-SN was significantly reduced. The 5-ethynyl-2'-deoxyuridine-positive INS-1 cells treated with ISC-SN were decreased. Propidium iodide- (PI-) positive INS-1 cells were 2.6-fold higher than those in control groups. Caspase-3 activity was increased. In conclusion, ISCs presented in fibrotic islet of GK rats might be special PSCs, which impaired beta-cell function and proliferation and increased beta-cell apoptosis. PMID:26697502

  13. Beta-cell mitochondrial carriers and the diabetogenic stress response.

    PubMed

    Brun, Thierry; Maechler, Pierre

    2016-10-01

    Mitochondria play a central role in pancreatic beta-cells by coupling metabolism of the secretagogue glucose to distal events of regulated insulin exocytosis. This process requires transports of both metabolites and nucleotides in and out of the mitochondria. The molecular identification of mitochondrial carriers and their respective contribution to beta-cell function have been uncovered only recently. In type 2 diabetes, mitochondrial dysfunction is an early event and may precipitate beta-cell loss. Under diabetogenic conditions, characterized by glucotoxicity and lipotoxicity, the expression profile of mitochondrial carriers is selectively modified. This review describes the role of mitochondrial carriers in beta-cells and the selective changes in response to glucolipotoxicity. In particular, we discuss the importance of the transfer of metabolites (pyruvate, citrate, malate, and glutamate) and nucleotides (ATP, NADH, NADPH) for beta-cell function and dysfunction. This article is part of a Special Issue entitled: Mitochondrial Channels edited by Pierre Sonveaux, Pierre Maechler and Jean-Claude Martinou. PMID:26979549

  14. The level of nitric oxide regulates lipocalin-2 expression under inflammatory condition in RINm5F beta-cells.

    PubMed

    Chang, Seo-Yoon; Kim, Dong-Bin; Ko, Seung-Hyun; Jang, Hyun-Jong; Jo, Yang-Hyeok; Kim, Myung-Jun

    2016-07-15

    We previously reported that proinflammatory cytokines (interleukin-1β and interferon-γ) induced the expression of lipocalin-2 (LCN-2) together with inducible nitric oxide synthase (iNOS) in RINm5F beta-cells. Therefore, we examined the effect of nitric oxide (NO) on LCN-2 expression in cytokines-treated RINm5F beta-cells. Additionally, we observed the effect of LCN-2 on cell viability. First, we found the existence of LCN-2 receptor and the internalization of exogenous recombinant LCN-2 peptide in RINm5F and INS-1 beta-cells. Next, the effects of NO on LCN-2 expression were evaluated. Aminoguanidine, an iNOS inhibitor and iNOS gene silencing significantly inhibited cytokines-induced LCN-2 expression while sodium nitroprusside (SNP), an NO donor potentiated it. Luciferase reporter assay showed that transcription factor NF-κB was not involved in LCN-2 expression. Both LCN-2 mRNA and protein stability assays were conducted. SNP did not affect LCN-2 mRNA stability, however, it significantly reduced LCN-2 protein degradation. The LCN-2 protein degradation was significantly attenuated by MG132, a proteasome inhibitor. Finally, the effect of LCN-2 on cell viability was evaluated. LCN-2 peptide treatment and LCN-2 overexpression significantly reduced cell viability. FACS analysis showed that LCN-2 induced the apoptosis of the cells. Collectively, NO level affects LCN-2 expression via regulation of LCN-2 protein stability under inflammatory condition and LCN-2 may reduce beta-cell viability by promoting apoptosis. PMID:27233602

  15. Beta-cell preservation…Is weight loss the answer?

    PubMed

    Mazza, Angela D; Pratley, Richard E; Smith, Steven R

    2011-01-01

    Obesity is associated with an increased risk of type 2 diabetes (T2D). Pancreatic beta-cell failure is an early event in the development of glucose dysregulation and diabetes. Interventions to halt beta-cell failure in T2D include diet modification, exercise, and use of pharmacologic agents. There is evidence that abdominal obesity may contribute to diabetes through insulin resistance and beta-cell impairment. Pivotal long-term studies into the prevention of T2D have shown the importance of weight loss beside diet, lifestyle, and medication. The Finnish Diabetes Prevention Program (DPP) showed that weight loss gradually reduces the risk of diabetes, and that even modest weight loss can significantly reduce the incidence of T2D. Similarly, in the US DPP, weight loss as part of intensive lifestyle modification was the major factor in reducing the incidence of T2D in high-risk subjects, being more effective than drug intervention. While understanding the relationship between obesity and diabetes is complex, we know that weight loss has positive effects on adipose tissue. It causes an increase in the beneficial fat cell hormone adiponectin, and a decrease in adipose tissue inflammation. Also, it is associated with reduced insulin resistance and a consequential reduction in glucolipotoxicity, which can improve beta-cell function. In summary, weight loss improves glycemic control and thereby mitigates diabetes symptoms and complications, possibly through the preservation of beta-cell function. Therefore, efforts to prevent diabetes and preserve beta-cell function in patients with T2D should more consequently emphasize and target weight loss. PMID:22580726

  16. On the coherent behavior of pancreatic beta cell clusters

    NASA Astrophysics Data System (ADS)

    Loppini, Alessandro; Capolupo, Antonio; Cherubini, Christian; Gizzi, Alessio; Bertolaso, Marta; Filippi, Simonetta; Vitiello, Giuseppe

    2014-09-01

    Beta cells in pancreas represent an example of coupled biological oscillators which via communication pathways, are able to synchronize their electrical activity, giving rise to pulsatile insulin release. In this work we numerically analyze scale free self-similarity features of membrane voltage signal power density spectrum, through a stochastic dynamical model for beta cells in the islets of Langerhans fine tuned on mouse experimental data. Adopting the algebraic approach of coherent state formalism, we show how coherent molecular domains can arise from proper functional conditions leading to a parallelism with “phase transition” phenomena of field theory.

  17. Nr2e1 Deficiency Augments Palmitate-Induced Oxidative Stress in Beta Cells

    PubMed Central

    Shi, Xiaoli; Deng, Haohua; Dai, Zhe; Xu, Yancheng; Xiong, Xiaokan; Ma, Pei; Cheng, Jing

    2016-01-01

    Nuclear receptor subfamily 2 group E member 1 (Nr2e1) has been regarded as an essential regulator of the growth of neural stem cells. However, its function elsewhere is unknown. In the present study, we generated Nr2e1 knockdown MIN6 cells and studied whether Nr2e1 knockdown affected basal beta cell functions such as proliferation, cell death, and insulin secretion. We showed that knockdown of Nr2e1 in MIN6 cells resulted in increased sensitivity to lipotoxicity, decreased proliferation, a partial G0/G1 cell-cycle arrest, and higher rates of apoptosis. Moreover, Nr2e1 deficiency exaggerates palmitate-induced impairment in insulin secretion. At the molecular level, Nr2e1 deficiency augments palmitate-induced oxidative stress. Nr2e1 deficiency also resulted in decreases in antioxidant enzymes and expression level of Nrf2. Together, this study indicated a potential protective effect of Nr2e1 on beta cells, which may serve as a target for the development of novel therapies for diabetes. PMID:26649147

  18. Regulation of. beta. -cell glucose transporter gene expression

    SciTech Connect

    Chen, Ling; Alam, Tausif; Johnson, J.H.; Unger, R.H. Department of Veterans Affairs Medical Center, Dallas, TX ); Hughes, S.; Newgard, C.B. )

    1990-06-01

    It has been postulated that a glucose transporter of {beta} cells (GLUT-2) may be important in glucose-stimulated insulin secretion. To determine whether this transporter is constitutively expressed or regulated, the authors subjected conscious unrestrained Wistar rats to perturbations in glucose homeostasis and quantitated {beta}-cell GLUT-2 mRNA by in situ hybridization. After 3 hr of hypoglycemia, GLUT-2 and proinsulin mRNA signal densities were reduced by 25% of the level in control rats. After 4 days, GLUT-2 and proinsulin mRNA densities were reduced by 85% and 65%, respectively. After 12 days of hypoglycemia, the K{sub m} for 3-O-methyl-D-glucose transport in isolated rat islets, normally 18-20 mM, was 2.5 mM. This provides functional evidence of a profound reduction of high K{sub m} glucose transporter in {beta} cells. In contrast, GLUT-2 was only slightly reduced by hypoglycemia in liver. To determine the effect of prolonged hyperglycemia, they also infused animals with 50% (wt/vol) glucose for 5 days. Hyperglycemic clamping increased GLUT-2 mRNA by 46% whereas proinsulin mRNA doubled. They conclude that GLUT-2 expression in {beta} cells, but not liver, is subject to regulation by certain perturbations in blood glucose homeostasis.

  19. Beta-cell Assembly for the Quad Gas Sampling Detector

    SciTech Connect

    Cooper, Matthew W.; Bowyer, Ted W.; McIntyre, Justin I.; Hayes, James C.; Heimbigner, Tom R.; Ripplinger, Michael D.; Thompson, Robert C.

    2008-05-05

    The beta-cells used in the beta-gamma detector have taken time to develop and to standardize the assembly of them. In making the assembly routine it is important to have step by step assembly instructions as well as a list of potential problems and their solutions. This document attempts to accomplish these goals.

  20. Beta cell adaptation in pregnancy: a tribute to Claes Hellerström

    PubMed Central

    2016-01-01

    Pregnancy is associated with a compensatory increase in beta cell mass. It is well established that somatolactogenic hormones contribute to the expansion both indirectly by their insulin antagonistic effects and directly by their mitogenic effects on the beta cells via receptors for prolactin and growth hormone expressed in rodent beta cells. However, the beta cell expansion in human pregnancy seems to occur by neogenesis of beta cells from putative progenitor cells rather than by proliferation of existing beta cells. Claes Hellerström has pioneered the research on beta cell growth for decades, but the mechanisms involved are still not clarified. In this review the information obtained in previous studies is recapitulated together with some of the current attempts to resolve the controversy in the field: identification of the putative progenitor cells, identification of the factors involved in the expansion of the beta cell mass in human pregnancy, and the relative roles of endocrine factors and nutrients. PMID:27055631

  1. IDO-Expressing Fibroblasts Protect Islet Beta Cells From Immunological Attack and Reverse Hyperglycemia in Non-Obese Diabetic Mice.

    PubMed

    Zhang, Yun; Jalili, Reza B; Kilani, Ruhangiz T; Elizei, Sanam Salimi; Farrokhi, Ali; Khosravi-Maharlooei, Mohsen; Warnock, Garth L; Ao, Ziliang; Marzban, Lucy; Ghahary, Aziz

    2016-09-01

    Indoleamine 2,3-dioxygenase (IDO) induces immunological tolerance in physiological and pathological conditions. Therefore, we used dermal fibroblasts with stable IDO expression as a cell therapy to: (i) Investigate the factors determining the efficacy of this cell therapy for autoimmune diabetes in non-obese diabetic (NOD) mice; (ii) Scrutinize the potential immunological mechanisms. Newly diabetic NOD mice were randomly injected with either 10 × 10(6) (10M) or 15 × 10(6) (15M) IDO-expressing dermal fibroblasts. Blood glucose levels (BGLs), body weight, plasma kynurenine levels, insulitis severity, islet beta cell function, autoreactive CD8(+) T cells, Th17 cells and regulatory T cells (Tregs) were then investigated in these mice. IL-1β and cleaved caspase-3 levels were assessed in islets co-cultured with IDO-expressing fibroblasts. BGLs in 83% mice treated with 15M IDO-expressing fibroblasts recovered to normal up to 120 days. However, only 17% mice treated with 10M IDO-expressing cells were reversed to normoglycemia. A 15M IDO-expressing fibroblasts significantly reduced infiltrated immune cells in islets and recovered the functionality of remaining islet beta cells in NOD mice. Additionally, they successfully inhibited autoreactive CD8(+) T cells and Th17 cells as well as increased Tregs in different organs of NOD mice. Islet beta cells co-cultured with IDO-expressing fibroblasts had reduced IL-1β levels and cell apoptosis. Both cell number and IDO enzymatic activity contributes to the efficiency of IDO cell therapy. Optimized IDO-expressing fibroblasts successfully reverse the progression of diabetes in NOD mice through induction of Tregs as well as inhibition of beta cell specific autoreactive CD8(+) T cells and Th17 cells. J. Cell. Physiol. 231: 1964-1973, 2016. © 2016 Wiley Periodicals, Inc. PMID:26743772

  2. The role of autophagy in pancreatic beta-cell and diabetes.

    PubMed

    Fujitani, Yoshio; Kawamori, Ryuzo; Watada, Hirotaka

    2009-02-01

    Pancreatic beta-cells play a key role in glucose homeostasis in mammals. Although large-scale protein synthesis and degradation occur in pancreatic beta-cells, the mechanism underlying dynamic protein turnover in beta-cells remains largely unknown. We found low-level constitutive autophagy in beta-cells of C57BL/6 mice fed a standard diet; however, autophagy was markedly upregulated in mice fed a high-fat diet. beta-cells of diabetic db/db mice contained large numbers of autophagosomes, compared with nondiabetic db/misty controls. The functional importance of autophagy was analyzed using beta-cell-specific Atg7 knockout mice. Autophagy-deficient mice showed degeneration of beta-cells and impaired glucose tolerance with reduced insulin secretion. While a high-fat diet stimulated beta-cell autophagy in control mice, it induced a profound deterioration of glucose intolerance in beta-cell autophagy-deficient mutants, partly because of the lack of a compensatory increase in beta-cell mass. These results suggest that the degradation of unnecessary cellular components by autophagy is essential for maintenance of the architecture and function of beta-cells. Autophagy also serves as a crucial element of stress responses to protect beta-cells under insulin-resistant states. Impairment of autophagic machinery could thus predispose individuals to type 2 diabetes. PMID:19158492

  3. Induction of human pancreatic beta cell replication by inhibitors of dual specificity tyrosine regulated kinase

    PubMed Central

    Wang, Peng; Alvarez-Perez, Juan-Carlos; Felsenfeld, Dan P.; Liu, Hongtao; Sivendran, Sharmila; Bender, Aaron; Kumar, Anil; Sanchez, Roberto; Scott, Donald K.; Garcia-Ocaña, Adolfo; Stewart, Andrew F.

    2015-01-01

    Types 1 and 2 diabetes affect some 380 million people worldwide. Both result ultimately from a deficiency of functional pancreatic insulin-producing beta cells. Beta cells proliferate in humans during a brief temporal window beginning around the time of birth, with peak beta cell labeling indices achieving approximately 2% in first year of life1-4. In embryonic life and after early childhood, beta cell replication rates are very low. While beta cell expansion seems an obvious therapeutic approach to beta cell deficiency, adult human beta cells have proven recalcitrant to such efforts1-8. Hence, there remains an urgent need for diabetes therapeutic agents that can induce regeneration and expansion of adult human beta cells in vivo or ex vivo. Here, we report the results of a high-throughput small molecule screen (HTS) revealing a novel class of human beta cell mitogenic compounds, analogues of the small molecule, harmine. We also define dual specificity tyrosine-regulated kinase-1a (DYRK1A) as the likely target of harmine, and the Nuclear Factors of activated T-cells (NFAT) family of transcription factors as likely mediators of human beta cell proliferation as well as beta cell differentiation. These observations suggest that harmine analogues (“harmalogs”) may have unique therapeutic promise for human diabetes therapy. Enhancing potency and beta cell specificity are important future challenges. PMID:25751815

  4. Increased perinatal remodelling of the pancreas in somatostatin-deficient mice: potential role of transforming growth factor-beta signalling in regulating beta cell growth in early life.

    PubMed

    Richardson, C C; To, K; Foot, V L; Hauge-Evans, A C; Carmignac, D; Christie, M R

    2015-01-01

    Early postnatal life is a critical period for development of the endocrine pancreas, involving remodelling of islet cells and maturation of secretory responses. Factors that regulate these processes are undefined. Somatostatin-secreting delta-cells are abundant in the developing pancreas and, because somatostatin inhibits growth, the hormone may regulate islet expansion in early life. The aim of this study was to investigate effects of somatostatin-deficiency on proliferation, apoptosis and pancreas expansion in the first 3 weeks of life in mice. Pancreases from control or somatostatin-knockout mice were analysed for beta cell, alpha cell and pancreatic volumes by morphometry, proliferation by BrdU incorporation and apoptosis by TUNEL labelling. Signalling pathways associated with proliferation and apoptosis were studied by immunohistochemistry and Western blotting. Knockout mice grew normally in the first 3 weeks of life, but had high circulating insulin that normalised by day 21. Beta cell, alpha cell and pancreatic volumes were decreased in knockout mice, accompanied by reduced proliferation and increased apoptosis in the pancreas. Decreased growth was not due to impaired Akt signalling, as Akt phosphorylation and nuclear cyclin-D2 increased in the knockout pancreas. Levels of TGF-β1, a factor implicated in tissue remodelling, together with SMAD phosphorylation through which TGF-β mediates its effects, were increased in the knockout pancreas. Beta cell expansion was impaired in knockout mice, potentially compensating for increased insulin secretion from islets lacking inhibitory effects of somatostatin, and was associated with increased TGF-β1 levels. TGF-β1 may represent an important regulator of beta cell mass in early life. PMID:25350519

  5. Protective role of sodium butyrate, a HDAC inhibitor on beta-cell proliferation, function and glucose homeostasis through modulation of p38/ERK MAPK and apoptotic pathways: study in juvenile diabetic rat.

    PubMed

    Khan, S; Jena, G B

    2014-04-25

    Type 1 diabetes (T1D) also known as juvenile diabetes is a chronic autoimmune disorder that precipitates in genetically susceptible individuals by environmental factors particularly during early age. Both genetic and epigenetic factors are implicated in the beta-cell development, proliferation, differentiation and function. Recent evidences suggested that there is a link between diabetes and histone deacetylases (HDACs), because HDAC inhibitors promote beta-cell development, proliferation and function as well as improve glucose homeostasis. Sodium butyrate (NaB) is a short chain fatty acid having HDAC inhibition activity. The present study was aimed to investigate the protective role of NaB treatment on the beta-cell proliferation, function and glucose homeostasis as well as apoptosis in juvenile diabetic rat. Diabetes was induced by single injection of STZ (60 mg/kg, i.p.) in chilled citrate buffer, while NaB (500 mg/kg/day) was administrated by i.p. route for 21 days as pre- and post-treatment schedule. Plasma glucose and insulin levels, HbA1c, glucose tolerance, apoptosis, and expression of proliferating cell nuclear antigen (PCNA), p38, p53, caspase-3, extracellular signal-regulated kinase-1/2 (ERK-1/2), forkhead box protein O1 (FOXO1) and insulin receptor substrate-1 (IRS-1) as well as histone acetylation were evaluated. NaB treatment decreased plasma glucose, HbA1c, beta-cell apoptosis and improved plasma insulin level and glucose homeostasis through HDAC inhibition and histone acetylation in diabetic animal as compared to control. NaB treatment improved the beta-cell proliferation, function and glucose homeostasis as well as reduced beta-cell apoptosis in juvenile diabetic rat by the modulation of p38/ERK MAPK and apoptotic pathway. PMID:24530320

  6. Circadian Transcription from Beta Cell Function to Diabetes Pathophysiology.

    PubMed

    Perelis, Mark; Ramsey, Kathryn Moynihan; Marcheva, Biliana; Bass, Joseph

    2016-08-01

    The mammalian circadian clock plays a central role in the temporal coordination of physiology across the 24-h light-dark cycle. A major function of the clock is to maintain energy constancy in anticipation of alternating periods of fasting and feeding that correspond with sleep and wakefulness. While it has long been recognized that humans exhibit robust variation in glucose tolerance and insulin sensitivity across the sleep-wake cycle, experimental genetic analysis has now revealed that the clock transcription cycle plays an essential role in insulin secretion and metabolic function within pancreatic beta cells. This review addresses how studies of the beta cell clock may elucidate the etiology of subtypes of diabetes associated with circadian and sleep cycle disruption, in addition to more general forms of the disease. PMID:27440914

  7. A Figure-of-Merit for Beta Cell Detector Characterization

    SciTech Connect

    Foxe, Michael P.; Miller, Brian W.; Suarez, Rey; Hayes, James C.

    2015-09-02

    In order to decrease the minimum detectable activities (MDAs) of beta-gamma radioxenon detectors, it is important to increase the ability to resolve the individual isotopes. One proposed method for doing this is to increase the energy resolution of the beta cell through the use of silicon detectors. While silicon detectors can improve the energy resolution, it is accompanied with a decrease in detection efficiency compared to plastic scintillator beta cells. Due to the uncertainty on the impact of the competing variables, we have developed a figure-of-merit (FOM) capable of determining the impact of detector parameters on the MDAs. By utilizing the FOM to analyze different detectors, we are able to directly compare current and future detectors and estimate their impact on the radioxenon MDAs.

  8. Boost for Alginate Encapsulation in Beta Cell Transplantation.

    PubMed

    Pipeleers, Daniel; Keymeulen, Bart

    2016-05-01

    A recent study reported that encapsulation of human embryonic stem cell (hESC)-derived beta cells by a novel alginate formula protects against foreign body reactivity in immune-competent mice. Intraperitoneal implants corrected a diabetic state for at least 6 months. These observations will stimulate the development of alginate encapsulation towards novel cell therapy protocols for treating type 1 diabetes (T1DM). PMID:27037212

  9. Measuring and Modeling Xenon Uptake in Plastic Beta-Cells

    NASA Astrophysics Data System (ADS)

    Suarez, R.; Hayes, J. C.; Harper, W. W.; Humble, P.; Ripplinger, M. D.; Stephenson, D. E.; Williams, R. M.

    2013-12-01

    The precision of the stable xenon volume measurement in atmospheric monitoring radio-xenon systems is a critical parameter used to determine the activity concentration of a radio-xenon sample. Typically these types of systems use a plastic scintillating beta-cell as part of a beta-gamma detection scheme to measure the radioactivity present in the gas sample. Challenges arise when performing the stable xenon calculation during or after radioactive counting of the sample due to xenon uptake into the plastic beta-cells. Plastic beta cells can adsorb as much as 5% of the sample during counting. If quantification is performed after counting, the uptake of xenon into the plastic results in an underestimation of the xenon volume measurement. This behavior also causes what is typically known as 'memory effect' in the cell. Experiments were conducted using a small volume low pressure range thermal conductivity sensor to quantify the amount of xenon uptake into the cell over a given period of time. Understanding the xenon uptake in the cell provides a better estimate of the stable volume which improves the overall measurement capability of the system. The results from these experiments along with modeling will be presented.

  10. Cell-type, allelic, and genetic signatures in the human pancreatic beta cell transcriptome.

    PubMed

    Nica, Alexandra C; Ongen, Halit; Irminger, Jean-Claude; Bosco, Domenico; Berney, Thierry; Antonarakis, Stylianos E; Halban, Philippe A; Dermitzakis, Emmanouil T

    2013-09-01

    Elucidating the pathophysiology and molecular attributes of common disorders as well as developing targeted and effective treatments hinges on the study of the relevant cell type and tissues. Pancreatic beta cells within the islets of Langerhans are centrally involved in the pathogenesis of both type 1 and type 2 diabetes. Describing the differentiated state of the human beta cell has been hampered so far by technical (low resolution microarrays) and biological limitations (whole islet preparations rather than isolated beta cells). We circumvent these by deep RNA sequencing of purified beta cells from 11 individuals, presenting here the first characterization of the human beta cell transcriptome. We perform the first comparison of gene expression profiles between beta cells, whole islets, and beta cell depleted islet preparations, revealing thus beta-cell-specific expression and splicing signatures. Further, we demonstrate that genes with consistent increased expression in beta cells have neuronal-like properties, a signal previously hypothesized. Finally, we find evidence for extensive allelic imbalance in expression and uncover genetic regulatory variants (eQTLs) active in beta cells. This first molecular blueprint of the human beta cell offers biological insight into its differentiated function, including expression of key genes associated with both major types of diabetes. PMID:23716500

  11. Oleate protects beta-cells from the toxic effect of palmitate by activating pro-survival pathways of the ER stress response.

    PubMed

    Sargsyan, Ernest; Artemenko, Konstantin; Manukyan, Levon; Bergquist, Jonas; Bergsten, Peter

    2016-09-01

    Long-term exposure of beta cells to saturated fatty acids impairs insulin secretion and increases apoptosis. In contrast, unsaturated fatty acids protect beta-cells from the long-term negative effects of saturated fatty acids. We aimed to identify the mechanisms underlying this protective action of unsaturated fatty acids. To address the aim, insulin-secreting MIN6 cells were exposed to palmitate in the absence or presence of oleate and analyzed by using nano-LC MS/MS based proteomic approach. Important findings were validated by using alternative approaches. Proteomic analysis identified 34 proteins differentially expressed in the presence of palmitate compared to control samples. These proteins play a role in insulin processing, mitochondrial function, metabolism of biomolecules, calcium homeostasis, exocytosis, receptor signaling, ER protein folding, antioxidant activity and anti-apoptotic function. When oleate was also present during culture, expression of 15 proteins was different from the expression in the presence of palmitate alone. Most of the proteins affected by oleate are targets of the ER stress response and play a pro-survival role in beta cells such as protein folding and antioxidative defence. We conclude that restoration of pro-survival pathways of the ER stress response is a major mechanism underlying the protective effect of unsaturated fatty acids in beta-cells treated with saturated fatty acids. PMID:27344025

  12. Mitochondrial network regulation and its potential interference with inflammatory signals in pancreatic beta cells.

    PubMed

    Baltrusch, Simone

    2016-04-01

    Mitochondria fulfil multiple tasks in nutrient metabolism, energy production, redox homeostasis and stress response, and are essential for pancreatic beta cell function. The dynamism and health of the mitochondrial network is regulated by fission- and fusion-triggering factors and by a quality control system that removes dysfunctional organelles. Alongside the role of mitochondria in regulating apoptotic cell death mediated primarily via production of reactive oxygen species and release of cytochrome c, there is evidence of other links between mitochondria and inflammation that have implications for cell viability. This review briefly outlines two pathways that are potentially vital for pancreatic beta cell function. The first concerns the regulation of Parkin, a protein that acts, not only as a central player in regulating mitophagy, but also as an activator of the NF-ĸB pathway. The fact that expression of optic atrophy protein 1 (OPA1), a mitochondrial fusion inducer and master mitochondrial cristae biogenetic factor, is increased following NF-ĸB activation highlights a point of mitochondrial control that might be influenced by TNFα signalling. A second axis of interest is suggested by IL-6-mediated upregulation of the fission inducer FIS1 alongside downregulation of mitofusin 2 (MFN2), a guard of mitochondrial fusion and metabolism and an inhibitor of apoptosis. This review summarises a presentation given at the 'Islet inflammation in type 2 diabetes' symposium at the 2015 annual meeting of the EASD. It is accompanied two other reviews on topics from this symposium (by Marc Donath, DOI: 10.1007/s00125-016-3873-z , and Jerry Nadler and colleagues, DOI: 10.1007/s00125-016-3890-y ) and a commentary by the Session Chair, Piero Marchetti (DOI: 10.1007/s00125-016-3875-x ). PMID:26873508

  13. Proliferation and plasticity of human beta cells on physiologically occurring laminin isoforms.

    PubMed

    Banerjee, Meenal; Virtanen, Ismo; Palgi, Jaan; Korsgren, Olle; Otonkoski, Timo

    2012-05-15

    We have previously characterized the molecular composition of human islet basement membranes and shown that human beta cells bind to laminin 511 (LM511) through integrin α3β1 and Lutheran glycoprotein. We have now investigated the impact of physical contact between cultured human beta cells and the laminin isoforms occurring in their natural niche. Human islet preparations derived from 15 donors were used, beta cells and duct cells were purified by magnetic sorting. Overall beta-cell proliferation was low or undetectable. However, in many experiments the only proliferating beta cells were detected in contact with the laminin isoforms that are found in the human islets in vivo (511 and 411). Purified ductal and beta cells underwent epithelial-mesenchymal transition (EMT). LM511 partially blocked this dedifferentiation of purified beta cells, and did not affect purified duct cells. Interactions with the surrounding basement membrane are important for the growth and function of human beta cells. However, only a very limited level of beta-cell proliferation can be induced by exogenous factors. LM511 may be a useful substrate for human beta-cell maintenance in vitro. PMID:22314207

  14. Regulating the beta cell mass as a strategy for type-2 diabetes treatment.

    PubMed

    Song, Imane; Muller, Christo; Louw, Johan; Bouwens, Luc

    2015-01-01

    The incidence of type 2 diabetes (T2D) increases dramatically worldwide and has created an enormous health care burden. Obesity, dyslipidemia and insulin resistance are major risk factors for the development of T2D, but the major factor leading to the disease is failure of the insulin-producing beta cell mass to compensate for increasing insulin demands of the body. Progression of the disease further diminishes the beta cell mass as a result of lipotoxicity and glucotoxicity for which beta cells are particularly sensitive. Hence, treatment aiming to prevent beta cell loss or increase the number of beta cells could inhibit diabetes progression or lead to restoration of normal metabolism. Whereas current and new antidiabetic drugs are mainly targeting insulin secretion and action or glucose uptake, newer interventions must be found that prevent beta cell loss or increase beta cell number. The targets for this are beta cell proliferation, neogenesis and survival. This review examines major evidence from animal experiments suggesting that it is feasible to regulate the beta cell mass by bioactive compounds like growth factors, cytokines, hormones, phytochemicals and small molecules. Often the mode of action remains unclear due to inadequate methods to assess the effects of the compounds on the beta cell dynamics. Furthermore, a major challenge is to identify compounds with sufficient specificity in order to avoid unwanted effects on other cell types. Provided such safety issues can be solved, this may provide a curative approach for diabetes treatment. PMID:25654737

  15. Role of microRNAs in islet beta-cell compensation and failure during diabetes.

    PubMed

    Plaisance, Valérie; Waeber, Gérard; Regazzi, Romano; Abderrahmani, Amar

    2014-01-01

    Pancreatic beta-cell function and mass are markedly adaptive to compensate for the changes in insulin requirement observed during several situations such as pregnancy, obesity, glucocorticoids excess, or administration. This requires a beta-cell compensation which is achieved through a gain of beta-cell mass and function. Elucidating the physiological mechanisms that promote functional beta-cell mass expansion and that protect cells against death, is a key therapeutic target for diabetes. In this respect, several recent studies have emphasized the instrumental role of microRNAs in the control of beta-cell function. MicroRNAs are negative regulators of gene expression, and are pivotal for the control of beta-cell proliferation, function, and survival. On the one hand, changes in specific microRNA levels have been associated with beta-cell compensation and are triggered by hormones or bioactive peptides that promote beta-cell survival and function. Conversely, modifications in the expression of other specific microRNAs contribute to beta-cell dysfunction and death elicited by diabetogenic factors including, cytokines, chronic hyperlipidemia, hyperglycemia, and oxidized LDL. This review underlines the importance of targeting the microRNA network for future innovative therapies aiming at preventing the beta-cell decline in diabetes. PMID:24734255

  16. Role of MicroRNAs in Islet Beta-Cell Compensation and Failure during Diabetes

    PubMed Central

    Plaisance, Valérie; Waeber, Gérard

    2014-01-01

    Pancreatic beta-cell function and mass are markedly adaptive to compensate for the changes in insulin requirement observed during several situations such as pregnancy, obesity, glucocorticoids excess, or administration. This requires a beta-cell compensation which is achieved through a gain of beta-cell mass and function. Elucidating the physiological mechanisms that promote functional beta-cell mass expansion and that protect cells against death, is a key therapeutic target for diabetes. In this respect, several recent studies have emphasized the instrumental role of microRNAs in the control of beta-cell function. MicroRNAs are negative regulators of gene expression, and are pivotal for the control of beta-cell proliferation, function, and survival. On the one hand, changes in specific microRNA levels have been associated with beta-cell compensation and are triggered by hormones or bioactive peptides that promote beta-cell survival and function. Conversely, modifications in the expression of other specific microRNAs contribute to beta-cell dysfunction and death elicited by diabetogenic factors including, cytokines, chronic hyperlipidemia, hyperglycemia, and oxidized LDL. This review underlines the importance of targeting the microRNA network for future innovative therapies aiming at preventing the beta-cell decline in diabetes. PMID:24734255

  17. Cdk5 inhibitory peptide (CIP) inhibits Cdk5/p25 activity induced by high glucose in pancreatic beta cells and recovers insulin secretion from p25 damage.

    PubMed

    Zheng, Ya-Li; Li, Congyu; Hu, Ya-Fang; Cao, Li; Wang, Hui; Li, Bo; Lu, Xiao-Hua; Bao, Li; Luo, Hong-Yan; Shukla, Varsha; Amin, Niranjana D; Pant, Harish C

    2013-01-01

    Cdk5/p25 hyperactivity has been demonstrated to lead to neuron apoptosis and degenerations. Chronic exposure to high glucose (HG) results in hyperactivity of Cdk5 and reduced insulin secretion. Here, we set out to determine whether abnormal upregulation of Cdk5/p25 activity may be induced in a pancreatic beta cell line, Min6 cells. We first confirmed that p25 were induced in overexpressed p35 cells treated with HG and increased time course dependence. Next, we showed that no p25 was detected under short time HG stimulation (4-12 hrs), however was detectable in the long exposure in HG cells (24 hrs and 48 hrs). Cdk5 activity in the above cells was much higher than low glucose treated cells and resulted in more than 50% inhibition of insulin secretion. We confirmed these results by overexpression of p25 in Min6 cells. As in cortical neurons, CIP, a small peptide, inhibited Cdk5/p25 activity and restored insulin secretion. The same results were detected in co-infection of dominant negative Cdk5 (DNCdk5) with p25. CIP also reduced beta cells apoptosis induced by Cdk5/p25. These studies indicate that Cdk5/p25 hyperactivation deregulates insulin secretion and induces cell death in pancreatic beta cells and suggests that CIP may serve as a therapeutic agent for type 2 diabetes. PMID:24039692

  18. Parathyroid Hormone-Related Peptide (1-36) Enhances Beta Cell Regeneration and Increases Beta Cell Mass in a Mouse Model of Partial Pancreatectomy

    PubMed Central

    Mozar, Anaïs; Lin, Hugo; Williams, Katoura; Chin, Connie; Li, Rosemary; Kondegowda, Nagesha Guthalu; Stewart, Andrew F.; Garcia-Ocaña, Adolfo; Vasavada, Rupangi Chhaya

    2016-01-01

    Aims/Hypothesis Finding ways to stimulate the regeneration of endogenous pancreatic beta cells is an important goal in the treatment of diabetes. Parathyroid hormone-related protein (PTHrP), the full-length (1–139) and amino-terminal (1–36) peptides, enhance beta cell function, proliferation, and survival. Therefore, we hypothesize that PTHrP(1–36) has the potential to regenerate endogenous beta cells. Methods The partial pancreatectomy (PPx) mouse model of beta cell injury was used to test this hypothesis. Male Balb/c mice underwent either sham-operation or PPx, and were subsequently injected with PTHrP(1–36) (160μg/kg) or vehicle (veh), for 7, 30, or 90 days. The four groups of mice, sham-veh, sham-PTHrP, PPx-veh, and PPx-PTHrP were assessed for PTHrP and receptor expression, and glucose and beta cell homeostasis. Results PTHrP-receptor, but not the ligand, was significantly up-regulated in islets from mice that underwent PPx compared to sham-operated mice. This suggests that exogenous PTHrP could further enhance beta cell regeneration after PPx. PTHrP did not significantly affect body weight, blood glucose, plasma insulin, or insulin sensitivity, in either sham or PPx mice. Glucose tolerance improved in the PPx-PTHrP versus PPx-veh mice only in the early stages of treatment. As hypothesized, there was a significant increase in beta cell proliferation in PPx-PTHrP mice at days 7 and 30; however, this was normalized by day 90, compared to PPx-veh mice. Enhanced beta cell proliferation translated to a marked increase in beta cell mass at day 90, in PPx-PTHrP versus PPx-veh mice. Conclusions PTHrP(1–36) significantly enhances beta cell regeneration through increased beta cell proliferation and beta cell mass after PPx. Future studies will determine the potential of PTHrP to enhance functional beta cell mass in the setting of diabetes. PMID:27391423

  19. Regulation of Beta-Cell Function and Mass by the Dual Leucine Zipper Kinase.

    PubMed

    Oetjen, Elke

    2016-06-01

    Diabetes mellitus is one of the most rapidly increasing diseases worldwide, whereby approximately 90-95% of patients suffer from type 2 diabetes. Considering its micro- and macrovascular complications like blindness and myocardial infarction, a reliable anti-diabetic treatment is needed. Maintaining the function and the mass of the insulin producing beta-cells despite elevated levels of beta-cell-toxic prediabetic signals represents a desirable mechanism of action of anti-diabetic drugs. The dual leucine zipper kinase (DLK) inhibits the action of two transcription factors within the beta-cell, thereby interfering with insulin secretion and production and the conservation of beta-cell mass. Furthermore, DLK action is regulated by prediabetic signals. Hence, the inhibition of this kinase might protect beta-cells against beta-cell-toxic prediabetic signals and prevent the development of diabetes. DLK might thus present a novel drug target for the treatment of diabetes mellitus type 2. PMID:27100796

  20. Inorganic mercury causes pancreatic beta-cell death via the oxidative stress-induced apoptotic and necrotic pathways

    SciTech Connect

    Chen Yawen; Huang Chunfa; Yang Chingyao; Yen Chengchieh; Tsai Kehsung; Liu Shinghwa

    2010-03-15

    Mercury is a well-known highly toxic metal. In this study, we characterize and investigate the cytotoxicity and its possible mechanisms of inorganic mercury in pancreatic beta-cells. Mercury chloride (HgCl{sub 2}) dose-dependently decreased the function of insulin secretion and cell viability in pancreatic beta-cell-derived HIT-T15 cells and isolated mouse pancreatic islets. HgCl{sub 2} significantly increased ROS formation in HIT-T15 cells. Antioxidant N-acetylcysteine effectively reversed HgCl{sub 2}-induced insulin secretion dysfunction in HIT-T15 cells and isolated mouse pancreatic islets. Moreover, HgCl{sub 2} increased sub-G1 hypodiploids and annexin-V binding in HIT-T15 cells, indicating that HgCl{sub 2} possessed ability in apoptosis induction. HgCl{sub 2} also displayed several features of mitochondria-dependent apoptotic signals including disruption of the mitochondrial membrane potential, increase of mitochondrial cytochrome c release and activations of poly (ADP-ribose) polymerase (PARP) and caspase 3. Exposure of HIT-T15 cells to HgCl{sub 2} could significantly increase both apoptotic and necrotic cell populations by acridine orange/ethidium bromide dual staining. Meanwhile, HgCl{sub 2} could also trigger the depletion of intracellular ATP levels and increase the LDH release from HIT-T15 cells. These HgCl{sub 2}-induced cell death-related signals could be significantly reversed by N-acetylcysteine. The intracellular mercury levels were markedly elevated in HgCl{sub 2}-treated HIT-T15 cells. Taken together, these results suggest that HgCl{sub 2}-induced oxidative stress causes pancreatic beta-cell dysfunction and cytotoxicity involved the co-existence of apoptotic and necrotic cell death.

  1. Nkx6.1 is essential for maintaining the functional state of pancreatic beta cells.

    PubMed

    Taylor, Brandon L; Liu, Fen-Fen; Sander, Maike

    2013-09-26

    Recently, loss of beta-cell-specific traits has been proposed as an early cause of beta cell failure in diabetes. However, the molecular mechanisms that underlie the loss of beta cell features remain unclear. Here, we identify an Nkx6.1-controlled gene regulatory network as essential for maintaining the functional and molecular traits of mature beta cells. Conditional Nkx6.1 inactivation in adult mice caused rapid-onset diabetes and hypoinsulinemia. Genome-wide analysis of Nkx6.1-regulated genes and functional assays further revealed a critical role for Nkx6.1 in the control of insulin biosynthesis, insulin secretion, and beta cell proliferation. Over time, Nkx6.1-deficient beta cells acquired molecular characteristics of delta cells, revealing a molecular link between impaired beta cell functional properties and loss of cell identity. Given that Nkx6.1 levels are reduced in human type 2 diabetic beta cells, our study lends support to the concept that loss of beta cell features could contribute to the pathogenesis of diabetes. PMID:24035389

  2. Quantitative-Proteomic Comparison of Alpha and Beta Cells to Uncover Novel Targets for Lineage Reprogramming

    PubMed Central

    Mertins, Philipp; Udeshi, Namrata D.; Dančík, Vlado; Fomina-Yadlin, Dina; Kubicek, Stefan; Clemons, Paul A.; Schreiber, Stuart L.; Carr, Steven A.; Wagner, Bridget K.

    2014-01-01

    Type-1 diabetes (T1D) is an autoimmune disease in which insulin-secreting pancreatic beta cells are destroyed by the immune system. An emerging strategy to regenerate beta-cell mass is through transdifferentiation of pancreatic alpha cells to beta cells. We previously reported two small molecules, BRD7389 and GW8510, that induce insulin expression in a mouse alpha cell line and provide a glimpse into potential intermediate cell states in beta-cell reprogramming from alpha cells. These small-molecule studies suggested that inhibition of kinases in particular may induce the expression of several beta-cell markers in alpha cells. To identify potential lineage reprogramming protein targets, we compared the transcriptome, proteome, and phosphoproteome of alpha cells, beta cells, and compound-treated alpha cells. Our phosphoproteomic analysis indicated that two kinases, BRSK1 and CAMKK2, exhibit decreased phosphorylation in beta cells compared to alpha cells, and in compound-treated alpha cells compared to DMSO-treated alpha cells. Knock-down of these kinases in alpha cells resulted in expression of key beta-cell markers. These results provide evidence that perturbation of the kinome may be important for lineage reprogramming of alpha cells to beta cells. PMID:24759943

  3. Maturation of Stem Cell-Derived Beta-cells Guided by the Expression of Urocortin 3

    PubMed Central

    van der Meulen, Talitha; Huising, Mark O.

    2014-01-01

    Type 1 diabetes (T1D) is a devastating disease precipitated by an autoimmune response directed at the insulin-producing beta-cells of the pancreas for which no cure exists. Stem cell-derived beta-cells show great promise for a cure as they have the potential to supply unlimited numbers of cells that could be derived from a patient's own cells, thus eliminating the need for immunosuppression. Current in vitro protocols for the differentiation of stem cell-derived beta-cells can successfully generate pancreatic endoderm cells. In diabetic rodents, such cells can differentiate further along the beta-cell lineage until they are eventually capable of restoring normoglycemia. While these observations demonstrate that stem cell-derived pancreatic endoderm has the potential to differentiate into mature, glucose-responsive beta-cells, the signals that direct differentiation and maturation from pancreatic endoderm onwards remain poorly understood. In this review, we analyze the sequence of events that culminates in the formation of beta-cells during embryonic development. and summarize how current protocols to generate beta-cells have sought to capitalize on this ontogenic template. We place particular emphasis on the current challenges and opportunities which occur in the later stages of beta-cell differentiation and maturation of transplantable stem cell-derived beta-cells. Another focus is on the question how the use of recently identified maturation markers such as urocortin 3 can be instrumental in guiding these efforts. PMID:25148370

  4. Oxidative Stress Type Influences the Properties of Antioxidants Containing Polyphenols in RINm5F Beta Cells

    PubMed Central

    Auberval, Nathalie; Dal, Stéphanie; Bietiger, William; Seyfritz, Elodie; Peluso, Jean; Muller, Christian; Zhao, Minjie; Marchioni, Eric; Pinget, Michel; Jeandidier, Nathalie; Maillard, Elisa; Schini-Kerth, Valérie; Sigrist, Séverine

    2015-01-01

    The in vitro methods currently used to screen bioactive compounds focus on the use of a single model of oxidative stress. However, this simplistic view may lead to conflicting results. The aim of this study was to evaluate the antioxidant properties of two natural extracts (a mix of red wine polyphenols (RWPs) and epigallocatechin gallate (EGCG)) with three models of oxidative stress induced with hydrogen peroxide (H2O2), a mixture of hypoxanthine and xanthine oxidase (HX/XO), or streptozotocin (STZ) in RINm5F beta cells. We employed multiple approaches to validate their potential as therapeutic treatment options, including cell viability, reactive oxygen species production, and antioxidant enzymes expression. All three oxidative stresses induced a decrease in cell viability and an increase in apoptosis, whereas the level of ROS production was variable depending on the type of stress. The highest level of ROS was found for the HX/XO-induced stress, an increase that was reflected by higher expression antioxidant enzymes. Further, both antioxidant compounds presented beneficial effects during oxidative stress, but EGCG appeared to be a more efficient antioxidant. These data indicate that the efficiency of natural antioxidants is dependent on both the nature of the compound and the type of oxidative stress generated. PMID:26508986

  5. Beta-cell deterioration during diabetes: what's in the gun?

    PubMed

    Robertson, R Paul

    2009-10-01

    Clinical management of patients with type 2 diabetes (T2D) successfully prevents extreme hyperglycemia but does not precisely control glucose levels throughout the day. The pathogenesis of T2D is akin to a double-barrel shotgun. The first trigger causes an explosion that sets genetic expression of the disease in motion; the second trigger discharges a host of environmental factors that worsen its clinical course. Candidate shells include glucolipotoxicity, cytokines, oxidative and endoplasmic reticulum stress and insulin resistance. This review considers how each candidate adversely impacts beta-cell function to create the downward spiral of glycemic control. Their roles in pathogenesis raise possibilities for new drug therapies designed to protect against adverse effects of residual hyperglycemia in patients treated with conventional drugs. PMID:19748794

  6. Glucose activates prenyltransferases in pancreatic islet {beta}-cells

    SciTech Connect

    Goalstone, Marc; Kamath, Vasudeva; Kowluru, Anjaneyulu

    2010-01-01

    A growing body of evidence implicates small G-proteins [e.g., Cdc42 and Rac1] in glucose-stimulated insulin secretion [GSIS] in the islet {beta}-cell. These signaling proteins undergo post-translational modifications [e.g., prenylation] at their C-terminal cysteine residue and appear to be essential for the transport and fusion of insulin-containing secretory granules with the plasma membrane and the exocytotic secretion of insulin. However, potential regulation of the prenylating enzymes by physiological insulin secretogues [e.g., glucose] has not been investigated thus far. Herein, we report immunological localization, sub-cellular distribution and regulation of farnesyltransferases [FTases] and geranylgeranyltransferase [GGTase] by glucose in insulin-secreting INS 832/13 {beta}-cells and normal rat islets. Our findings suggest that an insulinotropic concentration of glucose [20 mM] markedly stimulated the expression of the {alpha}-subunits of FTase/GGTase-1, but not the {beta}-subunits of FTase or GGTase-1 without significantly affecting the predominantly cytosolic distribution of these holoenzymes in INS 832/13 cells and rodent islets. Under these conditions, glucose significantly stimulated [2.5- to 4.0-fold over basal] the activities of both FTase and GGTase-1 in both cell types. Together, these findings provide the first evidence to suggest that GSIS involves activation of the endogenous islet prenyltransferases by glucose, culminating in the activation of their respective G-protein substrates, which is necessary for cytoskeletal rearrangement, vesicular transport, fusion and secretion of insulin.

  7. Cell therapies for pancreatic beta-cell replenishment.

    PubMed

    Okere, Bernard; Lucaccioni, Laura; Dominici, Massimo; Iughetti, Lorenzo

    2016-01-01

    The current treatment approach for type 1 diabetes is based on daily insulin injections, combined with blood glucose monitoring. However, administration of exogenous insulin fails to mimic the physiological activity of the islet, therefore diabetes often progresses with the development of serious complications such as kidney failure, retinopathy and vascular disease. Whole pancreas transplantation is associated with risks of major invasive surgery along with side effects of immunosuppressive therapy to avoid organ rejection. Replacement of pancreatic beta-cells would represent an ideal treatment that could overcome the above mentioned therapeutic hurdles. In this context, transplantation of islets of Langerhans is considered a less invasive procedure although long-term outcomes showed that only 10 % of the patients remained insulin independent five years after the transplant. Moreover, due to shortage of organs and the inability of islet to be expanded ex vivo, this therapy can be offered to a very limited number of patients. Over the past decade, cellular therapies have emerged as the new frontier of treatment of several diseases. Furthermore the advent of stem cells as renewable source of cell-substitutes to replenish the beta cell population, has blurred the hype on islet transplantation. Breakthrough cellular approaches aim to generate stem-cell-derived insulin producing cells, which could make diabetes cellular therapy available to millions. However, to date, stem cell therapy for diabetes is still in its early experimental stages. This review describes the most reliable sources of stem cells that have been developed to produce insulin and their most relevant experimental applications for the cure of diabetes. PMID:27400873

  8. Nanomaterial Solutions for the Protection of Insulin Producing Beta Cells

    NASA Astrophysics Data System (ADS)

    Atchison, Nicole Ann

    Islet transplantation is a promising treatment for type 1 diabetes. However, even with the many successes, islet transplantation has yet to reach its full potential. Limited islet sources, loss of cell viability during isolation and culture, and post-transplant graft loss are a few of the issues preventing extensive use of islet transplantation. The application of biomaterial systems to alleviate some of the stresses affecting islet viability has led to improvements in isolation and transplantation outcomes, but problems persist. In this work we approach two distinct issues affecting islet viability; ischemic conditions and immunological attack post-transplant. Ischemic conditions have been linked to a loss of islet graft function and occur during organ preservation, islet isolation and culture, and after islets are transplanted. We show that liposomal delivery of adenosine triphosphate (ATP) to beta cells can limit cell death and loss of function in ischemic conditions. We demonstrate that by functionalizing liposomes with the fibronectin-mimetic peptide PR_b, delivery of liposomes to porcine islets and rat beta cells is increased compared to nontargeted controls. Additionally, liposomes are shown to protect by providing both ATP and lipids to the ischemic cells. The delivery of ATP was investigated here but application of PR_b functionalized liposomes could be extended to other interesting cargos as well. The second area of investigation involves encapsulation of islets with silica nanoparticles to create a permselective barrier. Silica nanoparticles are an interesting material for encapsulation given their ability to be fine-tuned and further functionalized. We demonstrate that size-tunable, fluorescent silica nanoparticles can be assembled layer-by-layer on the surface of cells and that silica nanoparticle encapsulated islets are able to secrete insulin in response to a glucose challenge.

  9. Glucagon-Like Peptide-1 Receptor Agonists: Beta-Cell Protection or Exhaustion?

    PubMed

    van Raalte, Daniël H; Verchere, C Bruce

    2016-07-01

    Glucagon-like peptide (GLP)-1 receptor agonists enhance insulin secretion and may improve pancreatic islet cell function. However, GLP-1 receptor (GLP-1R) agonist treatment may have more complex, and sometimes deleterious, effects on beta cells. We discuss the concepts of beta cell protection versus exhaustion for different GLP-1R agonists based on recent data. PMID:27160799

  10. Specialized Hub Beta Cells Trade Maximal Insulin Production for Perfect Timing.

    PubMed

    Kolic, Jelena; Johnson, James D

    2016-09-13

    The pulsatility of insulin release is disturbed early in type 2 diabetes, but it is not clear whether specialized pacemaker cells drive islet oscillations. In this issue of Cell Metabolism, Johnston et al. (2016) show that specialized hubs, identified as 1%-10% of beta cells with more active mitochondria and less insulin, synchronize beta cell oscillations. PMID:27626196

  11. Genetic predisposition for beta cell fragility underlies type 1 and type 2 diabetes.

    PubMed

    Dooley, James; Tian, Lei; Schonefeldt, Susann; Delghingaro-Augusto, Viviane; Garcia-Perez, Josselyn E; Pasciuto, Emanuela; Di Marino, Daniele; Carr, Edward J; Oskolkov, Nikolay; Lyssenko, Valeriya; Franckaert, Dean; Lagou, Vasiliki; Overbergh, Lut; Vandenbussche, Jonathan; Allemeersch, Joke; Chabot-Roy, Genevieve; Dahlstrom, Jane E; Laybutt, D Ross; Petrovsky, Nikolai; Socha, Luis; Gevaert, Kris; Jetten, Anton M; Lambrechts, Diether; Linterman, Michelle A; Goodnow, Chris C; Nolan, Christopher J; Lesage, Sylvie; Schlenner, Susan M; Liston, Adrian

    2016-05-01

    Type 1 (T1D) and type 2 (T2D) diabetes share pathophysiological characteristics, yet mechanistic links have remained elusive. T1D results from autoimmune destruction of pancreatic beta cells, whereas beta cell failure in T2D is delayed and progressive. Here we find a new genetic component of diabetes susceptibility in T1D non-obese diabetic (NOD) mice, identifying immune-independent beta cell fragility. Genetic variation in Xrcc4 and Glis3 alters the response of NOD beta cells to unfolded protein stress, enhancing the apoptotic and senescent fates. The same transcriptional relationships were observed in human islets, demonstrating the role of beta cell fragility in genetic predisposition to diabetes. PMID:26998692

  12. Update on the protective molecular pathways improving pancreatic beta-cell dysfunction.

    PubMed

    Puddu, Alessandra; Sanguineti, Roberta; Mach, François; Dallegri, Franco; Viviani, Giorgio Luciano; Montecucco, Fabrizio

    2013-01-01

    The primary function of pancreatic beta-cells is to produce and release insulin in response to increment in extracellular glucose concentrations, thus maintaining glucose homeostasis. Deficient beta-cell function can have profound metabolic consequences, leading to the development of hyperglycemia and, ultimately, diabetes mellitus. Therefore, strategies targeting the maintenance of the normal function and protecting pancreatic beta-cells from injury or death might be crucial in the treatment of diabetes. This narrative review will update evidence from the recently identified molecular regulators preserving beta-cell mass and function recovery in order to suggest potential therapeutic targets against diabetes. This review will also highlight the relevance for novel molecular pathways potentially improving beta-cell dysfunction. PMID:23737653

  13. Direct Reprogramming for Pancreatic Beta-Cells Using Key Developmental Genes

    PubMed Central

    Cavelti-Weder, Claudia; Li, Weida; Zumsteg, Adrian; Stemann, Marianne; Yamada, Takatsugu; Bonner-Weir, Susan; Weir, Gordon

    2015-01-01

    Direct reprogramming is a promising approach for regenerative medicine whereby one cell type is directly converted into another without going through a multipotent or pluripotent stage. This reprogramming approach has been extensively explored for the generation of functional insulin-secreting cells from non-beta-cells with the aim of developing novel cell therapies for the treatment of people with diabetes lacking sufficient endogenous beta-cells. A common approach for such conversion studies is the introduction of key regulators that are important in controlling beta-cell development and maintenance. In this review, we will summarize the recent advances in the field of beta-cell reprogramming and discuss the challenges of creating functional and long-lasting beta-cells. PMID:26998407

  14. Marked over expression of uncoupling protein-2 in beta cells exerts minor effects on mitochondrial metabolism

    SciTech Connect

    Hals, Ingrid K.; Ogata, Hirotaka; Pettersen, Elin; Ma, Zuheng; Bjoerklund, Anneli; Skorpen, Frank; Egeberg, Kjartan Wollo; Grill, Valdemar

    2012-06-29

    Highlights: Black-Right-Pointing-Pointer The impact of UCP-2 over expression on mitochondrial function is controversial. Black-Right-Pointing-Pointer We tested mitochondrial functions at defined levels of overexpression. Black-Right-Pointing-Pointer We find minor increases of fatty acid oxidation and uncoupling. Black-Right-Pointing-Pointer Effects were seen only at high level (fourfold) of over expression. Black-Right-Pointing-Pointer Hence it is doubtful whether these effects are of importance in diabetes. -- Abstract: Evidence is conflicting as to the impact of elevated levels of uncoupling protein-2 (UCP-2) on insulin-producing beta cells. Here we investigated effects of a fourfold induction of UCP-2 protein primarily on mitochondrial parameters and tested for replication of positive findings at a lower level of induction. We transfected INS-1 cells to obtain a tet-on inducible cell line. A 48 h exposure to 1 {mu}g/ml of doxycycline (dox) induced UCP-2 fourfold (424 {+-} 113%, mean {+-} SEM) and 0.1 {mu}g/ml twofold (178 {+-} 29%, n = 3). Fourfold induced cells displayed normal viability (MTT, apoptosis), normal cellular insulin contents and, glucose-induced insulin secretion (+27 {+-} 11%) as well as D-[U-{sup 14}C]-glucose oxidation (+5 {+-} 9% at 11 mM glucose). Oxidation of [1-{sup 14}C]-oleate was increased from 4088 to 5797 fmol/{mu}g prot/2 h at 3.3 mM glucose, p < 0.03. Oxidation of L-[{sup 14}C(U)]-glutamine was unaffected. Induction of UCP-2 did not significantly affect measures of mitochondrial membrane potential (Rhodamine 123) or mitochondrial mass (Mitotracker Green) and did not affect ATP levels. Oligomycin-inhibited oxygen consumption (a measure of mitochondrial uncoupling) was marginally increased, the effect being significant in comparison with dox-only treated cells, p < 0.05. Oxygen radicals, assessed by dichlorofluorescin diacetate, were decreased by 30%, p < 0.025. Testing for the lower level of UCP-2 induction did not reproduce any of the

  15. Mafa expression enhances glucose-responsive insulin secretion in neonatal rat beta cells

    PubMed Central

    Aguayo-Mazzucato, C.; Koh, A.; El Khattabi, I.; Li, W.-C.; Toschi, E.; Jermendy, A.; Juhl, K.; Mao, K.; Weir, G. C.

    2011-01-01

    Aim/hypothesis Neonatal beta cells lack glucose-stimulated insulin secretion and are thus functionally immature. We hypothesised that this lack of glucose responsiveness results from a generalised low expression of genes characteristic of mature functional beta cells. Important glucose-responsive transcription factors, Mafa and Pdx1, regulate genes involved in insulin synthesis and secretion, and have been implicated in late beta cell development. The aim of this study was to assess whether Mafa and/or Pdx1 regulates the postnatal functional maturation of beta cells. Methods By quantitative PCR we evaluated expression of these and other beta cell genes over the first month compared with adult. After infection with adenovirus expressing MAFA, Pdx1 or green fluorescent protein (Gfp), P2 rat islets were evaluated by RT-PCR and insulin secretion with static incubation and reverse haemolytic plaque assay (RHPA). Results At P2 most beta cell genes were expressed at about 10% of adult, but by P7 Pdx1 and Neurod1 no longer differ from adult; by contrast, Mafa expression remained significantly lower than adult through P21. Overexpression of Pdx1 increased Mafa, Neurod1, glucokinase (Gck) mRNA and insulin content but failed to enhance glucose responsiveness. Similar overexpression of MAFA resulted in increased Neurod1, Nkx6-1, Gck and Glp1r mRNAs and no change in insulin content but, importantly, acquisition of glucose-responsive insulin secretion. Both the percentage of secreting beta cells and the amount of insulin secreted per beta cell increased, approaching that of adult beta cells. Conclusions/interpretation In the process of functional maturation acquiring glucose-responsive insulin secretion, neonatal beta cells undergo a coordinated gene expression programme in which Mafa plays a crucial role. PMID:21190012

  16. Interleukin-1 beta induces the expression and production of stem cell factor by epithelial cells: crucial involvement of the PI-3K/mTOR pathway and HIF-1 transcription complex

    PubMed Central

    Wyszynski, Rafal W; Gibbs, Bernhard F; Varani, Luca; Iannotta, Daniela; Sumbayev, Vadim V

    2016-01-01

    Potential crosslinks between inflammation and leukaemia have been discussed for some time, but experimental evidence to support this dogma is scarce. In particular, it is important to understand the mechanisms responsible for potential upregulation of proto-oncogenic growth factor expressions by inflammatory mediators. Here, we investigated the ability of the highly inflammatory cytokine interleukin-1 beta (IL-1β) to induce the production of stem cell factor (SCF), which is a major hematopoietic growth factor that controls the progression of acute myeloid leukaemia upon malignant transformation of haematopoietic myeloid cells. We found that human IL-1β induced the expression/secretion of SCF in MCF-7 human epithelial breast cancer cells and that this process depended on the hypoxia-inducible factor 1 (HIF-1) transcription complex. We also demonstrated a crucial role of the phosphatidylinositol-3 kinase (PI-3K)/mammalian target of rapamycin (mTOR) pathway in IL-1β-induced HIF-1α accumulation in MCF-7 cells. Importantly, mTOR was also found to play a role in IL-1β-induced SCF production. Furthermore, a tendency for a positive correlation of IL-1β and SCF levels in the plasma of healthy human donors was observed. Altogether, our results demonstrate that IL-1β, which normally bridges innate and adaptive immunity, induces the production of the major haematopoietic/proleukaemic growth factor SCF through the PI-3K/mTOR pathway and the HIF-1 transcription complex. These findings strongly support a cross-talk between inflammation and acute myeloid leukaemia. PMID:25418470

  17. Bmal1 and Beta cell clock are required for adaptation to circadian disruption, and their loss of function leads to oxidative stress-induced Beta cell failure in mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Circadian disruption has deleterious effects on metabolism. Global deletion of Bmal1, a core clock gene, results in Beta cell dysfunction and diabetes. However, it is unknown if this is due to loss of cell-autonomous function of Bmal1 in Beta cells. To address this, we generated mice with Beta cell ...

  18. Beta Cell Mass Restoration in Alloxan-Diabetic Mice Treated with EGF and Gastrin

    PubMed Central

    Song, Imane; Patel, Oelfah; Himpe, Eddy; Muller, Christo J. F.; Bouwens, Luc

    2015-01-01

    One week of treatment with EGF and gastrin (EGF/G) was shown to restore normoglycemia and to induce islet regeneration in mice treated with the diabetogenic agent alloxan. The mechanisms underlying this regeneration are not fully understood. We performed genetic lineage tracing experiments to evaluate the contribution of beta cell neogenesis in this model. One day after alloxan administration, mice received EGF/G treatment for one week. The treatment could not prevent the initial alloxan-induced beta cell mass destruction, however it did reverse glycemia to control levels within one day, suggesting improved peripheral glucose uptake. In vitro experiments with C2C12 cell line showed that EGF could stimulate glucose uptake with an efficacy comparable to that of insulin. Subsequently, EGF/G treatment stimulated a 3-fold increase in beta cell mass, which was partially driven by neogenesis and beta cell proliferation as assessed by beta cell lineage tracing and BrdU-labeling experiments, respectively. Acinar cell lineage tracing failed to show an important contribution of acinar cells to the newly formed beta cells. No appearance of transitional cells co-expressing insulin and glucagon, a hallmark for alpha-to-beta cell conversion, was found, suggesting that alpha cells did not significantly contribute to the regeneration. An important fraction of the beta cells significantly lost insulin positivity after alloxan administration, which was restored to normal after one week of EGF/G treatment. Alloxan-only mice showed more pronounced beta cell neogenesis and proliferation, even though beta cell mass remained significantly depleted, suggesting ongoing beta cell death in that group. After one week, macrophage infiltration was significantly reduced in EGF/G-treated group compared to the alloxan-only group. Our results suggest that EGF/G-induced beta cell regeneration in alloxan-diabetic mice is driven by beta cell neogenesis, proliferation and recovery of insulin. The

  19. Beta Cell Mass Restoration in Alloxan-Diabetic Mice Treated with EGF and Gastrin.

    PubMed

    Song, Imane; Patel, Oelfah; Himpe, Eddy; Muller, Christo J F; Bouwens, Luc

    2015-01-01

    One week of treatment with EGF and gastrin (EGF/G) was shown to restore normoglycemia and to induce islet regeneration in mice treated with the diabetogenic agent alloxan. The mechanisms underlying this regeneration are not fully understood. We performed genetic lineage tracing experiments to evaluate the contribution of beta cell neogenesis in this model. One day after alloxan administration, mice received EGF/G treatment for one week. The treatment could not prevent the initial alloxan-induced beta cell mass destruction, however it did reverse glycemia to control levels within one day, suggesting improved peripheral glucose uptake. In vitro experiments with C2C12 cell line showed that EGF could stimulate glucose uptake with an efficacy comparable to that of insulin. Subsequently, EGF/G treatment stimulated a 3-fold increase in beta cell mass, which was partially driven by neogenesis and beta cell proliferation as assessed by beta cell lineage tracing and BrdU-labeling experiments, respectively. Acinar cell lineage tracing failed to show an important contribution of acinar cells to the newly formed beta cells. No appearance of transitional cells co-expressing insulin and glucagon, a hallmark for alpha-to-beta cell conversion, was found, suggesting that alpha cells did not significantly contribute to the regeneration. An important fraction of the beta cells significantly lost insulin positivity after alloxan administration, which was restored to normal after one week of EGF/G treatment. Alloxan-only mice showed more pronounced beta cell neogenesis and proliferation, even though beta cell mass remained significantly depleted, suggesting ongoing beta cell death in that group. After one week, macrophage infiltration was significantly reduced in EGF/G-treated group compared to the alloxan-only group. Our results suggest that EGF/G-induced beta cell regeneration in alloxan-diabetic mice is driven by beta cell neogenesis, proliferation and recovery of insulin. The

  20. glucagon is essential for alpha cell transdifferentiation and beta cell neogenesis

    PubMed Central

    Ye, Lihua; Robertson, Morgan A.; Hesselson, Daniel; Stainier, Didier Y. R.; Anderson, Ryan M.

    2015-01-01

    The interconversion of cell lineages via transdifferentiation is an adaptive mode of tissue regeneration and an appealing therapeutic target. However, its clinical exploitation is contingent upon the discovery of contextual regulators of cell fate acquisition and maintenance. In murine models of diabetes, glucagon-secreting alpha cells transdifferentiate into insulin-secreting beta cells following targeted beta cell depletion, regenerating the form and function of the pancreatic islet. However, the molecular triggers of this mode of regeneration are unknown. Here, using lineage-tracing assays in a transgenic zebrafish model of beta cell ablation, we demonstrate conserved plasticity of alpha cells during islet regeneration. In addition, we show that glucagon expression is upregulated after injury. Through gene knockdown and rescue approaches, we also find that peptides derived from the glucagon gene are necessary for alpha-to-beta cell fate switching. Importantly, whereas beta cell neogenesis was stimulated by glucose, alpha-to-beta cell conversion was not, suggesting that transdifferentiation is not mediated by glucagon/GLP-1 control of hepatic glucose production. Overall, this study supports the hypothesis that alpha cells are an endogenous reservoir of potential new beta cells. It further reveals that glucagon plays an important role in maintaining endocrine cell homeostasis through feedback mechanisms that govern cell fate stability. PMID:25852199

  1. Co-culture of clonal beta cells with GLP-1 and glucagon-secreting cell line impacts on beta cell insulin secretion, proliferation and susceptibility to cytotoxins.

    PubMed

    Green, Alastair D; Vasu, Srividya; Moffett, R Charlotte; Flatt, Peter R

    2016-06-01

    We investigated the direct effects on insulin releasing MIN6 cells of chronic exposure to GLP-1, glucagon or a combination of both peptides secreted from GLUTag L-cell and αTC1.9 alpha-cell lines in co-culture. MIN6, GLUTag and αTC1.9 cell lines exhibited high cellular hormone content and release of insulin, GLP-1 and glucagon, respectively. Co-culture of MIN6 cells with GLUTag cells significantly increased cellular insulin content, beta-cell proliferation, insulin secretory responses to a range of established secretogogues and afforded protection against exposure cytotoxic concentrations of glucose, lipid, streptozotocin or cytokines. Benefits of co-culture of MIN6 cells with αTC1.9 alphacells were limited to enhanced beta-cell proliferation with marginal positive actions on both insulin secretion and cellular protection. In contrast, co-culture of MIN6 with GLUTag cells plus αTC1.9 cells, markedly enhanced both insulin secretory responses and protection against beta-cell toxins compared with co-culture with GLUTag cells alone. These data indicate important long-term effects of conjoint GLP-1 and glucagon exposure on beta-cell function. This illustrates the possible functional significance of alpha-cell GLP-1 production as well as direct beneficial effects of dual agonism at beta-cell GLP-1 and glucagon receptors. PMID:27015674

  2. Reduced Expression of the Liver/Beta-Cell Glucose Transporter Isoform in Glucose-Insensitive Pancreatic Beta Cells of Diabetic Rats

    NASA Astrophysics Data System (ADS)

    Thorens, Bernard; Weir, Gordon C.; Leahy, John L.; Lodish, Harvey F.; Bonner-Weir, Susan

    1990-09-01

    Rats injected with a single dose of streptozocin at 2 days of age develop non-insulin-dependent diabetes 6 weeks later. The pancreatic beta islet cells of these diabetic rats display a loss of glucose-induced insulin secretion while maintaining sensitivity to other secretagogues such as arginine. We analyzed the level of expression of the liver/beta-cell glucose transporter isoform in diabetic islets by immunofluorescence staining of pancreas sections and by Western blotting of islet lysates. Islets from diabetic animals have a reduced expression of this beta-cell-specific glucose transporter isoform and the extent of reduction is correlated with the severity of hyperglycemia. In contrast, expression of this transporter isoform in liver is minimally modified by the diabetes. Thus a decreased expression of the liver/beta-cell glucose transporter isoform in beta cells is associated with the impaired glucose sensing characteristic of diabetic islets; our data suggest that this glucose transporter may be part of the beta-cell glucose sensor.

  3. Pancreatic beta cell function in the fetal pig and sow.

    PubMed

    Fowden, A L; Comline, R S; Silver, M

    1982-04-01

    Insulin secretion was investigated in acutely anaesthetized and chronically catheterized sows and their fetuses during late gestation. In the conscious animals, the mean fetal concentration of plasma insulin was 8.4 +/- 1.5 microunits/ml which was significantly less than the corresponding maternal value of 33.9 +/- 6.5 microunits/ml (n = 12, P less than 0.01). The plasma concentrations of insulin and glucose in the new-born piglets from these litters were not significantly different from the values observed in utero. The plasma concentration of insulin in the anaesthetized fetuses was significantly less than that in the chronically catheterized piglets over the same range of glucose levels. In the chronically catheterized animals, both fetal and maternal levels of insulin rose with increasing concentrations of plasma glucose while under acute conditions there was no correlation between the endogenous concentrations of insulin and glucose in either the fetuses or their mothers. Infusion of exogenous glucose (0.5 g as a 50% solution in 0.9% NaCl) stimulated the release of insulin in all the chronically catheterized fetuses studied but rarely increased the concentration of insulin in the anaesthetized fetusus. The present findings show that anaesthesia and surgery depress pancreatic beta cell function in the pig, particularly in the fetus. PMID:7043523

  4. Cocoa Phenolic Extract Protects Pancreatic Beta Cells against Oxidative Stress

    PubMed Central

    Martín, María Ángeles; Ramos, Sonia; Cordero-Herrero, Isabel; Bravo, Laura; Goya, Luis

    2013-01-01

    Diabetes mellitus is associated with reductions in glutathione, supporting the critical role of oxidative stress in its pathogenesis. Antioxidant food components such as flavonoids have a protective role against oxidative stress-induced degenerative and age-related diseases. Flavonoids constitute an important part of the human diet; they can be found in most plant foods, including green tea, grapes or cocoa and possess multiple biological activities. This study investigates the chemo-protective effect of a cocoa phenolic extract (CPE) containing mainly flavonoids against oxidative stress induced by tert-butylhydroperoxide (t-BOOH) on Ins-1E pancreatic beta cells. Cell viability and oxidative status were evaluated. Ins-1E cells treatment with 5–20 μg/mL CPE for 20 h evoked no cell damage and did not alter ROS production. Addition of 50 μM t-BOOH for 2 h increased ROS and carbonyl groups content and decreased reduced glutathione level. Pre-treatment of cells with CPE significantly prevented the t-BOOH-induced ROS and carbonyl groups and returned antioxidant defences to adequate levels. Thus, Ins-1E cells treated with CPE showed a remarkable recovery of cell viability damaged by t-BOOH, indicating that integrity of surviving machineries in the CPE-treated cells was notably protected against the oxidative insult. PMID:23912326

  5. Present and future cell therapies for pancreatic beta cell replenishment

    PubMed Central

    Domínguez-Bendala, Juan; Ricordi, Camillo

    2012-01-01

    If only at a small scale, islet transplantation has successfully addressed what ought to be the primary endpoint of any cell therapy: the functional replenishment of damaged tissue in patients. After years of less-than-optimal approaches to immunosuppression, recent advances consistently yield long-term graft survival rates comparable to those of whole pancreas transplantation. Limited organ availability is the main hurdle that stands in the way of the widespread clinical utilization of this pioneering intervention. Progress in stem cell research over the past decade, coupled with our decades-long experience with islet transplantation, is shaping the future of cell therapies for the treatment of diabetes. Here we review the most promising avenues of research aimed at generating an inexhaustible supply of insulin-producing cells for islet regeneration, including the differentiation of pluripotent and multipotent stem cells of embryonic and adult origin along the beta cell lineage and the direct reprogramming of non-endocrine tissues into insulin-producing cells. PMID:23322984

  6. A role for G(z) in pancreatic islet beta-cell biology.

    PubMed

    Kimple, Michelle E; Nixon, Andrew B; Kelly, Patrick; Bailey, Candice L; Young, Kenneth H; Fields, Timothy A; Casey, Patrick J

    2005-09-01

    Glucose-stimulated insulin secretion and beta-cell growth are important facets of pancreatic islet beta-cell biology. As a result, factors that modulate these processes are of great interest for the potential treatment of Type 2 diabetes. Here, we present evidence that the heterotrimeric G protein G(z) and its effectors, including some previously thought to be confined in expression to neuronal cells, are present in pancreatic beta-cells, the largest cellular constituent of the islets of Langerhans. Furthermore, signaling pathways upon which G alpha(z) impacts are intact in beta-cells, and G alpha(z) activation inhibits both cAMP production and glucose-stimulated insulin secretion in the Ins-1(832/13) beta-cell-derived line. Inhibition of glucose-stimulated insulin secretion by prostaglandin E (PGE1) is pertussis-toxin insensitive, indicating that other G alpha(i) family members are not involved in this process in this beta-cell line. Indeed, overexpression of a selective deactivator of G alpha(z), the RGS domain of RGSZ1, blocks the inhibitory effect of PGE1 on glucose-stimulated insulin secretion. Finally, the inhibition of glucose-stimulated insulin secretion by PGE1 is substantially blunted by small interfering RNA-mediated knockdown of G alpha(z) expression. Taken together, these data strongly imply that the endogenous E prostanoid receptor in the Ins-1(832/13) beta-cell line couples to G(z) predominantly and perhaps even exclusively. These data provide the first evidence for G(z) signaling in pancreatic beta-cells, and identify an endogenous receptor-mediated signaling process in beta-cells that is dependent on G alpha(z) function. PMID:16157560

  7. Influence and timing of arrival of murine neural crest on pancreatic beta cell development and maturation.

    PubMed

    Plank, Jennifer L; Mundell, Nathan A; Frist, Audrey Y; LeGrone, Alison W; Kim, Thomas; Musser, Melissa A; Walter, Teagan J; Labosky, Patricia A

    2011-01-15

    Interactions between cells from the ectoderm and mesoderm influence development of the endodermally-derived pancreas. While much is known about how mesoderm regulates pancreatic development, relatively little is understood about how and when the ectodermally-derived neural crest regulates pancreatic development and specifically, beta cell maturation. A previous study demonstrated that signals from the neural crest regulate beta cell proliferation and ultimately, beta cell mass. Here, we expand on that work to describe timing of neural crest arrival at the developing pancreatic bud and extend our knowledge of the non-cell autonomous role for neural crest derivatives in the process of beta cell maturation. We demonstrated that murine neural crest entered the pancreatic mesenchyme between the 26 and 27 somite stages (approximately 10.0 dpc) and became intermingled with pancreatic progenitors as the epithelium branched into the surrounding mesenchyme. Using a neural crest-specific deletion of the Forkhead transcription factor Foxd3, we ablated neural crest cells that migrate to the pancreatic primordium. Consistent with previous data, in the absence of Foxd3, and therefore the absence of neural crest cells, proliferation of insulin-expressing cells and insulin-positive area are increased. Analysis of endocrine cell gene expression in the absence of neural crest demonstrated that, although the number of insulin-expressing cells was increased, beta cell maturation was significantly impaired. Decreased MafA and Pdx1 expression illustrated the defect in beta cell maturation; we discovered that without neural crest, there was a reduction in the percentage of insulin-positive cells that co-expressed Glut2 and Pdx1 compared to controls. In addition, transmission electron microscopy analyses revealed decreased numbers of characteristic insulin granules and the presence of abnormal granules in insulin-expressing cells from mutant embryos. Together, these data demonstrate that

  8. Influence and timing of arrival of murine neural crest on pancreatic beta cell development and maturation

    PubMed Central

    Plank, Jennifer L.; Mundell, Nathan A.; Frist, Audrey Y.; LeGrone, Alison W.; Kim, Thomas; Musser, Melissa A.; Walter, Teagan J.; Labosky, Patricia A.

    2010-01-01

    Interactions between cells from the ectoderm and mesoderm influence development of the endodermally-derived pancreas. While much is known about how mesoderm regulates pancreatic development, relatively little is understood about how and when the ectodermally-derived neural crest regulates pancreatic development and specifically, beta cell maturation. A previous study demonstrated that signals from the neural crest regulate beta cell proliferation and ultimately, beta cell mass. Here, we expand on that work to describe timing of neural crest arrival at the developing pancreatic bud and extend our knowledge of the non-cell autonomous role for neural crest derivatives in the process of beta cell maturation. We demonstrated that murine neural crest entered the pancreatic mesenchyme between the 26 and 27 somite stages (approximately 10.0 dpc) and became intermingled with pancreatic progenitors as the epithelium branched into the surrounding mesenchyme. Using a neural crest-specific deletion of the Forkhead transcription factor Foxd3, we ablated neural crest cells that migrate to the pancreatic primordium. Consistent with previous data, in the absence of Foxd3, and therefore the absence of neural crest cells, proliferation of Insulin-expressing cells and Insulin-positive area are increased. Analysis of endocrine cell gene expression in the absence of neural crest demonstrated that, although the number of Insulin-expressing cells was increased, beta cell maturation was significantly impaired. Decreased MafA and Pdx1 expression illustrated the defect in beta cell maturation; we discovered that without neural crest, there was a reduction in the percentage of Insulin-positive cells that co-expressed Glut2 and Pdx1 compared to controls. In addition, transmission electron microscopy analyses revealed decreased numbers of characteristic Insulin granules and the presence of abnormal granules in Insulin-expressing cells from mutant embryos. Together, these data demonstrate that

  9. Beta cell imaging - a key tool in optimized diabetes prevention and treatment.

    PubMed

    Gotthardt, Martin; Eizirik, Decio L; Cnop, Miriam; Brom, Maarten

    2014-08-01

    The prevalence of diabetes is 382 million worldwide, and is expected to rise to 592 million in 2035 (http://www.idf.org/diabetesatlas); 2.5-15% of national annual healthcare budgets are related to diabetes care, potentially increasing to 40% in high-prevalence countries. Beta cell dysfunction and death are central events in diabetes pathogenesis, but the natural history of beta cell loss remains unknown. Clinical imaging of beta cells will play a pivotal role in developing strategies for optimized diabetes prevention and treatment. PMID:24726483

  10. Isolation, characterization and potential role in beta cell-endothelium cross-talk of extracellular vesicles released from human pancreatic islets.

    PubMed

    Figliolini, Federico; Cantaluppi, Vincenzo; De Lena, Michela; Beltramo, Silvia; Romagnoli, Renato; Salizzoni, Mauro; Melzi, Raffaella; Nano, Rita; Piemonti, Lorenzo; Tetta, Ciro; Biancone, Luigi; Camussi, Giovanni

    2014-01-01

    The cross-talk between beta cells and endothelium plays a key role in islet physiopathology and in the revascularization process after islet transplantation. However, the molecular mechanisms involved in this cross-talk are not fully elucidated. Extracellular vesicles (EVs) are secreted membrane nanoparticles involved in inter-cellular communication through the transfer of proteins and nucleic acids. The aims of this study were: 1) isolation and characterization of EVs from human islets; 2) evaluation of the pro-angiogenic effect of islet-derived EVs on human islet endothelial cells (IECs). EVs were isolated by ultracentrifugation from conditioned medium of human islets and characterized by nanotrack analysis (Nanosight), FACS, western blot, bioanalyzer, mRNA/microRNA RT-PCR array. On IECs, we evaluated EV-induced insulin mRNA transfer, proliferation, resistance to apoptosis, in vitro angiogenesis, migration, gene and protein profiling. EVs sized 236±54 nm, expressed different surface molecules and islet-specific proteins (insulin, C-peptide, GLP1R) and carried several mRNAs (VEGFa, eNOS) and microRNAs (miR-27b, miR-126, miR-130 and miR-296) involved in beta cell function, insulin secretion and angiogenesis. Purified EVs were internalized into IECs inducing insulin mRNA expression, protection from apoptosis and enhancement of angiogenesis. Human islets release biologically active EVs able to shuttle specific mRNAs and microRNAs (miRNAs) into target endothelial cells. These results suggest a putative role for islet-derived EVs in beta cell-endothelium cross-talk and in the neoangiogenesis process which is critical for engraftment of transplanted islets. PMID:25028931

  11. Isolation, Characterization and Potential Role in Beta Cell-Endothelium Cross-Talk of Extracellular Vesicles Released from Human Pancreatic Islets

    PubMed Central

    De Lena, Michela; Beltramo, Silvia; Romagnoli, Renato; Salizzoni, Mauro; Melzi, Raffaella; Nano, Rita; Piemonti, Lorenzo; Tetta, Ciro; Biancone, Luigi; Camussi, Giovanni

    2014-01-01

    The cross-talk between beta cells and endothelium plays a key role in islet physiopathology and in the revascularization process after islet transplantation. However, the molecular mechanisms involved in this cross-talk are not fully elucidated. Extracellular vesicles (EVs) are secreted membrane nanoparticles involved in inter-cellular communication through the transfer of proteins and nucleic acids. The aims of this study were: 1) isolation and characterization of EVs from human islets; 2) evaluation of the pro-angiogenic effect of islet-derived EVs on human islet endothelial cells (IECs). EVs were isolated by ultracentrifugation from conditioned medium of human islets and characterized by nanotrack analysis (Nanosight), FACS, western blot, bioanalyzer, mRNA/microRNA RT-PCR array. On IECs, we evaluated EV-induced insulin mRNA transfer, proliferation, resistance to apoptosis, in vitro angiogenesis, migration, gene and protein profiling. EVs sized 236±54 nm, expressed different surface molecules and islet-specific proteins (insulin, C-peptide, GLP1R) and carried several mRNAs (VEGFa, eNOS) and microRNAs (miR-27b, miR-126, miR-130 and miR-296) involved in beta cell function, insulin secretion and angiogenesis. Purified EVs were internalized into IECs inducing insulin mRNA expression, protection from apoptosis and enhancement of angiogenesis. Human islets release biologically active EVs able to shuttle specific mRNAs and microRNAs (miRNAs) into target endothelial cells. These results suggest a putative role for islet-derived EVs in beta cell-endothelium cross-talk and in the neoangiogenesis process which is critical for engraftment of transplanted islets. PMID:25028931

  12. Adhesion of pancreatic beta cells to biopolymer films.

    PubMed

    Williams, S Janette; Wang, Qun; Macgregor, Ronal R; Siahaan, Teruna J; Stehno-Bittel, Lisa; Berkland, Cory

    2009-08-01

    Dramatic reversal of Type 1 diabetes in patients receiving pancreatic islet transplants continues to prompt vigorous research concerning the basic mechanisms underlying patient turnaround. At the most fundamental level, transplanted islets must maintain viability and function in vitro and in vivo and should be protected from host immune rejection. Our previous reports showed enhancement of islet viability and insulin secretion per tissue mass for small islets (<125 mum) as compared with large islets (>125 mum), thus, demonstrating the effect of enhancing the mass transport of islets (i.e. increasing tissue surface area to volume ratio). Here, we report the facile dispersion of rat islets into individual cells that are layered onto the surface of a biopolymer film towards the ultimate goal of improving mass transport in islet tissue. The tightly packed structure of intact islets was disrupted by incubating in calcium-free media resulting in fragmented islets, which were further dispersed into individual or small groups of cells by using a low concentration of papain. The dispersed cells were screened for adhesion to a range of biopolymers and the nature of cell adhesion was characterized for selected groups by quantifying adherent cells, measuring the surface area coverage of the cells, and immunolabeling cells for adhesion proteins interacting with selected biopolymers. Finally, beta cells in suspension were centrifuged to form controlled numbers of cell layers on films for future work determining the mass transport limitations in the adhered tissue constructs. (c) 2009 Wiley Periodicals, Inc. Biopolymers 91: 676-685, 2009.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com. PMID:19353639

  13. Beta cells transfer vesicles containing insulin to phagocytes for presentation to T cells.

    PubMed

    Vomund, Anthony N; Zinselmeyer, Bernd H; Hughes, Jing; Calderon, Boris; Valderrama, Carolina; Ferris, Stephen T; Wan, Xiaoxiao; Kanekura, Kohsuke; Carrero, Javier A; Urano, Fumihiko; Unanue, Emil R

    2015-10-01

    Beta cells from nondiabetic mice transfer secretory vesicles to phagocytic cells. The passage was shown in culture studies where the transfer was probed with CD4 T cells reactive to insulin peptides. Two sets of vesicles were transferred, one containing insulin and another containing catabolites of insulin. The passage required live beta cells in a close cell contact interaction with the phagocytes. It was increased by high glucose concentration and required mobilization of intracellular Ca2+. Live images of beta cell-phagocyte interactions documented the intimacy of the membrane contact and the passage of the granules. The passage was found in beta cells isolated from islets of young nonobese diabetic (NOD) mice and nondiabetic mice as well as from nondiabetic humans. Ultrastructural analysis showed intraislet phagocytes containing vesicles having the distinct morphology of dense-core granules. These findings document a process whereby the contents of secretory granules become available to the immune system. PMID:26324934

  14. Targeting Homeostatic T Cell Proliferation to Control Beta-Cell Autoimmunity.

    PubMed

    Vignali, Debora; Monti, Paolo

    2016-05-01

    Immunomodulation of the autoreactive T cell response is considered a major strategy to control beta-cell autoimmunity, both in the natural history of type 1 diabetes and in islet transplantation, which can be affected by autoimmunity recurrence. So far, these strategies have had modest results, prompting efforts to define novel cellular and molecular targets to control autoreactive T cell expansion and activation. Novel findings highlighted the important role of the homeostatic cytokine interleukin-7 in inducing proliferation and differentiation of autoreactive T cell clones that causes beta-cell autoimmunity. In this review, we discuss recent evidences and novel findings on the role of IL-7 mediated homeostatic T cell proliferation in the process of beta-cell destruction and evidences of how targeting IL-7 and its receptor could be an innovative and effective strategy to control beta-cell autoimmunity. PMID:26983628

  15. Radioiodinated Naphthylalanine Derivatives Targeting Pancreatic Beta Cells in Normal and Nonobese Diabetic Mice

    PubMed Central

    Amartey, John K.; Shi, Yufei; Al-Jammaz, Ibrahim; Esguerra, Celestina; Al-Otaibi, Basem; Al-Mohanna, Futwan

    2008-01-01

    An imaging method capable of using a signal from pancreatic beta cells to determine their mass would be of immense value in monitoring the progression of diabetes as well as response to treatment. Somatostatin receptors (SSTRs) are expressed on beta cells and are a potential target for imaging. The main objective of this study was to investigate whether pancreatic beta cells are a target for radiolabeled naphthylalanine derivatives. The molecules were subjected to in vitro and ex vivo evaluations. Pancreatic uptake of radioactivity was lower in nonobese diabetic (NOD) mice than normal mice at all time points investigated (P < .05) and correlated with the number of islets in tissue sections of both control and NOD mice. Immunohistochemical and confocal fluorescent microscopic studies showed colocalization of insulin and the conjugate radioligand in the pancreas. The results demonstrated that pancreatic uptake is receptor-mediated, and that beta cells are the primary target. PMID:18483609

  16. Connexin implication in the control of the murine beta-cell mass.

    PubMed

    Klee, Philippe; Lamprianou, Smaragda; Charollais, Anne; Caille, Dorothée; Sarro, Rossella; Cederroth, Manon; Haefliger, Jacques-Antoine; Meda, Paolo

    2011-08-01

    Diabetes develops when the insulin needs of peripheral cells exceed the availability or action of the hormone. This situation results from the death of most beta-cells in type 1 diabetes, and from an inability of the beta-cell mass to adapt to increasing insulin needs in type 2 and gestational diabetes. We analyzed several lines of transgenic mice and showed that connexins (Cxs), the transmembrane proteins that form gap junctions, are implicated in the modulation of the beta-cell mass. Specifically, we found that the native Cx36 does not alter islet size or insulin content, whereas the Cx43 isoform increases both parameters, and Cx32 has a similar effect only when combined with GH. These findings open interesting perspectives for the in vitro and in vivo regulation of the beta-cell mass. PMID:21527868

  17. Activation of the GLP-1 receptor signalling pathway: a relevant strategy to repair a deficient beta-cell mass.

    PubMed

    Portha, Bernard; Tourrel-Cuzin, Cécile; Movassat, Jamileh

    2011-01-01

    Recent preclinical studies in rodent models of diabetes suggest that exogenous GLP-1R agonists and DPP-4 inhibitors have the ability to increase islet mass and preserve beta-cell function, by immediate reactivation of beta-cell glucose competence, as well as enhanced beta-cell proliferation and neogenesis and promotion of beta-cell survival. These effects have tremendous implication in the treatment of T2D because they directly address one of the basic defects in T2D, that is, beta-cell failure. In human diabetes, however, evidence that the GLP-1-based drugs alter the course of beta-cell function remains to be found. Several questions surrounding the risks and benefits of GLP-1-based therapy for the diabetic beta-cell mass are discussed in this review and require further investigation. PMID:21716694

  18. Activation of the GLP-1 Receptor Signalling Pathway: A Relevant Strategy to Repair a Deficient Beta-Cell Mass

    PubMed Central

    Portha, Bernard; Tourrel-Cuzin, Cécile; Movassat, Jamileh

    2011-01-01

    Recent preclinical studies in rodent models of diabetes suggest that exogenous GLP-1R agonists and DPP-4 inhibitors have the ability to increase islet mass and preserve beta-cell function, by immediate reactivation of beta-cell glucose competence, as well as enhanced beta-cell proliferation and neogenesis and promotion of beta-cell survival. These effects have tremendous implication in the treatment of T2D because they directly address one of the basic defects in T2D, that is, beta-cell failure. In human diabetes, however, evidence that the GLP-1-based drugs alter the course of beta-cell function remains to be found. Several questions surrounding the risks and benefits of GLP-1-based therapy for the diabetic beta-cell mass are discussed in this review and require further investigation. PMID:21716694

  19. Maturation of adult beta-cells revealed using a Pdx1/insulin dual-reporter lentivirus.

    PubMed

    Szabat, Marta; Luciani, Dan S; Piret, James M; Johnson, James D

    2009-04-01

    The enigmatic process of beta-cell maturation has significant implications for diabetes pathogenesis, and potential diabetes therapies. This study examined the dynamics and heterogeneity of insulin and pancreatic duodenal homeobox (Pdx)-1 gene expression in adult beta-cells. Insulin and Pdx1 expression were monitored in human and mouse islet cells and MIN6 cells using a Pdx1-monomeric red fluorescent protein/insulin-enhanced green fluorescent protein dual-reporter lentivirus. The majority of fluorescent cells were highly positive for both Pdx1 and insulin. Cells expressing Pdx1 but little or no insulin (Pdx1(+)/Ins(low)) comprised 15-25% of the total population. Time-lapse imaging demonstrated that Pdx1(+)/Ins(low) primary beta-cells and MIN6 cells could convert to Pdx1(+)/Ins(+) cells without cell division. Genes involved in the mature beta-cell phenotype (Glut2, MafA) were expressed at higher levels in Pdx1(+)/Ins(+) cells relative to Pdx1(+)/Ins(low) cells. Conversely, genes implicated in early beta-cell development (MafB, Nkx2.2) were enriched in Pdx1(+)/Ins(low) cells. Sorted Pdx1(+)/Ins(low) MIN6 cells had a higher replication rate and secreted less insulin relative to double-positive cells. Long-term phenotype tracking of Pdx1(+)/Ins(low) cells showed two groups, one that matured into Pdx1(+)/Ins(+) cells and one that remained immature. These results demonstrate that adult beta-cells pass through distinct maturation states, which is consistent with previously observed heterogeneity in insulin and Pdx1 expression in adult beta-cells. At a given time, a proportion of adult beta-cells share similar characteristics to functionally immature embryonic beta-cell progenitors. The maturation of adult beta-cells recapitulates development in that Pdx1 expression precedes the robust expression of insulin and other mature beta-cell genes. These results have implications for harnessing the maturation process for therapeutic purposes. PMID:19095744

  20. ALDH Expression Characterizes G1-Phase Proliferating Beta Cells during Pregnancy

    PubMed Central

    Zhang, Lijuan; Wang, Lin; Liu, Xiaoliang; Zheng, Dongming; Liu, Sishi; Liu, Caixia

    2014-01-01

    High levels of aldehyde dehydrogenase (ALDH) activity have been detected in various progenitor and stem cells. Thus, Aldefluor fluorescence, which represents precisely the ALDH activity, has been widely used for the identification, evaluation, and isolation of stem and progenitor cells. Recently, ALDH activity was detected in embryonic and adult mouse pancreas, specifically in adult centroacinar and terminal duct cells supposed to harbor endocrine and exocrine progenitor cells in the adult pancreas. Nevertheless, ALDH activity and aldeflour fluorescence have not been examined in beta cells. Here, we report a dynamic increase in the number of aldeflour+ beta cells during pregnancy. Interestingly, nearly all these aldeflour+ beta cells are positive for Ki-67, suggesting that they are in an active cell cycle (G1, S and M phases). To determine precisely at which phase beta cells activate ALDH activity and thus become aldeflour+, we co-stained insulin with additional proliferation markers, phosphohistone3 (PHH3, a marker for M-phase proliferating cells) and Bromodeoxyuridine (BrdU, a marker for S-phase proliferating cells). Our data show little aldeflour+ beta cells that were positive for either PHH3, or BrdU, suggesting that beta cells activate ALDH and become Aldefluor+ when they enter G1-phase of active cell cycle, but may downregulate ALDH when they leave G1-phase and enter S phase. Our data thus reveal a potential change in ALDH activity of proliferating beta cells during pregnancy, which provides a novel method for isolation and analysis of proliferating beta cells. Moreover, our data also suggest that caution needs to be taken on interpretation of Aldefluor lineage-tracing data in pancreas. PMID:24787690

  1. The electrophysiology of the beta-cell based on single transmembrane protein characteristics.

    PubMed

    Meyer-Hermann, Michael E

    2007-10-15

    The electrophysiology of beta-cells is at the origin of insulin secretion. beta-Cells exhibit a complex behavior upon stimulation with glucose including repeated bursts and continuous spiking. Mathematical modeling is most suitable to improve knowledge about the function of various transmembrane currents provided the model is based on reliable data. This is the first attempt to build a mathematical model for the beta-cell electrophysiology in a bottom-up approach that relies on single protein conductance data. The results of previous whole-cell-based models are reconsidered. The full simulation including all prominent transmembrane proteins in beta-cells is used to provide a functional interpretation of their role in beta-cell bursting and an updated vantage point of beta-cell electrophysiology. As a result of a number of in silico knock-out and block experiments the novel model makes some unexpected predictions: single-channel conductance data imply that large-conductance calcium-gated potassium currents acquire the potential of driving oscillations at supralarge glucose levels. A more complex burst interruption model is presented. It also turns out that, depending on the species, sodium currents may be more relevant than considered so far. Experiments are proposed to verify these predictions. PMID:17573431

  2. The expression and function of histamine H3 receptors in pancreatic beta cells

    PubMed Central

    Nakamura, T; Yoshikawa, T; Noguchi, N; Sugawara, A; Kasajima, A; Sasano, H; Yanai, K

    2014-01-01

    BACKGROUND AND PURPOSE Histamine and its receptors in the CNS play important roles in energy homeostasis. Here, we have investigated the expression and role of histamine receptors in pancreatic beta cells, which secrete insulin. EXPERIMENTAL APPROACH The expression of histamine receptors in pancreatic beta cells was examined by RT-PCR, Western blotting and immunostaining. Insulin secretion assay, ATP measurement and calcium imaging studies were performed to determine the function and signalling pathway of histamine H3 receptors in glucose-induced insulin secretion (GIIS) from MIN6 cells, a mouse pancreatic beta cell line. The function and signalling pathway of H3 receptors in MIN6 cell proliferation were examined using pharmacological assay and Western blotting. KEY RESULTS Histamine H3 receptors were expressed in pancreatic beta cells. A selective H3 receptor agonist, imetit, and a selective inverse H3 receptor agonist, JNJ-5207852, had inhibitory and facilitatory effects, respectively, on GIIS in MIN6 cells. Neither imetit nor JNJ-5207852 altered intracellular ATP concentration, or intracellular calcium concentration stimulated by glucose and KCl, indicating that GIIS signalling was affected by H3 receptor signalling downstream of the increase in intracellular calcium concentration. Moreover, imetit attenuated bromodeoxyuridine incorporation in MIN6 cells. The phosphorylation of cAMP response element-binding protein (CREB), which facilitated beta cell proliferation, was inhibited, though not significantly, by imetit, indicating that activated H3 receptors inhibited MIN6 cell proliferation, possibly by decreasing CREB phosphorylation. CONCLUSIONS AND IMPLICATIONS Histamine H3 receptors were expressed in mouse beta cells and could play a role in insulin secretion and, possibly, beta cell proliferation. PMID:24117016

  3. Sustained Beta-Cell Dysfunction but Normalized Islet Mass in Aged Thrombospondin-1 Deficient Mice

    PubMed Central

    Emanuelsson, Hanna; Christoffersson, Gustav; Carlsson, Per-Ola

    2012-01-01

    Pancreatic islet endothelial cells have in recent years been shown to support beta-cell mass and function by paracrine interactions. Recently, we identified an islets endothelial-specific glycoprotein, thrombospondin-1 (TSP-1), that showed to be of importance for islet angiogenesis and beta-cell function in young mice. The present study aimed to investigate long-term consequences for islet morphology and beta-cell function of TSP-1 deficiency. Islet and beta-cell mass were observed increased at 10–12 weeks of age in TSP-1 deficient mice, but were normalized before 16 weeks of age when compared to wild-type controls. Islet vascularity was normal in 10–12 and 16-week-old TSP-1 deficient animals, whereas islets of one-year-old animals lacking TSP-1 were hypervascular. Beta-cell dysfunction in TSP-1 deficient animals was present at similar magnitudes between 10–12 and 52 weeks of age, as evaluated by glucose tolerance tests. The insulin secretion capacity in vivo of islets in one-year-old TSP-1 deficient animals was only ∼15% of that in wild-type animals. Using a transplantation model, we reconstituted TSP-1 in adult TSP-deficient islets. In contrast to neonatal TSP-1 deficient islets that we previously reported to regain function after TSP-1 reconstitution, adult islets failed to recover. We conclude that TSP-1 deficiency in islets causes changing vascular and endocrine morphological alterations postnatally, but is coupled to a chronic beta-cell dysfunction. The beta-cell dysfunction induced by TSP-1 deficiency is irreversible if not substituted early in life. PMID:23094049

  4. The effect of smoking cessation pharmacotherapies on pancreatic beta cell function

    SciTech Connect

    Woynillowicz, Amanda K.; Raha, Sandeep; Nicholson, Catherine J.; Holloway, Alison C.

    2012-11-15

    The goal of our study was to evaluate whether drugs currently used for smoking cessation (i.e., nicotine replacement therapy, varenicline [a partial agonist at nicotinic acetylcholine receptors (nAChR)] and bupropion [which acts in part as a nAChR antagonist]) can affect beta cell function and determine the mechanism(s) of this effect. INS-1E cells, a rat beta cell line, were treated with nicotine, varenicline and bupropion to determine their effects on beta cell function, mitochondrial electron transport chain enzyme activity and cellular/oxidative stress. Treatment of INS-1E cells with equimolar concentrations (1 μM) of three test compounds resulted in an ablation of normal glucose-stimulated insulin secretion by the cells. This disruption of normal beta cell function was associated with mitochondrial dysfunction since all three compounds tested significantly decreased the activity of mitochondrial electron transport chain enzyme activity. These results raise the possibility that the currently available smoking cessation pharmacotherapies may also have adverse effects on beta cell function and thus glycemic control in vivo. Therefore whether or not the use of nicotine replacement therapy, varenicline and bupropion can cause endocrine changes which are consistent with impaired pancreatic function warrants further investigation. -- Highlights: ► Smoking cessation drugs have the potential to disrupt beta cell function in vitro. ► The effects of nicotine, varenicline and bupropion are similar. ► The impaired beta cell function is mediated by mitochondrial dysfunction. ► If similar effects are seen in vivo, these drugs may increase the risk of diabetes.

  5. A Simple Matter of Life and Death—The Trials of Postnatal Beta-Cell Mass Regulation

    PubMed Central

    Tarabra, Elena; Pelengaris, Stella; Khan, Michael

    2012-01-01

    Pancreatic beta-cells, which secrete the hormone insulin, are the key arbiters of glucose homeostasis. Defective beta-cell numbers and/or function underlie essentially all major forms of diabetes and must be restored if diabetes is to be cured. Thus, the identification of the molecular regulators of beta-cell mass and a better understanding of the processes of beta-cell differentiation and proliferation may provide further insight for the development of new therapeutic targets for diabetes. This review will focus on the principal hormones and nutrients, as well as downstream signalling pathways regulating beta-cell mass in the adult. Furthermore, we will also address more recently appreciated regulators of beta-cell mass, such as microRNAs. PMID:22577380

  6. Joe Doupe lecture: emerging strategies for the preservation of pancreatic beta-cell function in early type 2 diabetes.

    PubMed

    Retnakaran, Ravi

    2014-01-01

    A fundamental problem in the clinical management of type 2 diabetes is the inability to prevent the ongoing deterioration of pancreatic beta-cell function over time that underlies the chronic progressive nature of this condition. Importantly, beta-cell dysfunction has both reversible and irreversible components. Furthermore, the amelioration of reversible beta-cell dysfunction through the early institution of short-term insulin-based therapy has emerged as a strategy that can yield temporary remission of type 2 diabetes. In this context, we have forwarded a novel therapeutic paradigm consisting of initial induction therapy to improve beta-cell function early in the course of diabetes followed by maintenance therapy aimed at preserving this beneficial beta-cell effect. Ultimately, this approach may yield an optimized therapeutic strategy for the durable preservation of beta-cell function and consequent modification of the natural history of type 2 diabetes. PMID:25618275

  7. ROS signaling, oxidative stress and Nrf2 in pancreatic beta-cell function

    SciTech Connect

    Pi Jingbo; Zhang Qiang; Fu Jingqi; Woods, Courtney G.; Hou Yongyong; Corkey, Barbara E.; Collins, Sheila; Andersen, Melvin E.

    2010-04-01

    This review focuses on the emerging evidence that reactive oxygen species (ROS) derived from glucose metabolism, such as H{sub 2}O{sub 2}, act as metabolic signaling molecules for glucose-stimulated insulin secretion (GSIS) in pancreatic beta-cells. Particular emphasis is placed on the potential inhibitory role of endogenous antioxidants, which rise in response to oxidative stress, in glucose-triggered ROS and GSIS. We propose that cellular adaptive response to oxidative stress challenge, such as nuclear factor E2-related factor 2 (Nrf2)-mediated antioxidant induction, plays paradoxical roles in pancreatic beta-cell function. On the one hand, induction of antioxidant enzymes protects beta-cells from oxidative damage and possible cell death, thus minimizing oxidative damage-related impairment of insulin secretion. On the other hand, the induction of antioxidant enzymes by Nrf2 activation blunts glucose-triggered ROS signaling, thus resulting in reduced GSIS. These two premises are potentially relevant to impairment of beta-cells occurring in the late and early stage of Type 2 diabetes, respectively. In addition, we summarized our recent findings that persistent oxidative stress due to absence of uncoupling protein 2 activates cellular adaptive response which is associated with impaired pancreatic beta-cell function.

  8. Assessment of beta-cell function and insulin secretion in subjects that underwent renal transplantation.

    PubMed

    Tura, Andrea; Hecking, Manfred; Wolzt, Michael; Saemann, Marcus D; Pacini, Giovanni

    2015-08-01

    In this study we aimed to assess the performance of various indices of beta-cell function derived from oral glucose tolerance test (OGTT) in subjects that underwent renal transplantation. Impaired insulin secretion seems in fact central for development of new onset diabetes after transplantation, but its assessment has not been systematically evaluated. Twenty subjects underwent a 75 g 2h-OGTT for measurement of glucose, insulin, C-peptide. OGTT indices of beta-cell function were either derived by mathematical modeling (yielding the reference index: glucose sensitivity) or were empirical: insulinogenic index (IGI), IGI derived indices, whole shape C-peptide (WHOSH_CP). Indices of beta cell function, showed significant correlation with glucose sensitivity (R(2)=0.40-0.86, all P<;0.003). The majority of beta-cell function indices provided comparable results also when subjects were divided into subgroups according to sex, age, body mass index, mean glycemia. In conclusion, in transplanted subjects OGTT empirical indices are typically acceptable for the estimation of beta-cell function. PMID:26736770

  9. New therapies aimed at the preservation or restoration of beta cell function in type 1 diabetes.

    PubMed

    Keymeulen, B

    2006-01-01

    Type 1 diabetes is caused by an immune-mediated destruction of the insulin-secreting beta cells in the pancreas. The disease can become clinically apparent at any age. At diagnosis, there is invariably some residual beta cell function and more so in adults than in children. Recent studies--including one conducted mainly in Belgium--have provided proof of principle that short-term anti-T-cell antibody treatment is able to preserve residual beta cell function for at least 18 months. The resultant stabilizing effect on metabolic control is expected to delay or limit chronic complications in these patients. With a similar goal in mind, nonuremic C-peptide negative patients are offered beta cell transplantation. The outcome of these implants looks promising but their final applicability hinges on finding ways to induce immune tolerance to the donor beta cells. A widespread application, however, will only occur if the shortage of viable human donor cells can be overcome. Both xenotransplantation and stem cell therapy provide possible strategies to solve this problem and represent areas of intense investigation. The ultimate goal is prevention of clinical disease. Studies by the Belgian Diabetes Registry and others in first degree family members of type 1 diabetic patients have refined the identification of individuals at very high risk of hyperglycaemia so that new immunological treatments can be tested in the prediabetic phase. PMID:17240745

  10. Transcriptome landmarks of the functional maturity of rat beta-cells, from lactation to adulthood.

    PubMed

    Larqué, Carlos; Velasco, Myrian; Barajas-Olmos, Francisco; García-Delgado, Neyvis; Chávez-Maldonado, Juan Pablo; García-Morales, Jazmín; Orozco, Lorena; Hiriart, Marcia

    2016-07-01

    Research on the postnatal development of pancreatic beta-cells has become an important subject in recent years. Understanding the mechanisms that govern beta-cell postnatal maturation could bring new opportunities to therapeutic approaches for diabetes. The weaning period consists of a critical postnatal window for structural and physiologic maturation of rat beta-cells. To investigate transcriptome changes involved in the maturation of beta-cells neighboring this period, we performed microarray analysis in fluorescence-activated cell-sorted (FACS) beta-cell-enriched populations. Our results showed a variety of gene sets including those involved in the integration of metabolism, modulation of electrical activity, and regulation of the cell cycle that play important roles in the maturation process. These observations were validated using reverse hemolytic plaque assay, electrophysiological recordings, and flow cytometry analysis. Moreover, we suggest some unexplored pathways such as sphingolipid metabolism, insulin-vesicle trafficking, regulation of transcription/transduction by miRNA-30, trafficking proteins, and cell cycle proteins that could play important roles in the process mentioned above for further investigation. PMID:27220619

  11. Species-Related Differences in the Proteome of Rat and Human Pancreatic Beta Cells

    PubMed Central

    Martens, G. A.

    2015-01-01

    The core proteomes of human and rat pancreatic beta cells were compared by label-free LC-MS/MS: this resulted in quantification of relative molar abundances of 707 proteins belonging to functional pathways of intermediary metabolism, protein synthesis, and cytoskeleton. Relative molar abundances were conserved both within and between pathways enabling the selection of a housekeeping network for geometric normalization and the analysis of potentially relevant differential expressions. Human beta cells differed from rat beta cells in their lower level of enzymes involved in glucose sensing (MDH1, PC, and ACLY) and upregulation of lysosomal enzymes. Human cells also expressed more heat shock proteins and radical scavenging systems: apart from SOD2, they expressed high levels of H2O2-scavenger peroxiredoxin 3 (PRDX3), confirmed by microarray, Western blotting, and microscopy. Besides conferring lower susceptibility to oxidative stress to human cells PRDX3 might also play a role in physiological redox regulation as, in rat, its expression was restricted to a beta cell subset with higher metabolic glucose responsiveness. In conclusion, although their core proteomic architecture is conserved, human and rat beta cells differ in their molar expression of key enzymes involved in glucose sensing and redox control. PMID:26064985

  12. Glucagon-like peptide-1 counteracts the detrimental effects of Advanced Glycation End-Products in the pancreatic beta cell line HIT-T 15

    SciTech Connect

    Puddu, A.; Storace, D.; Durante, A.; Odetti, P.; Viviani, G.L.

    2010-07-30

    Research highlights: {yields} GLP-1 prevents AGEs-induced cell death. {yields} GLP-1 prevents AGEs-induced oxidative stress. {yields} GLP-1 ameliorated AGEs-induced cell dysfunction. {yields} GLP-1 attenuates AGEs-induced RAGE increment. {yields} GLP-1 counteracts AGEs-induced pancreatic cell death and dysfunction. -- Abstract: Advanced Glycation End-Products (AGEs), a group of compounds resulting from the non-enzymatic reaction of reducing sugars with the free amino group of proteins, are implicated in diabetic complications. We previously demonstrated that exposure of the pancreatic islet cell line HIT-T 15 to high concentrations of AGEs significantly decreases cell proliferation and insulin secretion, and affects transcription factors regulating insulin gene transcription. The glucagon-like peptide-1 (GLP-1) is an incretin hormone that increases proinsulin biosynthesis, stimulates insulin secretion, and improves pancreatic beta-cell viability. The aim of this work was to investigate the effects of GLP-1 on the function and viability of HIT-T 15 cells cultured with AGEs. HIT-T 15 cells were cultured for 5 days in presence of AGEs alone, or supplemented with 10 nmol/l GLP-1. Cell viability, insulin secretion, redox balance, and expression of the AGEs receptor (RAGE) were then determined. The results showed that GLP-1 protected beta cell against AGEs-induced cell death preventing both apoptosis and necrosis. Moreover, addition of GLP-1 to the AGEs culture medium restored the redox balance, improved the responsiveness to glucose, and attenuated AGEs-induced RAGE expression. These findings provide evidence that GLP-1 protects beta cells from the dangerous effects of AGEs.

  13. Beta cells transfer vesicles containing insulin to phagocytes for presentation to T cells

    PubMed Central

    Vomund, Anthony N.; Zinselmeyer, Bernd H.; Hughes, Jing; Calderon, Boris; Valderrama, Carolina; Ferris, Stephen T.; Wan, Xiaoxiao; Kanekura, Kohsuke; Carrero, Javier A.; Urano, Fumihiko; Unanue, Emil R.

    2015-01-01

    Beta cells from nondiabetic mice transfer secretory vesicles to phagocytic cells. The passage was shown in culture studies where the transfer was probed with CD4 T cells reactive to insulin peptides. Two sets of vesicles were transferred, one containing insulin and another containing catabolites of insulin. The passage required live beta cells in a close cell contact interaction with the phagocytes. It was increased by high glucose concentration and required mobilization of intracellular Ca2+. Live images of beta cell–phagocyte interactions documented the intimacy of the membrane contact and the passage of the granules. The passage was found in beta cells isolated from islets of young nonobese diabetic (NOD) mice and nondiabetic mice as well as from nondiabetic humans. Ultrastructural analysis showed intraislet phagocytes containing vesicles having the distinct morphology of dense-core granules. These findings document a process whereby the contents of secretory granules become available to the immune system. PMID:26324934

  14. Pluripotent stem cells, a potential source of beta-cells for diabetes therapy.

    PubMed

    Hansson, Mattias; Madsen, Ole D

    2010-04-01

    Although the reconstitution of a functional beta-cell mass by transplantation of isolated islets can restore euglycemia in the absence of insulin treatment, a shortage of donor material is one of the factors preventing the general use of cell replacement therapy for the treatment of type 1 diabetes mellitus (T1DM). Advances in the directed differentiation of pluripotent stem cells toward beta-cells via the stepwise recapitulation of embryonic development have generated proof of concept demonstrating that stem cells may be an appropriate source of cells for the generation of therapeutic beta-cells. However, progress toward a clinical application of this technology is slow and challenging. This review highlights some of the critical issues impeding the translation of stem cell-based diabetes therapies to the clinic. PMID:20336589

  15. Zip4 Mediated Zinc Influx Stimulates Insulin Secretion in Pancreatic Beta Cells

    PubMed Central

    Hardy, Alexandre B.; Prentice, Kacey J.; Froese, Sean; Liu, Ying; Andrews, Glen K.; Wheeler, Michael B.

    2015-01-01

    Zinc has an important role in normal pancreatic beta cell physiology as it regulates gene transcription, insulin crystallization and secretion, and cell survival. Nevertheless, little is known about how zinc is transported through the plasma membrane of beta cells and which of the class of zinc influx transporters (Zip) is involved. Zip4 was previously shown to be expressed in human and mouse beta cells; however, its function there is still unknown. Therefore, the aim of this study was to define the zinc transport role of Zip4 in beta cells. To investigate this, Zip4 was over-expressed in MIN6 beta cells using a pCMV6-Zip4GFP plasmid. Organelle staining combined with confocal microscopy showed that Zip4 exhibits a widespread localization in MIN6 cells. Time-lapse zinc imaging experiments showed that Zip4 increases cytoplasmic zinc levels. This resulted in increased granular zinc content and glucose-stimulated insulin secretion. Interestingly, it is unlikely that the increased glucose stimulated insulin secretion was triggered by a modulation of mitochondrial function, as mitochondrial membrane potential remained unchanged. To define the role of Zip4 in-vivo, we generated a beta cell-specific knockout mouse model (Zip4BKO). Deletion of the Zip4 gene was confirmed in Zip4BKO islets by PCR, RT-PCR, and immuno-histochemistry. Zip4BKO mice showed slightly improved glucose homeostasis but no change in insulin secretion during an oral glucose tolerance test. While Zip4 was not found to be essential for proper glucose homeostasis and insulin secretion in vivo in mice, this study also found that Zip4 mediates increases in cytoplasmic and granular zinc pools and stimulates glucose dependant insulin secretion in-vitro. PMID:25806541

  16. Precise expression of Fis1 is important for glucose responsiveness of beta cells.

    PubMed

    Schultz, Julia; Waterstradt, Rica; Kantowski, Tobias; Rickmann, Annekatrin; Reinhardt, Florian; Sharoyko, Vladimir; Mulder, Hindrik; Tiedge, Markus; Baltrusch, Simone

    2016-07-01

    Mitochondrial network functionality is vital for glucose-stimulated insulin secretion in pancreatic beta cells. Altered mitochondrial dynamics in pancreatic beta cells are thought to trigger the development of type 2 diabetes mellitus. Fission protein 1 (Fis1) might be a key player in this process. Thus, the aim of this study was to investigate mitochondrial morphology in dependence of beta cell function, after knockdown and overexpression of Fis1. We demonstrate that glucose-unresponsive cells with impaired glucose-stimulated insulin secretion (INS1-832/2) showed decreased mitochondrial dynamics compared with glucose-responsive cells (INS1-832/13). Accordingly, mitochondrial morphology visualised using MitoTracker staining differed between the two cell lines. INS1-832/2 cells formed elongated and clustered mitochondria, whereas INS1-832/13 cells showed a homogenous mitochondrial network. Fis1 overexpression using lentiviral transduction significantly improved glucose-stimulated insulin secretion and mitochondrial network homogeneity in glucose-unresponsive cells. Conversely, Fis1 downregulation by shRNA, both in primary mouse beta cells and glucose-responsive INS1-832/13 cells, caused unresponsiveness and significantly greater numbers of elongated mitochondria. Overexpression of FIS1 in primary mouse beta cells indicated an upper limit at which higher FIS1 expression reduced glucose-stimulated insulin secretion. Thus, FIS1 was overexpressed stepwise up to a high concentration in RINm5F cells using the RheoSwitch system. Moderate FIS1 expression improved glucose-stimulated insulin secretion, whereas high expression resulted in loss of glucose responsiveness and in mitochondrial artificial loop structures and clustering. Our data confirm that FIS1 is a key regulator in pancreatic beta cells, because both glucose-stimulated insulin secretion and mitochondrial dynamics were clearly adapted to precise expression levels of this fission protein. PMID:27179109

  17. A human beta cell line with drug inducible excision of immortalizing transgenes

    PubMed Central

    Benazra, Marion; Lecomte, Marie-José; Colace, Claire; Müller, Andreas; Machado, Cécile; Pechberty, Severine; Bricout-Neveu, Emilie; Grenier-Godard, Maud; Solimena, Michele; Scharfmann, Raphaël; Czernichow, Paul; Ravassard, Philippe

    2015-01-01

    Objectives Access to immortalized human pancreatic beta cell lines that are phenotypically close to genuine adult beta cells, represent a major tool to better understand human beta cell physiology and develop new therapeutics for Diabetes. Here we derived a new conditionally immortalized human beta cell line, EndoC-βH3 in which immortalizing transgene can be efficiently removed by simple addition of tamoxifen. Methods We used lentiviral mediated gene transfer to stably integrate a tamoxifen inducible form of CRE (CRE-ERT2) into the recently developed conditionally immortalized EndoC βH2 line. The resulting EndoC-βH3 line was characterized before and after tamoxifen treatment for cell proliferation, insulin content and insulin secretion. Results We showed that EndoC-βH3 expressing CRE-ERT2 can be massively amplified in culture. We established an optimized tamoxifen treatment to efficiently excise the immortalizing transgenes resulting in proliferation arrest. In addition, insulin expression raised by 12 fold and insulin content increased by 23 fold reaching 2 μg of insulin per million cells. Such massive increase was accompanied by enhanced insulin secretion upon glucose stimulation. We further observed that tamoxifen treated cells maintained a stable function for 5 weeks in culture. Conclusions EndoC βH3 cell line represents a powerful tool that allows, using a simple and efficient procedure, the massive production of functional non-proliferative human beta cells. Such cells are close to genuine human beta cells and maintain a stable phenotype for 5 weeks in culture. PMID:26909308

  18. Ryanodine receptors are involved in nuclear calcium oscillation in primary pancreatic {beta}-cells

    SciTech Connect

    Zheng, Ji; Chen, Zheng; Yin, Wenxuan; Miao, Lin; Zhou, Zhansong; Ji, Guangju

    2012-06-29

    Highlights: Black-Right-Pointing-Pointer We found that RyRs are expressed on the nuclear envelope in single primary pancreatic {beta}-cells and isolated nuclei. Black-Right-Pointing-Pointer We showed that the pattern of glucose-induced Ca{sup 2+} oscillation in the nucleus and cytosol was similar. Black-Right-Pointing-Pointer Our results demonstrate that ryanodine-sensitive Ca{sup 2+} stores exist and have function in the pancreatic {beta}-cell nucleus. -- Abstract: Ryanodine receptors (RyRs) are mainly located on the endoplasmic reticulum (ER) and play an important role in regulating glucose-induced cytosolic Ca{sup 2+} oscillation in pancreatic {beta}-cells. However, subcellular locations and functions of RyRs on other cell organelles such as nuclear envelope are not well understood. In order to investigate the role of RyRs in nuclear Ca{sup 2+} oscillation we designed and conducted experiments in intact primary pancreatic {beta}-cells. Immunocytochemistry was used to examine the expression of RYRs on the nuclear envelope. Confocal microscopy was used to evaluate the function of RYRs on the nuclear envelope. We found that RyRs are expressed on the nuclear envelope in single primary pancreatic {beta}-cells and isolated nuclei. Laser scanning confocal microscopy studies indicated that application of glucose to the cells co-incubated with Ca{sup 2+} indicator Fluo-4 AM and cell-permeable nuclear indicator Hoechst 33342 resulted in nuclear Ca{sup 2+} oscillation. The pattern of glucose-induced Ca{sup 2+} oscillation in the nucleus and cytosol was similar. The reduction of Ca{sup 2+} oscillation amplitude by ryanodine was much greater in the nucleus though both the cytosol and the nucleus Ca{sup 2+} amplitude decreased by ryanodine. Our results suggest that functional ryanodine receptors not only exist in endoplasmic reticulum but are also expressed in nuclear envelope of pancreatic {beta}-cells.

  19. Tissue-Specific Methylation of Human Insulin Gene and PCR Assay for Monitoring Beta Cell Death

    PubMed Central

    Husseiny, Mohamed I.; Kaye, Alexander; Zebadua, Emily; Kandeel, Fouad; Ferreri, Kevin

    2014-01-01

    The onset of metabolic dysregulation in type 1 diabetes (T1D) occurs after autoimmune destruction of the majority of pancreatic insulin-producing beta cells. We previously demonstrated that the DNA encoding the insulin gene is uniquely unmethylated in these cells and then developed a methylation-specific PCR (MSP) assay to identify circulating beta cell DNA in streptozotocin-treated mice prior to the rise in blood glucose. The current study extends to autoimmune non-obese diabetic (NOD) mice and humans, showing in NOD mice that beta cell death occurs six weeks before the rise in blood sugar and coincides with the onset of islet infiltration by immune cells, demonstrating the utility of MSP for monitoring T1D. We previously reported unique patterns of methylation of the human insulin gene, and now extend this to other human tissues. The methylation patterns of the human insulin promoter, intron 1, exon 2, and intron 2 were determined in several normal human tissues. Similar to our previous report, the human insulin promoter was unmethylated in beta cells, but methylated in all other tissues tested. In contrast, intron 1, exon 2 and intron 2 did not exhibit any tissue-specific DNA methylation pattern. Subsequently, a human MSP assay was developed based on the methylation pattern of the insulin promoter and human islet DNA was successfully detected in circulation of T1D patients after islet transplantation therapy. Signal levels of normal controls and pre-transplant samples were shown to be similar, but increased dramatically after islet transplantation. In plasma the signal declines with time but in whole blood remains elevated for at least two weeks, indicating that association of beta cell DNA with blood cells prolongs the signal. This assay provides an effective method to monitor beta cell destruction in early T1D and in islet transplantation therapy. PMID:24722187

  20. In vitro effects of mycophenolic acid on survival, function, and gene expression of pancreatic beta-cells.

    PubMed

    Gallo, R; Natale, M; Vendrame, F; Boggi, U; Filipponi, F; Marchetti, P; Laghi Pasini, F; Dotta, F

    2012-12-01

    Post-transplant diabetes mellitus represents an important complication of prolonged immunosuppressive treatment after solid organ transplantation. The immunosuppressive toxicity, responsible for a persistent impairment of glucose metabolism in pancreatic islet-transplanted patients, is mainly attributed to calcineurin inhibitors and steroids, while other immunosuppressive molecules (azathioprine and mycophenolic acid, MPA) are considered not to have a toxic effect. In the present study, in vitro effects of MPA have been investigated in mouse beta-cell lines (βTC-1 and βTC-6) and in purified human pancreatic islets. βTC-1, βTC-6, and human pancreatic islets were exposed to various concentrations of MPA for different times. Consequently, we evaluated the viability, the induction of apoptosis, the glucose-stimulated insulin secretion, and the expression of β-cell function genes (Isl1, Pax6, Glut-2, glucokinase) and apoptosis-related genes (Bax and Bcl2). βTC-1, βTC-6, and human islets treated, respectively, for 48 and 72 h with 15-30 nM MPA showed altered islet architecture, as compared with control cells. We observed for βTC-1 and βTC-6 almost 70% reduction in cell viability; three to sixfold induction of TUNEL/apoptotic-positive cells quantified by FACS analysis. A twofold increase in apoptotic cells was observed in human islets after MPA exposure associated with strong inhibition of glucose-stimulated insulin secretion. Furthermore, we showed significant down-regulation of gene expression of molecules involved in β-cell function and increase rate between Bax/Bcl2. Our data demonstrate that MPA has an in vitro diabetogenic effect interfering at multiple levels with survival and function of murine and human pancreatic β-cells. PMID:22249339

  1. Role of Ink4a/Arf Locus in Beta Cell Mass Expansion under Physiological and Pathological Conditions

    PubMed Central

    Salas, Elisabet; Rabhi, Nabil; Froguel, Philippe; Annicotte, Jean-Sébastien

    2014-01-01

    The ARF/INK4A (Cdkn2a) locus includes the linked tumour suppressor genes p16INK4a and p14ARF (p19ARF in mice) that trigger the antiproliferative activities of both RB and p53. With beta cell self-replication being the primary source for new beta cell generation in adult animals, the network by which beta cell replication could be increased to enhance beta cell mass and function is one of the approaches in diabetes research. In this review, we show a general view of the regulation points at transcriptional and posttranslational levels of Cdkn2a locus. We describe the molecular pathways and functions of Cdkn2a in beta cell cycle regulation. Given that aging reveals increased p16Ink4a levels in the pancreas that inhibit the proliferation of beta cells and decrease their ability to respond to injury, we show the state of the art about the role of this locus in beta cell senescence and diabetes development. Additionally, we focus on two approaches in beta cell regeneration strategies that rely on Cdkn2a locus negative regulation: long noncoding RNAs and betatrophin. PMID:24672805

  2. Role of Ink4a/Arf locus in beta cell mass expansion under physiological and pathological conditions.

    PubMed

    Salas, Elisabet; Rabhi, Nabil; Froguel, Philippe; Annicotte, Jean-Sébastien

    2014-01-01

    The ARF/INK4A (Cdkn2a) locus includes the linked tumour suppressor genes p16INK4a and p14ARF (p19ARF in mice) that trigger the antiproliferative activities of both RB and p53. With beta cell self-replication being the primary source for new beta cell generation in adult animals, the network by which beta cell replication could be increased to enhance beta cell mass and function is one of the approaches in diabetes research. In this review, we show a general view of the regulation points at transcriptional and posttranslational levels of Cdkn2a locus. We describe the molecular pathways and functions of Cdkn2a in beta cell cycle regulation. Given that aging reveals increased p16Ink4a levels in the pancreas that inhibit the proliferation of beta cells and decrease their ability to respond to injury, we show the state of the art about the role of this locus in beta cell senescence and diabetes development. Additionally, we focus on two approaches in beta cell regeneration strategies that rely on Cdkn2a locus negative regulation: long noncoding RNAs and betatrophin. PMID:24672805

  3. Nuclear SREBP-1a causes loss of pancreatic {beta}-cells and impaired insulin secretion

    SciTech Connect

    Iwasaki, Yuko; Iwasaki, Hitoshi; Yatoh, Shigeru; Ishikawa, Mayumi; Kato, Toyonori; Matsuzaka, Takashi; Nakagawa, Yoshimi; Yahagi, Naoya; Kobayashi, Kazuto; Takahashi, Akimitsu; Suzuki, Hiroaki; Yamada, Nobuhiro; Shimano, Hitoshi

    2009-01-16

    Transgenic mice expressing nuclear sterol regulatory element-binding protein-1a under the control of the insulin promoter were generated to determine the role of SREBP-1a in pancreatic {beta}-cells. Only low expressors could be established, which exhibited mild hyperglycemia, impaired glucose tolerance, and reduced plasma insulin levels compared to C57BL/6 controls. The islets isolated from the transgenic mice were fewer and smaller, and had decreased insulin content and unaltered glucagon staining. Both glucose- and potassium-stimulated insulin secretions were decreased. The transgenic islets consistently expressed genes for fatty acids and cholesterol synthesis, resulting in accumulation of triglycerides but not cholesterol. PDX-1, {beta}{epsilon}{tau}{alpha}2, MafA, and IRS-2 were suppressed, partially explaining the loss and dysfunction of {beta}-cell mass. The transgenic mice on a high fat/high sucrose diet still exhibited impaired insulin secretion and continuous {beta}-cell growth defect. Therefore, nuclear SREBP-1a, even at a low level, strongly disrupts {beta}-cell mass and function.

  4. Coating nanofiber scaffolds with beta cell membrane to promote cell proliferation and function

    NASA Astrophysics Data System (ADS)

    Chen, Wansong; Zhang, Qiangzhe; Luk, Brian T.; Fang, Ronnie H.; Liu, Younian; Gao, Weiwei; Zhang, Liangfang

    2016-05-01

    The cell membrane cloaking technique has emerged as an intriguing strategy in nanomaterial functionalization. Coating synthetic nanostructures with natural cell membranes bestows the nanostructures with unique cell surface antigens and functions. Previous studies have focused primarily on development of cell membrane-coated spherical nanoparticles and the uses thereof. Herein, we attempt to extend the cell membrane cloaking technique to nanofibers, a class of functional nanomaterials that are drastically different from nanoparticles in terms of dimensional and mechanophysical characteristics. Using pancreatic beta cells as a model cell line, we demonstrate successful preparation of cell membrane-coated nanofibers and validate that the modified nanofibers possess an antigenic exterior closely resembling that of the source beta cells. When such nanofiber scaffolds are used to culture beta cells, both cell proliferation rate and function are significantly enhanced. Specifically, glucose-dependent insulin secretion from the cells is increased by near five-fold compared with the same beta cells cultured in regular, unmodified nanofiber scaffolds. Overall, coating cell membranes onto nanofibers could add another dimension of flexibility and controllability in harnessing cell membrane functions and offer new opportunities for innovative applications.

  5. Coating nanofiber scaffolds with beta cell membrane to promote cell proliferation and function.

    PubMed

    Chen, Wansong; Zhang, Qiangzhe; Luk, Brian T; Fang, Ronnie H; Liu, Younian; Gao, Weiwei; Zhang, Liangfang

    2016-05-21

    The cell membrane cloaking technique has emerged as an intriguing strategy in nanomaterial functionalization. Coating synthetic nanostructures with natural cell membranes bestows the nanostructures with unique cell surface antigens and functions. Previous studies have focused primarily on development of cell membrane-coated spherical nanoparticles and the uses thereof. Herein, we attempt to extend the cell membrane cloaking technique to nanofibers, a class of functional nanomaterials that are drastically different from nanoparticles in terms of dimensional and mechanophysical characteristics. Using pancreatic beta cells as a model cell line, we demonstrate successful preparation of cell membrane-coated nanofibers and validate that the modified nanofibers possess an antigenic exterior closely resembling that of the source beta cells. When such nanofiber scaffolds are used to culture beta cells, both cell proliferation rate and function are significantly enhanced. Specifically, glucose-dependent insulin secretion from the cells is increased by near five-fold compared with the same beta cells cultured in regular, unmodified nanofiber scaffolds. Overall, coating cell membranes onto nanofibers could add another dimension of flexibility and controllability in harnessing cell membrane functions and offer new opportunities for innovative applications. PMID:27139582

  6. Cellular Origins of Beta Cell Regeneration: a Legacy view of Historical Controversies

    PubMed Central

    Granger, Anne; Kushner, Jake A.

    2013-01-01

    Beta cell regeneration represents a major goal of therapy for diabetes. Unraveling the origin of beta cells during pancreatic regeneration could help restore a functional beta cell mass in diabetes patients. This scientific question has represented a longstanding interest still intensively investigated today. This review focuses on pioneering observations and subsequent theories made hundred years ago and describes how technical innovation helped resolve some, but not all, of the controversies generated by these early investigators. At the end of the nineteenth century, complete pancreatectomy demonstrated the crucial physiological role of the pancreas and its link to diabetes. Pancreatic injury models, including pancreatectomy and ductal ligation, allowed investigators to describe islet function and to assess the regenerative capacity of the pancreas. Three main theories were proposed to explain the origins of newly formed islets: 1) transdifferentiation of acinar cells into islets, 2) islet neogenesis, a process reminiscent of islet formation during embryonic development, and 3) replication of preexisting islet cells. Despite considerable technical innovation in the last fifty years, the origin of new adult beta cells remains highly controversial and the same three theories are still debated today. PMID:19765178

  7. Beta-cell metabolic alterations under chronic nutrient overload in rat and human islets

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The aim of this study was to assess multifactorial Beta-cell responses to metabolic perturbations in primary rat and human islets. Treatment of dispersed rat islet cells with elevated glucose and free fatty acids (FFAs, oleate:palmitate = 1:1 v/v) resulted in increases in the size and the number of ...

  8. Ataxin-10 interacts with O-GlcNAc transferase OGT in pancreatic {beta} cells

    SciTech Connect

    Andrali, Sreenath S.; Maerz, Pia; Oezcan, Sabire . E-mail: sozcan@uky.edu

    2005-11-11

    Several nuclear and cytoplasmic proteins in metazoans are modified by O-linked N-acetylglucosamine (O-GlcNAc). This modification is dynamic and reversible similar to phosphorylation and is catalyzed by the O-linked GlcNAc transferase (OGT). Hyperglycemia has been shown to increase O-GlcNAc levels in pancreatic {beta} cells, which appears to interfere with {beta}-cell function. To obtain a better understanding of the role of O-linked GlcNAc modification in {beta} cells, we have isolated OGT interacting proteins from a cDNA library made from the mouse insulinoma MIN6 cell line. We describe here the identification of Ataxin-10, encoded by the SCA10 (spinocerebellar ataxia type 10) gene as an OGT interacting protein. Mutations in the SCA10 gene cause progressive cerebellar ataxias and seizures. We demonstrate that SCA10 interacts with OGT in vivo and is modified by O-linked glycosylation in MIN6 cells, suggesting a novel role for the Ataxin-10 protein in pancreatic {beta} cells.

  9. Beta cell function and BMI in ethnically diverse children with newly diagnosed autoimmune type 1 diabetes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of our study was to examine the relationship between BMI and beta-cell function at diagnosis of autoimmune type 1 diabetes (T1D) in a large group of ethnically diverse children. Cross-sectional analysis of 524 children (60.8% White, 19.5% Hispanic, 14.5% African-American, 5.2% other n...

  10. Metabolomic profiling of amino acids and beta-cell function relative to insulin sensitivity in youth

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In longitudinal studies of adults, elevated amino acid (AA) concentrations predicted future type 2 diabetes mellitus (T2DM). The aim of the present investigation was to examine whether increased plasma AA concentrations are associated with impaired beta-cell function relative to insulin sensitivity ...

  11. Nitric oxide stimulates insulin gene transcription in pancreatic {beta}-cells

    SciTech Connect

    Campbell, S.C. . E-mail: s.c.campbell@ncl.ac.uk; Richardson, H.; Ferris, W.F.; Butler, C.S.; Macfarlane, W.M.

    2007-02-23

    Recent studies have identified a positive role for nitric oxide (NO) in the regulation of pancreatic {beta}-cell function. The aim of this study was to determine the effects of short-term exposure to NO on {beta}-cell gene expression and the activity of the transcription factor PDX-1. NO stimulated the activity of the insulin gene promoter in Min6 {beta}-cells and endogenous insulin mRNA levels in both Min6 and isolated islets of Langerhans. Addition of wortmannin prior to NO stimulation blocked the observed increases in insulin gene promoter activity. Although NO addition stimulated the phosphorylation of p38, inhibition by SB203580 did not block the effect of NO on the insulin gene promoter. NO addition also stimulated both the nuclear accumulation and the DNA binding activity of PDX-1. This study has shown that over 24 h, NO stimulates insulin gene expression, PI-3-kinase activity and the activity of the critical {beta}-cell transcription factor PDX-1.

  12. Generating pancreatic beta-cells from embryonic stem cells by manipulating signaling pathways.

    PubMed

    Champeris Tsaniras, Spyridon; Jones, Peter M

    2010-07-01

    Type 1 diabetes results from an insufficiency of insulin production as a result of autoimmune destruction of the insulin-secreting pancreatic beta-cells. It can be treated by transplantation of islets of Langerhans from human donors, but widespread application of this therapy is restricted by the scarcity of donor tissue. Generation of functional beta-cells from embryonic stem (ES) cells in vitro could provide a source of an alternative graft material. Several ES cell differentiation protocols have reported the production of insulin-producing cells by mimicking the in vivo developmental stages of pancreatic organogenesis in which cells are transitioned through mesendoderm, definitive endoderm, foregut endoderm, pancreatic endoderm, and the endocrine precursor stage, until mature beta-cells are obtained. These studies provide proof of concept that recapitulating pancreatic development in vitro offers a useful strategy for generating beta-cells, but current differentiation protocols employ a bewildering variety of growth factors, mitogens, and pharmacological agents. In this review, we will attempt to clarify the functions of these agents in in vitro differentiation strategies by focusing on the intracellular signaling pathways through which they operate - phosphatidylinositol 3-kinase, transforming growth factor beta, Wnt/beta-catenin, Hedgehog, and Notch. PMID:20385725

  13. TRPV6 channel modulates proliferation of insulin secreting INS-1E beta cell line.

    PubMed

    Skrzypski, M; Khajavi, N; Mergler, S; Szczepankiewicz, D; Kołodziejski, P A; Metzke, D; Wojciechowicz, T; Billert, M; Nowak, K W; Strowski, M Z

    2015-12-01

    Transient receptor potential channel vanilloid type 6 (TRPV6) is a non-selective cation channel with high permeability for Ca²⁺ ions. So far, the role of TRPV6 in pancreatic beta cells is unknown. In the present study, we characterized the role of TRPV6 in controlling calcium signaling, cell proliferation as well as insulin expression, and secretion in experimental INS-1E beta cell model. TRPV6 protein production was downregulated using siRNA by approx. 70%, as detected by Western blot. Intracellular free Ca²⁺ ([Ca²⁺]i) was measured by fluorescence Ca²⁺ imaging using fura-2. Calcineurin/NFAT signaling was analyzed using a NFAT reporter assay as well as a calcineurin activity assay. TRPV6 downregulation resulted in impaired cellular calcium influx. Its downregulation also reduced cell proliferation and decreased insulin mRNA expression. These changes were companied by the inhibition of the calcineurin/NFAT signaling. In contrast, insulin exocytosis was not affected by TRPV6 downregulation. In conclusion, this study demonstrates for the first time the expression of TRPV6 in INS-1E cells and rat pancreatic beta cells and describes its role in modulating calcium signaling, beta cell proliferation and insulin mRNA expression. In contrast, TRPV6 fails to influence insulin secretion. PMID:26384871

  14. Measuring beta-cell function relative to insulin sensitivity in youth: Does the hyperglycemic clamp suffice?

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To compare beta-cell function relative to insulin sensitivity, disposition index (DI), calculated from two clamps (2cDI, insulin sensitivity from the hyperinsulinemic-euglycemic clamp and first-phase insulin from the hyperglycemic clamp) with the DI calculated from the hyperglycemic clamp alone (hcD...

  15. Ethnic differences in insulin sensitivity and beta-cell function among Asian men

    PubMed Central

    Tan, V M H; Lee, Y S; Venkataraman, K; Khoo, E Y H; Tai, E S; Chong, Y S; Gluckman, P; Leow, M K S; Khoo, C M

    2015-01-01

    Background and objectives: Lean Asian Indians are less insulin sensitive compared with Chinese and Malays, but the pancreatic beta-cell function among these ethnic groups has yet to be studied in depth. We aimed to study beta-cell function in relation to insulin sensitivity among individuals of Chinese, Malay and Asian-Indian ethnicity living in Singapore. Subjects and methods: This is a sub-group analysis of 59 normoglycemic lean (body mass index (BMI) <23 kg m−2) adult males (14 Chinese, 21 Malays and 24 Asian Indians) from the Singapore Adults Metabolism Study. Insulin sensitivity was determined using fasting state indices (homeostatic model assessment—insulin resistance), the euglycemic-hyperinsulinemic clamp (ISI-clamp) and a liquid mixed-meal tolerance test (LMMTT) (Matsuda insulin sensitivity index (ISI-Mat)). Beta-cell function was assessed using fasting state indices (homeostatic model assessment—beta-cell function) and from the LMMTT (insulinogenic index and insulin secretion index). The oral disposition index (DI), a measure of beta-cell function relative to insulin sensitivity during the LMMTT, was calculated as a product of ISI-Mat and insulin secretion index. Results: Asian Indians had higher waist circumference and percent body fat than Chinese and Malays despite similar BMI. Overall, Asian Indians were the least insulin sensitive whereas the Chinese were most insulin sensitive. Asian Indians had higher beta-cell function compared with Chinese or Malays but these were not statistically different. Malays had the highest incremental area under the curve for glucose during LMMTT compared with Asian Indians and Chinese. However, there were no significant ethnic differences in the incremental insulin area under the curve. The oral DI was the lowest in Malays, followed by Asian Indians and Chinese. Conclusion: Among lean Asians, Chinese are the most insulin sensitive whereas Asian Indians are the least insulin sensitive. However, Malays

  16. Insulin-producing cells could not mimic the physiological regulation of insulin secretion performed by pancreatic beta cells

    PubMed Central

    2013-01-01

    Objective The aim of this study was to compare the difference between insulin-producing cells (IPCs) and normal human pancreatic beta cells both in physiological function and morphological features in cellular level. Methods The levels of insulin secretion were measured by enzyme-linked immunosorbent assay. The insulin gene expression was determined by real-time quantitative polymerase chain reaction. The morphological features were detected by atomic force microscopy (AFM) and laser confocal scanning microscopy. Results IPCs and normal human pancreatic beta cells were similar to each other under the observation in AFM with the porous structure features in the cytoplasm. Both number of membrane particle size and average roughness of normal human beta cells were higher than those of IPCs. Conclusions Our results firstly revealed that the cellular ultrastructure of IPCs was closer to that of normal human pancreatic beta cells, but they still could not mimic the physiological regulation of insulin secretion performed by pancreatic beta cells. PMID:23421382

  17. Therapies aimed at preservation or restoration of beta cell function in type 1 diabetes.

    PubMed

    Keymeulen, B

    2008-01-01

    Type 1 diabetes is caused by an immune mediated destruction of the insulin-secreting beta cells in the pancreas. The disease can become clinically apparent at any age. At clinical diagnosis, there is invariably some residual beta cell function. Recent studies--including one mainly conducted in Belgium--have provided proof of principle that short-term humanized anti-T-cell antibody treatment is able to preserve residual beta cell function for at least 18 months in adult type 1 diabetic patients with a recent clinical onset of disease. The effect of anti-T-cell antibody treatment is more pronounced among patients with initial higher residual beta-cell function. The resultant stabilizing effect on metabolic control is expected to delay chronic complications and avoid hypoglycemia in these patients. With a similar goal in mind, non-uremic C-peptide negative type 1 diabetic patients are offered beta cell transplantation. During the last years the one year survival of these grafts under immune suppression with Anti-Thymocyte-Globulin, tacrolimus and mycophenolate mofetil exceeds 80% with virtually no cases of primary non-function. Widespread application will however only occur if ways are found to induce operational graft tolerance and the shortage of viable human donor cells can be overcome. Both islet xenotransplantation and stem cell therapy provide possible strategies to solve this problem and represent areas of intense investigation. The ultimate goal is prevention of clinical disease. Studies by the Belgian Diabetes Registry and others in first degree family members of type 1 diabetic patients have refined identification of individuals at very high risk of hyperglycemia so that new immunological treatments can be tested in the prediabetic phase. PMID:18630722

  18. Minireview: beta-cell replacement therapy for diabetes in the 21st century: manipulation of cell fate by directed differentiation.

    PubMed

    Yechoor, Vijay; Chan, Lawrence

    2010-08-01

    Pancreatic beta-cell failure underlies type 1 diabetes; it also contributes in an essential way to type 2 diabetes. beta-Cell replacement is an important component of any cure for diabetes. The current options of islet and pancreas transplantation are not satisfactory as definitive forms of therapy. Here, we review strategies for induced de novo pancreatic beta-cell formation, which depend on the targeted differentiation of cells into pancreatic beta-cells. With this objective in mind, one can manipulate the fate of three different types of cells: 1) from terminally differentiated cells, e.g. exocrine pancreatic cells, into beta-cells; 2) from multipotent adult stem cells, e.g. hepatic oval cells, into pancreatic islets; and 3) from pluripotent stem cells, e.g. embryonic stem cells and induced pluripotent stem cells, into beta-cells. We will examine the pros and cons of each strategy as well as the hurdles that must be overcome before these approaches to generate new beta-cells will be ready for clinical application. PMID:20219891

  19. Effects of antidiabetic agents on pancreatic beta-cell function in gestational diabetes: is there enough evidence?

    PubMed

    Tura, Andrea; Göbl, Christian; Pacini, Giovanni

    2016-01-01

    Gestational diabetes mellitus (GDM) is typically characterized by the presence of insulin resistance. However, recent studies showed that both insulin resistance and pancreatic beta-cell function impairment may contribute to the development of type 2 diabetes in women with history of GDM. In fact, beta-cell function decline was found as significant predictor of later disease in former GDM women progressing towards type 2 diabetes. Despite the evidence of the relevance of beta-cell function quantification in GDM, a low number of studies focused on the effects of GDM treatments on beta-cell function. We briefly present the evidence of the effects on beta-cell function of pharmacological agents, as well as nutrition supplements or medical nutrition therapy, used in the management of GDM. We found that few studies reported information on beta-cell function effects in GDM, despite some agents, such as glyburide, are well known insulin secretagogues. Therefore, further studies should be carried out to clearly assess the effects on beta-cell function of the treatments in GDM women. PMID:26609764

  20. Transient cytokine treatment induces acinar cell reprogramming and regenerates functional beta cell mass in diabetic mice

    PubMed Central

    Baeyens, Luc; Lemper, Marie; Leuckx, Gunter; De Groef, Sofie; Bonfanti, Paola; Stangé, Geert; Shemer, Ruth; Nord, Christoffer; Scheel, David W.; Pan, Fong C.; Ahlgren, Ulf; Gu, Guoqiang; Stoffers, Doris A.; Dor, Yuval; Ferrer, Jorge; Gradwohl, Gerard; Wright, Christopher VE; Van de Casteele, Mark; German, Michael S.; Bouwens, Luc; Heimberg, Harry

    2014-01-01

    Reprogramming of pancreatic exocrine cells into cells resembling beta cells may provide a strategy for treating diabetes. Here we show that transient administration of epidermal growth factor and ciliary neurotrophic factor to adult mice with chronic hyperglycemia efficiently stimulates the conversion of terminally differentiated acinar cells to beta-like cells. Newly generated beta-like cells are epigenetically reprogrammed, functional and glucose-responsive, and reinstate normal glycemic control for up to 248 days. The regenerative process depends on Stat3 signaling and requires a threshold number of Neurogenin 3 (Ngn3) expressing acinar cells. In contrast to previous work demonstrating in vivo conversion of acinar cells to beta-like cells by viral delivery of exogenous transcription factors, our approach achieves acinar-to-beta cell reprogramming through transient cytokine exposure rather than genetic modification. PMID:24240391

  1. Immune responses to an encapsulated allogeneic islet {beta}-cell line in diabetic NOD mice

    SciTech Connect

    Black, Sasha P. . E-mail: Sasha.Black@ca.crl.com; Constantinidis, Ioannis; Cui, Hong; Tucker-Burden, Carol; Weber, Collin J.; Safley, Susan A.

    2006-02-03

    Our goal is to develop effective islet grafts for treating type 1 diabetes. Since human islets are scarce, we evaluated the efficacy of a microencapsulated insulin-secreting conditionally transformed allogeneic {beta}-cell line ({beta}TC-tet) in non-obese diabetic mice treated with tetracycline to inhibit cell growth. Relatively low serum levels of tetracycline controlled proliferation of {beta}TC-tet cells without inhibiting effective control of hyperglycemia in recipients. There was no significant host cellular reaction to the allografts or host cell adherence to microcapsules, and host cytokine levels were similar to those of sham-operated controls. We conclude that encapsulated allogeneic {beta}-cell lines may be clinically relevant, because they effectively restore euglycemia and do not elicit a strong cellular immune response following transplantation. To our knowledge, this is First extensive characterization of the kinetics of host cellular and cytokine responses to an encapsulated islet cell line in an animal model of type 1 diabetes.

  2. Can we make surrogate beta-cells better than the original?

    PubMed

    Weir, Gordon C

    2004-06-01

    Insufficient pancreatic beta-cell mass is fundamental to the pathogenesis of both types 1 and 2 diabetes and constitutes the basis for the goal of beta-cell replacement therapy. Current methods for isolating islets from organ donor pancreases do not come close to supplying all in need, thus providing a compelling need to find new sources of insulin-producing cells. Possible sources include generation of cells from embryonic stem cells (ESC), adult stem/precursor cells, transdifferentiation of other cell types and xenodonors. Bioengineering can be used to improve secretory performance and strengthen cells to better withstand the challenges of transplantation. Strategies include protection against hypoxia, inflammation, and immune attack. PMID:15125898

  3. ER stress in pancreatic beta cells: the thin red line between adaptation and failure.

    PubMed

    Eizirik, Decio L; Cnop, Miriam

    2010-01-01

    Secretory cells, such as pancreatic beta cells, face the challenge of increasing protein synthesis severalfold during acute or chronic stimulation. This poses a burden on the endoplasmic reticulum (ER), the organelle where proinsulin synthesis and folding takes place. Thus, beta cells use various adaptive mechanisms to adjust the functional capacity of the ER to the prevailing demand. These check-and-balance mechanisms are collectively known as the unfolded protein response (UPR). It remains unclear how UPR signaling is ultimately regulated and what delineates the boundaries between a physiological and a pathological response. New discoveries point to the divergent effects of acute and chronic metabolic fluxes and chemical ER stressors on the formation of complexes among UPR transducers, scaffold proteins, and phosphatases. These and other findings provide a first glimpse on how different signals trigger diverging UPR outcomes. PMID:20179270

  4. Sodium arsenite impairs insulin secretion and transcription in pancreatic {beta}-cells

    SciTech Connect

    Diaz-Villasenor, Andrea; Sanchez-Soto, M. Carmen; Cebrian, Mariano E.; Ostrosky-Wegman, Patricia; Hiriart, Marcia . E-mail: mhiriart@ifc.unam.mx

    2006-07-01

    Human studies have shown that chronic inorganic arsenic (iAs) exposure is associated with a high prevalence and incidence of type 2 diabetes. However, the mechanism(s) underlying this effect are not well understood, and practically, there is no information available on the effects of arsenic on pancreatic {beta}-cells functions. Thus, since insulin secreted by the pancreas plays a crucial role in maintaining glucose homeostasis, our aim was to determine if sodium arsenite impairs insulin secretion and mRNA expression in single adult rat pancreatic {beta}-cells. Cells were treated with 0.5, 1, 2, 5 and 10 {mu}M sodium arsenite and incubated for 72 and 144 h. The highest dose tested (10 {mu}M) decreased {beta}-cell viability, by 33% and 83%, respectively. Insulin secretion and mRNA expression were evaluated in the presence of 1 and 5 {mu}M sodium arsenite. Basal insulin secretion, in 5.6 mM glucose, was not significantly affected by 1 or 5 {mu}M treatment for 72 h, but basal secretion was reduced when cells were exposed to 5 {mu}M sodium arsenite for 144 h. On the other hand, insulin secretion in response to 15.6 mM glucose decreased with sodium arsenite in a dose-dependent manner in such a way that cells were no longer able to distinguish between different glucose concentrations. We also showed a significant decrease in insulin mRNA expression of cells exposed to 5 {mu}M sodium arsenite during 72 h. Our data suggest that arsenic may contribute to the development of diabetes mellitus by impairing pancreatic {beta}-cell functions, particularly insulin synthesis and secretion.

  5. Biophysical properties of gap junctions between freshly dispersed pairs of mouse pancreatic beta cells.

    PubMed Central

    Pérez-Armendariz, M; Roy, C; Spray, D C; Bennett, M V

    1991-01-01

    Coupling between beta cells through gap junctions has been postulated as a principal mechanism of electrical synchronization of glucose-induced activity throughout the islet of Langerhans. We characterized junctional conductance between isolated pairs of mouse pancreatic beta cells by whole-cell recording with two independent patch-clamp circuits. Most pairs were coupled (67%, n = 155), although the mean junctional conductance (gj) (215 +/- 110 pS) was lower than reported in other tissues. Coupling could be recorded for long periods, up to 40 min. Voltage imposed across the junctional or nonjunctional membranes had no effect on gj. Up to several hours of treatment to increase intracellular cAMP levels did not affect gj. Electrically coupled pairs did not show transfer of the dye Lucifer yellow. Octanol (2 mM) reversibly decreased gj. Lower concentrations of octanol (0.5 mM) and heptanol (0.5 mM) than required to uncouple beta cells decreased voltage-dependent K+ and Ca2+ currents in nonjunctional membranes. Although gj recorded in these experiments would be expected to be provided by current flowing through only a few channels of the unitary conductance previously reported for other gap junctions, no unitary junctional currents were observed even during reversible suppression of gj by octanol. This result suggests either that the single channel conductance of gap junction channels between beta cells is smaller than in other tissues (less than 20 pS) or that the small mean conductance is due to transitions between open and closed states that are too rapid or too slow to be resolved. Images FIGURE 1 FIGURE 5 PMID:2015391

  6. Glucocorticoids Inhibit Basal and Hormone-Induced Serotonin Synthesis in Pancreatic Beta Cells

    PubMed Central

    Hasni Ebou, Moina; Singh-Estivalet, Amrit; Launay, Jean-Marie; Callebert, Jacques; Tronche, François; Ferré, Pascal; Gautier, Jean-François; Guillemain, Ghislaine; Bréant, Bernadette

    2016-01-01

    Diabetes is a major complication of chronic Glucocorticoids (GCs) treatment. GCs induce insulin resistance and also inhibit insulin secretion from pancreatic beta cells. Yet, a full understanding of this negative regulation remains to be deciphered. In the present study, we investigated whether GCs could inhibit serotonin synthesis in beta cell since this neurotransmitter has been shown to be involved in the regulation of insulin secretion. To this aim, serotonin synthesis was evaluated in vitro after treatment with GCs of either islets from CD1 mice or MIN6 cells, a beta-cell line. We also explored the effect of GCs on the stimulation of serotonin synthesis by several hormones such as prolactin and GLP 1. We finally studied this regulation in islet in two in vivo models: mice treated with GCs and with liraglutide, a GLP1 analog, and mice deleted for the glucocorticoid receptor in the pancreas. We showed in isolated islets and MIN6 cells that GCs decreased expression and activity of the two key enzymes of serotonin synthesis, Tryptophan Hydroxylase 1 (Tph1) and 2 (Tph2), leading to reduced serotonin contents. GCs also blocked the induction of serotonin synthesis by prolactin or by a previously unknown serotonin activator, the GLP-1 analog exendin-4. In vivo, activation of the Glucagon-like-Peptide-1 receptor with liraglutide during 4 weeks increased islet serotonin contents and GCs treatment prevented this increase. Finally, islets from mice deleted for the GR in the pancreas displayed an increased expression of Tph1 and Tph2 and a strong increased serotonin content per islet. In conclusion, our results demonstrate an original inhibition of serotonin synthesis by GCs, both in basal condition and after stimulation by prolactin or activators of the GLP-1 receptor. This regulation may contribute to the deleterious effects of GCs on beta cells. PMID:26901633

  7. Age-related differences in the pancreatic beta-cell response to hyperglycemia after eccentric exercise.

    PubMed

    Krishnan, R K; Hernandez, J M; Williamson, D L; O'Gorman, D J; Evans, W J; Kirwan, J P

    1998-09-01

    Eccentric exercise (ECC) causes muscle damage, insulin resistance, and increased pancreatic beta-cell secretion in young individuals. However, the effects of age on the pancreatic beta-cell response to glucose after ECC are unknown. Hyperglycemic clamps (180 min, 10.0 mM) were performed on eight young (age 22 +/- 1 yr) and eight older (age 66 +/- 2 yr) healthy sedentary males without exercise (CONT) and 48 h after ECC. ECC increased (P < 0.02) muscle soreness ratings and plasma creatine kinase concentrations in both groups. Insulin and C-peptide secretions were similar between young and older subjects during CONT clamps. ECC increased (P < 0.05) first-phase (0-10 min) C-peptide area under the curve in young (4.2 +/- 0.4 vs. 3.7 +/- 0.6 nM . min; ECC vs. CONT, respectively) but not in older subjects (3.2 +/- 0.7 vs. 3.5 +/- 0.7 nM . min; ECC vs. CONT), with significant group differences (P < 0.02). Indeed, ECC repressed (P < 0.05) first-phase peak C-peptide concentrations in older subjects (0. 93 +/- 0.16 vs. 1.12 +/- 0.11 nM; ECC vs. CONT). Moreover, first-phase C-peptide-to-insulin molar ratios suggest age-related differences (P < 0.05) in insulin/C-peptide clearance after ECC. Furthermore, the observed C-peptide response after ECC was related to abdominal adiposity [r = -0.62, P < 0.02, and r = -0.66, P < 0. 006, for first and second (10-180 min) phases, respectively]. In conclusion, older individuals did not exhibit the compensatory increase in beta-cell secretion observed among young individuals after ECC. Thus, with increasing age, the pancreatic beta-cell may be less responsive to the physiological stress associated with ECC. PMID:9725813

  8. Properties of the Ca-activated K+ channel in pancreatic beta-cells.

    PubMed

    Atwater, I; Rosario, L; Rojas, E

    1983-12-01

    The existence of [Ca2+]i-activated K+-channels in the pancreatic beta-cell membrane is based in two observations: quinine inhibits K+-permeability and, increasing intracellular Ca2+ stimulates it. The changes in K+-permeability of the beta-cell have been monitored electrically by combining measurements of the dependence of the membrane potential on external K+ concentration and input resistance. The changes in the passive 42K and 86Rb efflux from the whole islet have been measured directly. Intracellular Ca2+ has been increased by various means, including increasing extracellular Ca2+, addition of the Ca2+-ionophore A23187 or noradrenaline and application of mitochondrial uncouplers and blockers. In addition to quinine, many other substances have been found to inhibit or modulate the [Ca2+]i-activated K+-channel. The most important of these is the natural stimulus for insulin secretion, glucose. Glucose may inhibit K+-permeability by lowering intracellular Ca2+. Glibenclamide, a hypoglycaemic sulphonylurea, is about 25 times more active than quinine in blocking the K+-channel in beta-cells. The methylxanthines, c-AMP, various calmodulin inhibitors and Ba2+ also inhibit K+-permeability. Genetically diabetic mice have been studied and show an alteration in the [Ca2+]i-activated K+-channel. It is concluded that the [Ca2+]i-activated K+-channel plays a major role in the normal function of the pancreatic beta-cell. The study of its properties should prove valuable for the understanding and treatment of diabetes. PMID:6323007

  9. The Microtubule-Associated Protein Tau and Its Relevance for Pancreatic Beta Cells

    PubMed Central

    Maj, Magdalena; Hoermann, Gregor; Rasul, Sazan; Base, Wolfgang; Wagner, Ludwig; Attems, Johannes

    2016-01-01

    Structural and biochemical alterations of the microtubule-associated protein tau (MAPT) are associated with degenerative disorders referred to as tauopathies. We have previously shown that MAPT is present in human islets of Langerhans, human insulinomas, and pancreatic beta-cell line models, with biophysical similarities to the pathological MAPT in the brain. Here, we further studied MAPT in pancreatic endocrine tissue to better understand the mechanisms that lead to functional dysregulation of pancreatic beta cells. We found upregulation of MAPT protein expression in human insulinomas when compared to human pancreatic islets of Langerhans and an imbalance between MAPT isoforms in insulinomas tissue. We cloned one 3-repeat domain MAPT and transduced this into a beta-cell derived rodent cell line Rin-5F. Proliferation experiments showed higher growth rates and metabolic activities of cells overexpressing MAPT protein. We observed that a MAPT overexpressing cell line demonstrates altered insulin transcription, translation, and insulin secretion rates. We found the relative insulin secretion rates were significantly decreased in a MAPT overexpressing cell line and these findings could be confirmed using partial MAPT knock-down cell lines. Our findings support that MAPT may play an important role in insulin granule trafficking and indicate the importance of balanced MAPT phosphorylation and dephosphorylation for adequate insulin release. PMID:26824039

  10. GLP-1 receptor antagonist as a potential probe for pancreatic {beta}-cell imaging

    SciTech Connect

    Mukai, Eri; Toyoda, Kentaro; Kimura, Hiroyuki; Kawashima, Hidekazu; Fujimoto, Hiroyuki; Ueda, Masashi; Temma, Takashi; Hirao, Konomu; Nagakawa, Kenji; Saji, Hideo; Inagaki, Nobuya

    2009-11-20

    We examined exendin(9-39), an antagonist of glucagon-like peptide-1 (GLP-1) receptor (GLP-1R), as a potential probe for imaging of pancreatic {beta}-cells. To evaluate in vitro receptor specificity, binding assay was performed using dispersed mouse islet cells. Binding assay showed competitive inhibition of [{sup 125}I]BH-exendin(9-39) binding by non-radioactive exendin(9-39). To assess in vivo selectivity, the biodistribution was evaluated by intravenous administration of [{sup 125}I]BH-exendin(9-39) to mice. Radioactivity of harvested pancreas reached highest levels at 60 and 120 min among organs examined except lung. Pre-administration of excess non-radioactive exendin(9-39) remarkably and specifically blocked the radioactivity of pancreas. After [{sup 125}I]BH-exendin(9-39) injection into transgenic mice with pancreatic {beta}-cells expressing GFP, fluorescent and radioactive signals of sections of pancreas were evaluated with an image analyzer. Imaging analysis showed that the fluorescent GFP signals and the radioactive signals were correspondingly located. Thus, the GLP-1R antagonist exendin(9-39) may serve as a useful probe for pancreatic {beta}-cell imaging.

  11. Modulation of Ionic Channels and Insulin Secretion by Drugs and Hormones in Pancreatic Beta Cells.

    PubMed

    Velasco, Myrian; Díaz-García, Carlos Manlio; Larqué, Carlos; Hiriart, Marcia

    2016-09-01

    Pancreatic beta cells, unique cells that secrete insulin in response to an increase in glucose levels, play a significant role in glucose homeostasis. Glucose-stimulated insulin secretion (GSIS) in pancreatic beta cells has been extensively explored. In this mechanism, glucose enters the cells and subsequently the metabolic cycle. During this process, the ATP/ADP ratio increases, leading to ATP-sensitive potassium (KATP) channel closure, which initiates depolarization that is also dependent on the activity of TRP nonselective ion channels. Depolarization leads to the opening of voltage-gated Na(+) channels (Nav) and subsequently voltage-dependent Ca(2+) channels (Cav). The increase in intracellular Ca(2+) triggers the exocytosis of insulin-containing vesicles. Thus, electrical activity of pancreatic beta cells plays a central role in GSIS. Moreover, many growth factors, incretins, neurotransmitters, and hormones can modulate GSIS, and the channels that participate in GSIS are highly regulated. In this review, we focus on the principal ionic channels (KATP, Nav, and Cav channels) involved in GSIS and how classic and new proteins, hormones, and drugs regulate it. Moreover, we also discuss advances on how metabolic disorders such as metabolic syndrome and diabetes mellitus change channel activity leading to changes in insulin secretion. PMID:27436126

  12. In vivo imaging of beta cells with radiotracers: state of the art, prospects and recommendations for development and use.

    PubMed

    Eriksson, Olof; Laughlin, Maren; Brom, Maarten; Nuutila, Pirjo; Roden, Michael; Hwa, Albert; Bonadonna, Riccardo; Gotthardt, Martin

    2016-07-01

    Radiotracer imaging is characterised by high in vivo sensitivity, with a detection limit in the lower picomolar range. Therefore, radiotracers represent a valuable tool for imaging pancreatic beta cells. High demands are made of radiotracers for in vivo imaging of beta cells. Beta cells represent only a small fraction of the volume of the pancreas (usually 1-3%) and are scattered in the tiny islets of Langerhans throughout the organ. In order to be able to measure a beta cell-specific signal, one has to rely on highly specific tracer molecules because current in vivo imaging technologies do not allow the resolution of single islets in humans non-invasively. Currently, a considerable amount of preclinical data are available for several radiotracers and three are under clinical evaluation. We summarise the current status of the evaluation of these tracer molecules and put forward recommendations for their further evaluation. PMID:27094935

  13. Nkx6.1 controls a gene regulatory network required for establishing and maintaining pancreatic Beta cell identity.

    PubMed

    Schaffer, Ashleigh E; Taylor, Brandon L; Benthuysen, Jacqueline R; Liu, Jingxuan; Thorel, Fabrizio; Yuan, Weiping; Jiao, Yang; Kaestner, Klaus H; Herrera, Pedro L; Magnuson, Mark A; May, Catherine Lee; Sander, Maike

    2013-01-01

    All pancreatic endocrine cell types arise from a common endocrine precursor cell population, yet the molecular mechanisms that establish and maintain the unique gene expression programs of each endocrine cell lineage have remained largely elusive. Such knowledge would improve our ability to correctly program or reprogram cells to adopt specific endocrine fates. Here, we show that the transcription factor Nkx6.1 is both necessary and sufficient to specify insulin-producing beta cells. Heritable expression of Nkx6.1 in endocrine precursors of mice is sufficient to respecify non-beta endocrine precursors towards the beta cell lineage, while endocrine precursor- or beta cell-specific inactivation of Nkx6.1 converts beta cells to alternative endocrine lineages. Remaining insulin(+) cells in conditional Nkx6.1 mutants fail to express the beta cell transcription factors Pdx1 and MafA and ectopically express genes found in non-beta endocrine cells. By showing that Nkx6.1 binds to and represses the alpha cell determinant Arx, we identify Arx as a direct target of Nkx6.1. Moreover, we demonstrate that Nkx6.1 and the Arx activator Isl1 regulate Arx transcription antagonistically, thus establishing competition between Isl1 and Nkx6.1 as a critical mechanism for determining alpha versus beta cell identity. Our findings establish Nkx6.1 as a beta cell programming factor and demonstrate that repression of alternative lineage programs is a fundamental principle by which beta cells are specified and maintained. Given the lack of Nkx6.1 expression and aberrant activation of non-beta endocrine hormones in human embryonic stem cell (hESC)-derived insulin(+) cells, our study has significant implications for developing cell replacement therapies. PMID:23382704

  14. Evidence for glucagon-like peptide-1 receptor signaling to activate ATP-sensitive potassium channels in pancreatic beta cells.

    PubMed

    Kwon, Hye-Jung; Park, Hyun-Sun; Park, Sung-Hee; Park, Jae-Hyung; Shin, Su-Kyung; Song, Seung Eun; Hwang, Meeyul; Cho, Ho-Chan; Song, Dae-Kyu

    2016-01-01

    Glucagon-like peptide-1 (GLP-1) is a gut peptide that promotes insulin release from pancreatic beta cells. GLP-1 has been shown to confer glucose-insensitive beta cells with glucose sensitivity by modulation of the activity of the ATP-sensitive potassium (KATP) channel. The channel closing effect of GLP-1, interacting with corresponding G-protein-coupled receptors, has been well established; however, to our knowledge, no study has shown whether GLP-1 directly induces activation of beta-cell KATP channels. Here, we aimed to evaluate whether the activation of beta-cell KATP channels by GLP-1 exists and affects intracellular Ca(2+) levels ([Ca(2+)]i). KATP channel activity was measured in isolated rat pancreatic beta cells by whole-cell perforated patch-clamp recordings with a diazoxide-containing pipette solution. Changes in [Ca(2+)]i and the subcellular localization of KATP channels were observed using the calcium-sensitive dye fura-4/AM and anti-Kir6.2 antibodies in INS-1 beta cells, respectively. To eliminate the well-known inhibitory effects of GLP-1 on KATP channel activity, channels were fully inhibited by pretreatment with methyl pyruvate and epigallocatechin-3-gallate. In the pretreated beta cells, GLP-1 and exendin-4 promptly activated the channels, reducing [Ca(2+)]i. The phosphoinositide 3-kinase (PI3K) inhibitor LY294002 blocked the effects of GLP-1 on channel activity. Moreover, phosphatidylinositol-3,4,5-trisphosphate mimicked the effects of GLP-1. These results suggested that beta-cell GLP-1 receptor signaling involved activation of KATP channels via a PI3K-dependent pathway. This alternative mechanism of GLP-1 function may act as a negative feedback pathway, modulating the glucose-dependent GLP-1 inhibition on KATP channel activity. PMID:26655814

  15. Increased androgen levels in rats impair glucose-stimulated insulin secretion through disruption of pancreatic beta cell mitochondrial function.

    PubMed

    Wang, Hongdong; Wang, Xiaping; Zhu, Yunxia; Chen, Fang; Sun, Yujie; Han, Xiao

    2015-11-01

    Although insulin resistance is recognized to contribute to the reproductive and metabolic phenotypes of polycystic ovary syndrome (PCOS), pancreatic beta cell dysfunction plays an essential role in the progression from PCOS to the development of type 2 diabetes. However, the role of insulin secretory abnormalities in PCOS has received little attention. In addition, the precise changes in beta cells and the underlying mechanisms remain unclear. In this study, we therefore attempted to elucidate potential mechanisms involved in beta cell alterations in a rat model of PCOS. Glucose-induced insulin secretion was measured in islets isolated from DHT-treated and control rats. Oxygen consumption rate (OCR), ATP production, and mitochondrial copy number were assayed to evaluate mitochondrial function. Glucose-stimulated insulin secretion is significantly decreased in islets from DHT-treated rats. On the other hand, significant reductions are observed in the expression levels of several key genes involved in mitochondrial biogenesis and in mitochondrial OCR and ATP production in DHT-treated rat islets. Meanwhile, we found that androgens can directly impair beta cell function by inducing mitochondrial dysfunction in vitro in an androgen receptor dependent manner. For the first time, our study demonstrates that increased androgens in female rats can impair glucose-stimulated insulin secretion partly through disruption of pancreatic beta cell mitochondrial function. This work has significance for hyperandrogenic women with PCOS: excess activation of the androgen receptor by androgens may provoke beta cell dysfunction via mitochondrial dysfunction. PMID:26348137

  16. Ascorbic acid recycling by cultured beta cells: effects of increased glucose metabolism.

    PubMed

    Steffner, Robert J; Wu, Lan; Powers, Alvin C; May, James M

    2004-11-15

    Ascorbic acid is necessary for optimal insulin secretion from pancreatic islets. We evaluated ascorbate recycling and whether it is impaired by increased glucose metabolism in the rat beta-cell line INS-1. INS-1 cells, engineered with the potential for overexpression of glucokinase under the control of a tetracycline-inducible gene expression system, took up and reduced dehydroascorbic acid to ascorbate in a concentration-dependent manner that was optimal in the presence of physiologic D-glucose concentrations. Ascorbate uptake did not affect intracellular GSH concentrations. Whereas depletion of GSH in culture to levels about 25% of normal also did not affect the ability of the cells to reduce dehydroascorbic acid, more severe acute GSH depletion to less than 10% of normal levels did impair dehydroascorbic acid reduction. Culture of inducible cells in 11.8 mM D-glucose and doxycycline for 48 h enhanced glucokinase activity, increased glucose utilization, abolished D-glucose-dependent insulin secretion, and increased generation of reactive oxygen species. The latter may have contributed to subsequent decreases in the ability of the cells both to maintain intracellular ascorbate and to recycle it from dehydroascorbic acid. Cultured beta cells have a high capacity to recycle ascorbate, but this is sensitive to oxidant stress generated by increased glucose metabolism due to culture in high glucose concentrations and increased glucokinase expression. Impaired ascorbate recycling as a result of increased glucose metabolism may have implications for the role of ascorbate in insulin secretion in diabetes mellitus and may partially explain glucose toxicity in beta cells. PMID:15477012

  17. Effect of Hypertriglyceridemia on Beta Cell Mass and Function in ApoC3 Transgenic Mice.

    PubMed

    Liu, Yun-Zi; Cheng, Xiaoyun; Zhang, Ting; Lee, Sojin; Yamauchi, Jun; Xiao, Xiangwei; Gittes, George; Qu, Shen; Jiang, Chun-Lei; Dong, H Henry

    2016-07-01

    Hypertriglyceridemia results from increased production and decreased clearance of triglyceride-rich very low-density lipoproteins, a pathological condition that accounts for heightened risk of ischemic vascular diseases in obesity and type 2 diabetes. Despite its intimate association with insulin resistance, whether hypertriglyceridemia constitutes an independent risk for beta cell dysfunction in diabetes is unknown. Answering this fundamental question is stymied by the fact that hypertriglyceridemia is intertwined with hyperglycemia and insulin resistance in obese and diabetic subjects. To circumvent this limitation, we took advantage of apolipoprotein C3 (ApoC3)-transgenic mice, a model with genetic predisposition to hypertriglyceridemia. We showed that ApoC3-transgenic mice, as opposed to age/sex-matched wild-type littermates, develop hypertriglyceridemia with concomitant elevations in plasma cholesterol and non-esterified fatty acid levels. Anti-insulin and anti-glucagon dual immunohistochemistry in combination with morphometric analysis revealed that ApoC3-transgenic and wild-type littermates had similar beta cell and alpha cell masses as well as islet size and architecture. These effects correlated with similar amplitudes of glucose-stimulated insulin secretion and similar degrees of postprandial glucose excursion in ApoC3-transgenic versus wild-type littermates. Oil Red O histology did not visualize lipid infiltration into islets, correlating with the lack of ectopic triglyceride and cholesterol depositions in the pancreata of ApoC3-transgenic versus wild-type littermates. ApoC3-transgenic mice, despite persistent hypertriglyceridemia, maintained euglycemia under both fed and fasting conditions without manifestation of insulin resistance and fasting hyperinsulinemia. Thus, hypertriglyceridemia per se is not an independent risk factor for beta cell dysfunction in ApoC3 transgenic mice. PMID:27226540

  18. Junctophilin 3 expresses in pancreatic beta cells and is required for glucose-stimulated insulin secretion.

    PubMed

    Li, L; Pan, Z-F; Huang, X; Wu, B-W; Li, T; Kang, M-X; Ge, R-S; Hu, X-Y; Zhang, Y-H; Ge, L-J; Zhu, D-Y; Wu, Y-L; Lou, Y-J

    2016-01-01

    It is well accepted that junctophilin (JPHs) isoforms act as a physical bridge linking plasma membrane and endoplasmic reticulum (ER) for channel crosstalk in excitable cells. Our purpose is to investigate whether JPHs are involved in the proper communication between Ca(2+) influx and subsequent Ca(2+) amplification in pancreatic beta cells, thereby participating in regulating insulin secretion. The expression of JPH isoforms was examined in human and mouse pancreatic tissues, and JPH3 expression was found in both the beta cells. In mice, knockdown of Jph3 (si-Jph3) in islets decreased glucose-stimulated insulin secretion (GSIS) accompanied by mitochondrial function impairment. Si-Jph3 lowered the insulin secretory response to Ca(2+) signaling in the presence of glucose, and reduced [Ca(2+)]c transient amplitude triggered by caffeine. Si-Jph3 also attenuated mitofusin 2 expression, thereby disturbing the spatial organization of ER-mitochondria contact in islets. These results suggest that the regulation of GSIS by the KATP channel-independent pathways is partly impaired due to decrease of JPH3 expression in mouse islets. JPH3 also binds to type 2 ryanodine receptors (RyR2) in mouse and human pancreatic tissues, which might contribute to Ca(2+) release amplification in GSIS. This study demonstrates some previously unrecognized findings in pancreatic tissues: (1) JPH3 expresses in mouse and human beta cells; (2) si-Jph3 in mouse primary islets impairs GSIS in vitro; (3) impairment in GSIS in si-Jph3 islets is due to changes in RyR2-[Ca(2+)]c transient amplitude and ER-mitochondria contact. PMID:27336719

  19. Glucagon-like peptide-1 improves beta-cell antioxidant capacity via extracellular regulated kinases pathway and Nrf2 translocation.

    PubMed

    Fernández-Millán, E; Martín, M A; Goya, L; Lizárraga-Mollinedo, E; Escrivá, F; Ramos, S; Álvarez, C

    2016-06-01

    Oxidative stress plays an important role in the development of beta-cell dysfunction and insulin resistance, two major pathophysiological abnormalities of type 2 diabetes. Expression levels of antioxidant enzymes in beta cells are very low, rendering them more susceptible to damage caused by reactive oxygen species (ROS). Although the antioxidant effects of glucagon-like peptide-1 (GLP-1) and its analogs have been previously reported, the exact mechanisms involved are still unclear. In this study, we demonstrated that GLP-1 was able to effectively inhibit oxidative stress and cell death of INS-1E beta cells induced by the pro-oxidant tert-butyl hydroperoxide (tert-BOOH). Incubation with GLP-1 enhanced cellular levels of glutathione and the activity of its related enzymes, glutathione-peroxidase (GPx) and -reductase (GR) in beta cells. However, inhibition of ERK, but not of the PI3K/AKT pathway abolished, at least in part, the antioxidant effect of GLP-1. Moreover, ERK activation seems to be protein kinase A (PKA)-dependent because inhibition of PKA with H-89 was sufficient to block the GLP-1-derived protective effect on beta cells. GLP-1 likewise increased the synthesis of GR and favored the translocation of the nuclear transcription factor erythroid 2p45-related factor (Nrf2), a transcription factor implicated in the expression of several antioxidant/detoxificant enzymes. Glucose-stimulated insulin secretion was also preserved in beta-cells challenged with tert-BOOH but pre-treated with GLP-1, probably through the down-regulation of the mitochondrial uncoupling-protein2 (UCP2). Thus, our results provide additional mechanisms of action of GLP-1 to prevent oxidative damage in beta cells through the modulation of signaling pathways involved in antioxidant enzyme regulation. PMID:26968794

  20. Molecular basis for the regulation of islet beta cell mass in mice: the role of E-cadherin

    PubMed Central

    Wakae-Takada, N.; Xuan, S.; Watanabe, K.; Meda, P.; Leibel, R. L.

    2014-01-01

    Aims/hypothesis In rodents and humans, the rate of beta cell proliferation declines rapidly after birth; formation of the islets of Langerhans begins perinatally and continues after birth. Here, we tested the hypothesis that increasing levels of E-cadherin during islet formation mediate the decline in beta cell proliferation rate by contributing to a reduction of nuclear β-catenin and D-cyclins. Methods We examined E-cadherin, nuclear β-catenin, and D-cyclin levels, as well as cell proliferation during in vitro and in vivo formation of islet cell aggregates, using β-TC6 cells and transgenic mice with green fluorescent protein (GFP)-labelled beta cells, respectively. We tested the role of E-cadherin using antisense-mediated reductions of E-cadherin in β-TC6 cells, and mice segregating for a beta cell-specific E-cadherin knockout (Ecad [also known as Cdh1] βKO). Results In vitro, pseudo-islets of β-TC6 cells displayed increased E-cadherin but decreased nuclear β-catenin and cyclin D2, and reduced rates of cell proliferation, compared with monolayers. Antisense knockdown of E-cadherin increased cell proliferation and levels of cyclins D1 and D2. After birth, beta cells showed increased levels of E-cadherin, but decreased levels of D-cyclin, whereas islets of Ecad βKO mice showed increased levels of D-cyclins and nuclear β-catenin, as well as increased beta cell proliferation. These islets were significantly larger than those of control mice and displayed reduced levels of connexin 36. These changes correlated with reduced insulin response to ambient glucose, both in vitro and in vivo. Conclusions/interpretation The findings support our hypothesis by indicating an important role of E-cadherin in the control of beta cell mass and function. PMID:23354125

  1. Measurements of insulin responses as predictive markers of pancreatic beta-cell mass in normal and beta-cell-reduced lean and obese Göttingen minipigs in vivo.

    PubMed

    Larsen, Marianne O; Rolin, Bidda; Sturis, Jeppe; Wilken, Michael; Carr, Richard D; Pørksen, Niels; Gotfredsen, Carsten F

    2006-04-01

    At present, the best available estimators of beta-cell mass in humans are those based on measurement of insulin levels or appearance rates in the circulation. In several animal models, these estimators have been validated against beta-cell mass in lean animals. However, as many diabetic humans are obese, a correlation between in vivo tests and beta-cell mass must be evaluated over a range of body weights to include different levels of insulin sensitivity. For this purpose, obese (n = 10) and lean (n = 25) Göttingen minipigs were studied. Beta-cell mass had been reduced (n = 16 lean, n = 5 obese) with a combination of nicotinamide (67 mg/kg) and streptozotocin (125 mg/kg), acute insulin response (AIR) to intravenous glucose and/or arginine was tested, pulsatile insulin secretion was evaluated by deconvolution (n = 30), and beta-cell mass was determined histologically. AIR to 0.3 (r(2) = 0.4502, P < 0.0001) or 0.6 g/kg glucose (r(2) = 0.6806, P < 0.0001), 67 mg/kg arginine (r(2) = 0.5730, P < 0.001), and maximum insulin concentration (r(2) = 0.7726, P < 0.0001) were all correlated to beta-cell mass when evaluated across study groups, and regression lines were not different between lean and obese groups except for AIR to 0.3 g/kg glucose. Baseline pulse mass was not significantly correlated to beta-cell mass across the study groups (r(2) = 0.1036, NS), whereas entrained pulse mass did show a correlation across groups (r(2) = 0.4049, P < 0.001). This study supports the use of in vivo tests of insulin responses to evaluate beta-cell mass over a range of body weights in the minipig. Extensive stimulation of insulin secretion by a combination of glucose and arginine seems to give the best correlation to beta-cell mass. PMID:16278249

  2. Beta Cell Formation in vivo Through Cellular Networking, Integration and Processing (CNIP) in Wild Type Adult Mice.

    PubMed

    Doiron, Bruno; Hu, Wenchao; DeFronzo, Ralph A

    2016-01-01

    Insulin replacement therapy is essential in type 1 diabetic individuals and is required in ~40- 50% of type 2 diabetics during their lifetime. Prior attempts at beta cell regeneration have relied upon pancreatic injury to induce beta cell proliferation, dedifferentiation and activation of the embryonic pathway, or stem cell replacement. We report an alternative method to transform adult non-stem (somatic) cells into pancreatic beta cells. The Cellular Networking, Integration and Processing (CNIP) approach targets cellular mechanisms involved in pancreatic function in the organ's adult state and utilizes a synergistic mechanism that integrates three important levels of cellular regulation to induce beta cell formation: (i) glucose metabolism, (ii) membrane receptor function, and (iii) gene transcription. The aim of the present study was to induce pancreatic beta cell formation in vivo in adult animals without stem cells and without dedifferentiating cells to recapitulate the embryonic pathway as previously published (1-3). Our results employing CNIP demonstrate that: (i) insulin secreting cells can be generated in adult pancreatic tissue in vivo and circumvent the problem of generating endocrine (glucagon and somatostatin) cells that exert deleterious effects on glucose homeostasis, and (ii) longterm normalization of glucose tolerance and insulin secretion can be achieved in a wild type diabetic mouse model. The CNIP cocktail has the potential to be used as a preventative or therapeutic treatment or cure for both type 1 and type 2 diabetes. PMID:26696016

  3. Chemiluminescence Imaging of Superoxide Anion Detects Beta-Cell Function and Mass

    PubMed Central

    Bronsart, Laura L.; Stokes, Christian; Contag, Christopher H.

    2016-01-01

    Superoxide anion is produced during normal cellular respiration and plays key roles in cellular physiology with its dysregulation being associated with a variety of diseases. Superoxide anion is a short-lived molecule and, therefore, its homeostatic regulation and role in biology and disease requires dynamic quantification with fine temporal resolution. Here we validated coelenterazine as a reporter of intracellular superoxide anion concentration and used it as a dynamic measure both in vitro and in vivo. Chemiluminescence was dependent upon superoxide anion levels, including those produced during cellular respiration, and concentrations varied both kinetically and temporally in response to physiologically relevant fluctuations in glucose levels. In vivo imaging with coelenterazine revealed that beta cells of the pancreas have increased levels of superoxide anion, which acted as a measure of beta-cell function and mass and could predict the susceptibility of mice to diabetes mellitus. Glucose response and regulation are key elements of cellular physiology and organismal biology, and superoxide anion appears to play a fundamental and dynamic role in both of these processes. PMID:26752052

  4. Detection of C-Peptide in Urine as a Measure of Ongoing Beta Cell Function.

    PubMed

    McDonald, T J; Perry, M H

    2016-01-01

    C-peptide is a protein secreted by the pancreatic beta cells in equimolar quantities with insulin, following the cleavage of proinsulin into insulin. Measurement of C-peptide is used as a surrogate marker of endogenous insulin secretory capacity. Assessing C-peptide levels can be useful in classifying the subtype of diabetes as well as assessing potential treatment choices in the management of diabetes.Standard measures of C-peptide involve blood samples collected either fasted or, most often, after a fixed stimulus (such as oral glucose, mixed meal, or IV glucagon). Despite the established clinical utility of blood C-peptide measurement, its widespread use is limited. In many instances this is due to perceived practical restrictions associated with sample collection.Urine C-peptide measurement is an attractive noninvasive alternative to blood measures of beta-cell function. Urine C-peptide creatinine ratio measured in a single post stimulated sample has been shown to be a robust, reproducible measure of endogenous C-peptide which is stable for three days at room temperature when collected in boric acid. Modern high sensitivity immunoassay technologies have facilitated measurement of C-peptide down to single picomolar concentrations. PMID:27083170

  5. Maternal diabetes, programming of beta-cell disorders and intergenerational risk of type 2 diabetes.

    PubMed

    Chavey, A; Ah Kioon, M-D; Bailbé, D; Movassat, J; Portha, B

    2014-11-01

    A substantial body of evidence suggests that an abnormal intra-uterine milieu elicited by maternal metabolic disturbances as diverse as malnutrition, placental insufficiency, diabetes and obesity may be able to programme susceptibility of the foetus to later develop chronic degenerative diseases such as obesity, hypertension, cardiovascular diseases and type 2 diabetes (T2D). As insulin-producing cells have been placed centre stage in the development of T2D, this review examines developmental programming of the beta-cell mass (BCM) in various rodent models of maternal protein restriction, calorie restriction, overnutrition and diabetes. The main message is that whatever the initial maternal insult (F0 generation) and whether alone or in combination, it gives rise to the same programmed BCM outcome in the daughter generation (F1). The altered BCM phenotype in F1 females prohibits normal BCM adaptation during pregnancy and, thus, diabetes (gestational diabetes) ensues. This gestational diabetes is then passed from one generation (F1) to the next (F2, F3 and so on). This review highlights a number of studies that have identified epigenetic mechanisms that may contribute to altered BCM development and beta-cell failure, as observed in diabetes. In addition to their role in instilling the programmed defect, these non-genomic mechanisms may also be involved in its intergenerational transmission. PMID:24948417

  6. Evidence for a slowly exchangeable pool of calcium in the pancreatic beta cell plasma membrane.

    PubMed Central

    Gylfe, E; Hellman, B

    1982-01-01

    1. Exposure to media deprived of Ca2+ resulted in prompt and transient stimulation of 45Ca efflux from beta cell-rich pancreatic islets microdissected from ob/ob-mice and to some extent also from the isolated neurohypophysis. 2. Particular high efflux rates were reached when the Ca2+-deficient medium contained EGTA, but there was no effect of the chelator on the total amount of radioactivity mobilized from the islets. 3. The removal of extracellular Ca2+ was less effective in promoting the 45Ca efflux in the absence of Na+ and no stimulatory response was seen in the presence of 1 mM-La3+. 4. The 45Ca washout was stimulated whether or not the media used for the loading or subsequent perifusion of the islets were supplemented with 20 mM-D-glucose. However, there was no response to a second exposure to a Ca2+-deficient medium even subsequent to redistribution of intracellular calcium induced by temporary lowering of the temperature. 5. It is suggested that the islet 45Ca released by the removal of extracellular Ca2+ originates from a distinct plasma membrane pool which is exchanged slowly compared to most of the calcium at the beta cell periphery. PMID:6752376

  7. Iron Regulation of Pancreatic Beta-Cell Functions and Oxidative Stress.

    PubMed

    Backe, Marie Balslev; Moen, Ingrid Wahl; Ellervik, Christina; Hansen, Jakob Bondo; Mandrup-Poulsen, Thomas

    2016-07-17

    Dietary advice is the cornerstone in first-line treatment of metabolic diseases. Nutritional interventions directed at these clinical conditions mainly aim to (a) improve insulin resistance by reducing energy-dense macronutrient intake to obtain weight loss and (b) reduce fluctuations in insulin secretion through avoidance of rapidly absorbable carbohydrates. However, even in the majority of motivated patients selected for clinical trials, massive efforts using this approach have failed to achieve lasting efficacy. Less attention has been given to the role of micronutrients in metabolic diseases. Here, we review the evidence that highlights (a) the importance of iron in pancreatic beta-cell function and dysfunction in diabetes and (b) the integrative pathophysiological effects of tissue iron levels in the interactions among the beta cell, gut microbiome, hypothalamus, innate and adaptive immune systems, and insulin-sensitive tissues. We propose that clinical trials are warranted to clarify the impact of dietary or pharmacological iron reduction on the development of metabolic disorders. PMID:27146016

  8. Cereulide food toxin, beta cell function and diabetes: Facts and hypotheses.

    PubMed

    Vangoitsenhoven, Roman; Maris, Michael; Overbergh, Lut; Van Loco, Joris; Mathieu, Chantal; Van der Schueren, Bart

    2015-07-01

    The incidence of both type 1 and type 2 diabetes is increasing and although environmental pollutants are believed to be potential culprits, the extent to which they can be held responsible remains uncertain. Some bacterial strains of the Bacillus cereus produce a toxin, cereulide, which is frequently found in starchy meals and which is difficult to eradicate from the food chain as it is highly resistant to heat, acidity and proteolysis. While cereulide is well known to cause acute emetic toxicity when ingested at high doses, several in vitro studies have shown that also extremely low doses of cereulide can be toxic, with beta cells being particularly sensitive. Mechanistically, such low doses impair the mitochondrial activity of the beta cells thereby leading to hampered insulin secretion and cell death, both key traits in the pathophysiology of diabetes. In vivo studies of chronic or repeated low dose exposure to cereulide are currently lacking, but should be performed to further clarify the true relevance of cereulide as a potential environmental contributor to the ongoing diabetes epidemic. PMID:25998918

  9. Beta cell antigens in type 1 diabetes: triggers in pathogenesis and therapeutic targets

    PubMed Central

    Mauvais, François-Xavier; Diana, Julien; van Endert, Peter

    2016-01-01

    Research focusing on type 1 diabetes (T1D) autoantigens aims to explore our understanding of these beta cell proteins in order to design assays for monitoring the pathogenic autoimmune response, as well as safe and efficient therapies preventing or stopping it. In this review, we will discuss progress made in the last 5 years with respect to mechanistic understanding, diagnostic monitoring, and therapeutic modulation of the autoantigen-specific cellular immune response in T1D. Some technical progress in monitoring tools has been made; however, the potential of recent technologies for highly multiplexed exploration of human cellular immune responses remains to be exploited in T1D research, as it may be the key to the identification of surrogate markers of disease progression that are still wanting. Detailed analysis of autoantigen recognition by T cells suggests an important role of non-conventional antigen presentation and processing in beta cell-directed autoimmunity, but the impact of this in human T1D has been little explored. Finally, therapeutic administration of autoantigens to T1D patients has produced disappointing results. The application of novel modes of autoantigen administration, careful translation of mechanistic understanding obtained in preclinical studies and in vitro with human cells, and combination therapies including CD3 antibodies may help to make autoantigen-based immunotherapy for T1D a success story in the future. PMID:27158463

  10. BACE2 is stored in secretory granules of mouse and rat pancreatic beta cells.

    PubMed

    Finzi, Giovanna; Franzi, Francesca; Placidi, Claudia; Acquati, Francesco; Palumbo, Elisa; Russo, Antonella; Taramelli, Roberto; Sessa, Fausto; La Rosa, Stefano

    2008-01-01

    BACE2 is a protease homologous to BACE1 protein, an enzyme involved in the amyloid formation of Alzheimer disease (AD). However, despite the high homology between these two proteins, the biological role of BACE2 is still controversial, even though a few studies have suggested a pathogenetic role in sporadic inclusion-body myositis and hereditary inclusion-body myopathy, which are characterized by vacuolization of muscular fibers with intracellular deposits of proteins similar to those found in the brain of AD patients. Although BACE2 has also been identified in the pancreas, its function remains unknown and its specific localization in different pancreatic cell types has not been definitively ascertained. For these reasons, the authors have investigated the cellular and subcellular localization of BACE2 in normal rodent pancreases. BACE2 immunoreactivity was found in secretory granules of beta cells, co-stored with insulin and IAPP, while it was lacking in the other endocrine and exocrine cell types. The presence of BACE2 in secretory granules of beta cells suggests that it may play a role in diabetes-associated amyloidogenesis. PMID:19117266

  11. Relationship Between Beta Cell Dysfunction and Severity of Disease Among Critically Ill Children

    PubMed Central

    Liu, Ping-Ping; Lu, Xiu-Lan; Xiao, Zheng-Hui; Qiu, Jun; Zhu, Yi-Min

    2016-01-01

    Abstract Although beta cell dysfunction has been proved to predict prognosis among humans and animals, its prediction on severity of disease remains unclear among children. The present study was aimed to examine the relationship between beta cell dysfunction and severity of disease among critically ill children. This prospective study included 1146 critically ill children, who were admitted to Pediatric Intensive Care Unit (PICU) of Hunan Children's Hospital from November 2011 to August 2013. Information on characteristics, laboratory tests, and prognostic outcomes was collected. Homeostasis model assessment (HOMA)-β, evaluating beta cell function, was used to divide all participants into 4 groups: HOMA-β = 100% (group I, n = 339), 80% ≤ HOMA-β < 100% (group II, n = 71), 40% ≤ HOMA-β < 80% (group III, n = 293), and HOMA-β < 40% (group IV, n = 443). Severity of disease was assessed using the worst Sequential Organ Failure Assessment (SOFA) score, Pediatric Risk of Mortality (PRISM) III score, incidence of organ damage, septic shock, multiple organ dysfunction syndrome (MODS), mechanical ventilation (MV) and mortality. Logistic regression analysis was used to evaluate the risk of developing poor outcomes among patients in different HOMA-β groups, with group I as the reference group. Among 1146 children, incidence of HOMA-β < 100% was 70.41%. C-peptide and insulin declined with the decrement of HOMA-β (P < 0.01). C-reactive protein and procalcitonin levels, rather than white blood cell, were significantly different among 4 groups (P < 0.01). In addition, the worst SOFA score and the worst PRISMIII score increased with declined HOMA-β. For example, the worst SOFA score in group I, II, III, and IV was 1.55 ± 1.85, 1.71 ± 1.93, 1.92 ± 1.63, and 2.18 ± 1.77, respectively. Furthermore, patients with declined HOMA-β had higher risk of developing septic shock, MODS, MV, and mortality, even after adjusting age

  12. Bulk-like endocytosis plays an important role in the recycling of insulin granules in pancreatic beta cells.

    PubMed

    Wen, Du; Xue, Yanhong; Liang, Kuo; Yuan, Tianyi; Lu, Jingze; Zhao, Wei; Xu, Tao; Chen, Liangyi

    2012-08-01

    Although bulk endocytosis has been found in a number of neuronal and endocrine cells, the molecular mechanism and physiological function of bulk endocytosis remain elusive. In pancreatic beta cells, we have observed bulk-like endocytosis evoked both by flash photolysis and trains of depolarization. Bulk-like endocytosis is a clathrin-independent process that is facilitated by enhanced extracellular Ca(2+) entry and suppressed by the inhibition of dynamin function. Moreover, defects in bulk-like endocytosis are accompanied by hyperinsulinemia in primary beta cells dissociated from diabetic KKAy mice, which suggests that bulk-like endocytosis plays an important role in maintaining the exo-endocytosis balance and beta cell secretory capability. PMID:22729398

  13. Phosphorylation events implicating p38 and PI3K mediate tungstate-effects in MIN6 beta cells

    SciTech Connect

    Piquer, Sandra; Gomis, Ramon . E-mail: rgomis@clinic.ub.es

    2007-06-29

    Oral administration of sodium tungstate is an effective treatment for diabetes in animal models. Several lines of evidence indicate the pancreatic beta cell as one of the targets of tungstate action. Here, we examined the molecular mechanism by which this compound exerts its effects on the beta cell line MIN6. Tungstate treatment induced phosphorylation and subsequent activation of p38 and PI3K which in turn are implicated in tungstate PDX-1 nuclear localization and activation. Although no effect was observed in glucose-induced insulin secretion we found that tungstate activates basal insulin release, a process driven, at least in part, by activation of p38. These results show a direct involvement of p38 and PI3K phosphorylation in the mechanism of action of tungstate in the beta cell.

  14. [A preliminary study on the mechanism of impaired beta cell function in monosodium glutamate obese rat with insulin resistance].

    PubMed

    Liu, Shuai-Nan; Liu, Quan; Shen, Zhu-Fang

    2008-11-01

    This study is to evaluate beta cell function and investigate the mechanism of impaired pancreatic islet beta cell function in monosodium glutamate (MSG) obese rat with insulin resistance, an animal model of metabolic syndrome. Insulin tolerance test was used to screen MSG obese rats with insulin resistance. Blood concentrations of glucose, triglyceride, total cholesterol and insulin were determined. Beta cell function was assessed with hyperglycemic clamp technique. The morphological alterations in pancreas and changes of islet beta cell mass were evaluated by hematoxylin-eosin (HE) and Gomori aldehyde fuchsin staining. Lipid, oxidative stress relevant factors, nitric oxide (NO) level and activity of ATPase in pancreas and pancreatic mitochondrial were tested. The MSG obese rats with insulin resistance could be validated as a typical metabolic syndrome animal model possessing increased fasting plasma triglycerides and insulin (P < 0. 001), markedly decreased weight indices of pancreas and impaired glucose-stimulated insulin secretion. Hematoxylin-eosin (HE) and Gomori aldehyde fuchsin staining showed increased adipocytes and fibroplasia deposition in pancreas and reduced beta cell mass. The increased contents of triglyceride and NO level, the decreased SOD levels and activities of total ATPase (P < 0.001), Na+-K+-ATPase (P < 0.001) and Ca2+-Mg2+-ATPase (P < 0.01) were observed in pancreas and its mitochondria versus normal rat. The study demonstrates that accumulation of lipids in pancreas could lead to increased systemic indicators of inflammation, such as NO, which may influence the activities of several kinds of ATPase in cell membranes and interfere the ion transport, substance metabolism and energy production in pancreas. Finally the MSG obese rats characterized with metabolic syndrome displayed an impairment of beta cell function. PMID:19239028

  15. Differential effects of three echovirus strains on cell lysis and insulin secretion in beta cell derived lines.

    PubMed

    Sarmiento, Luis; Medina, Anya; Aziz, Kosrat; Anagandula, Mahesh; Cabrera-Rode, Eduardo; Fex, Malin; Frisk, Gun; Cilio, Corrado M

    2016-06-01

    In an earlier study, infection of human pancreatic islets with epidemic strains of echovirus (E4, E16, E30), with proven but differently ability to induce islet autoimmunity, resulted either in a severe damage (i.e., E16 and E30) or proceeded without visible changes in infected islets (i.e., E4). In this study, the ability of these strains to replicate in beta cells and the consequence of such an infection for beta cell lysis and beta cell function was studied in the pancreatic beta cell lines INS-1, MIN6, and NIT-1. The strains of E16 and E30 did replicate in INS1, MIN6, and NIT1 cells and resulted in a pronounced cytopathic effect within 3 days following infection. By contrast, E4 replicated in all examined insulinoma cells with no apparent cell destruction. The insulin release in response to high glucose stimulation was hampered in all infected cells (P < 0.05) when no evidence of cytolysis was present; however, the adverse effect of E16 and E30 on insulin secretion appeared to be higher than that of the E4 strain. The differential effects of echovirus infection on cell lysis, and beta cell function in the rodent insulinoma INS1, MIN6, and NIT 1 cells reflect those previously obtained in primary human islets and support the notion that the insulin-producing beta cells can harbor a non-cytopathic viral infection. J. Med. Virol. 88:971-978, 2016. © 2015 Wiley Periodicals, Inc. PMID:26629879

  16. Hepatocyte nuclear factor 3beta is involved in pancreatic beta-cell-specific transcription of the pdx-1 gene.

    PubMed Central

    Wu, K L; Gannon, M; Peshavaria, M; Offield, M F; Henderson, E; Ray, M; Marks, A; Gamer, L W; Wright, C V; Stein, R

    1997-01-01

    The mammalian homeobox gene pdx-1 is expressed in pluripotent precursor cells in the dorsal and ventral pancreatic bud and duodenal endoderm, which will produce the pancreas and the rostral duodenum. In the adult, pdr-1 is expressed principally within insulin-secreting pancreatic islet beta cells and cells of the duodenal epithelium. Our objective in this study was to localize sequences within the mouse pdx-1 gene mediating selective expression within the islet. Studies of transgenic mice in which a genomic fragment of the mouse pdx-1 gene from kb -4.5 to +8.2 was used to drive a beta-galactosidase reporter showed that the control sequences sufficient for appropriate developmental and adult specific expression were contained within this region. Three nuclease-hypersensitive sites, located between bp -2560 and -1880 (site 1), bp -1330 and -800 (site 2), and bp -260 and +180 (site 3), were identified within the 5'-flanking region of the endogenous pdx-1 gene. Pancreatic beta-cell-specific expression was shown to be controlled by sequences within site 1 from an analysis of the expression pattern of various pdr-1-herpes simplex virus thymidine kinase promoter expression constructs in transfected beta-cell and non-beta-cell lines. Furthermore, we also established that this region was important in vivo by demonstrating that expression from a site 1-driven beta-galactosidase reporter construct was directed to islet beta-cells in transgenic mice. The activity of the site 1-driven constructs was reduced substantially in beta-cell lines by mutating a hepatocyte nuclear factor 3 (HNF3)-like site located between nucleotides -2007 and -1996. Gel shift analysis indicated that HNF3beta present in islet beta cells binds to this element. Immunohistochemical studies revealed that HNF3beta was present within the nuclei of almost all islet beta cells and subsets of pancreatic acinar cells. Together, these results suggest that HNF3beta, a key regulator of endodermal cell lineage

  17. Activation of transmembrane bile acid receptor TGR5 stimulates insulin secretion in pancreatic {beta} cells

    SciTech Connect

    Kumar, Divya P.; Rajagopal, Senthilkumar; Mahavadi, Sunila; Mirshahi, Faridoddin; Grider, John R.; Murthy, Karnam S.; Sanyal, Arun J.

    2012-10-26

    Highlights: Black-Right-Pointing-Pointer G protein coupled receptor TGR5 is expressed in mouse and human islets. Black-Right-Pointing-Pointer TGR5 is coupled to activation of Gs and Ca{sup 2+} release via cAMP/Epac/PLC-{epsilon} pathway. Black-Right-Pointing-Pointer Activation of TGR5 by bile salts and selective ligands causes insulin secretion. Black-Right-Pointing-Pointer TGR5 could be a potential therapeutic target to treat diabetes. -- Abstract: Bile acids act as signaling molecules and stimulate the G protein coupled receptor, TGR5, in addition to nuclear farnesoid X receptor to regulate lipid, glucose and energy metabolism. Bile acid induced activation of TGR5 in the enteroendocrine cells promotes glucagon like peptide-1 (GLP-1) release, which has insulinotropic effect in the pancreatic {beta} cells. In the present study, we have identified the expression of TGR5 in pancreatic {beta} cell line MIN6 and also in mouse and human pancreatic islets. TGR5 selective ligands, oleanolic acid (OA) and INT-777 selectively activated G{alpha}{sub s} and caused an increase in intracellular cAMP and Ca{sup 2+}. OA and INT-777 also increased phosphoinositide (PI) hydrolysis and the increase was blocked by NF449 (a selective G{alpha}{sub s} inhibitor) or (U73122) (PI hydrolysis inhibitor). OA, INT-777 and lithocholic acid increased insulin release in MIN6 and human islets and the increase was inhibited by treatment with NF449, (U73122) or BAPTA-AM (chelator of calcium), but not with myristoylated PKI (PKA inhibitor), suggesting that the release is dependent on G{sub s}/cAMP/Ca{sup 2+} pathway. 8-pCPT-2 Prime -O-Me-cAMP, a cAMP analog, which activates Epac, but not PKA also stimulated PI hydrolysis. In conclusion, our study demonstrates that the TGR5 expressed in the pancreatic {beta} cells regulates insulin secretion and highlights the importance of ongoing therapeutic strategies targeting TGR5 in the control of glucose homeostasis.

  18. Lack of lipotoxicity effect on {beta}-cell dysfunction in ketosis-prone type 2 diabetes.

    PubMed

    Umpierrez, Guillermo E; Smiley, Dawn; Robalino, Gonzalo; Peng, Limin; Gosmanov, Aidar R; Kitabchi, Abbas E

    2010-03-01

    OBJECTIVE Over half of newly diagnosed obese African Americans with diabetic ketoacidosis (DKA) discontinue insulin therapy and go through a period of near-normoglycemia remission. This subtype of diabetes is known as ketosis-prone type 2 diabetes (KPDM). RESEARCH DESIGN AND METHODS To investigate the role of lipotoxicity on beta-cell function, eight obese African Americans with KPDM, eight obese subjects with type 2 diabetes with severe hyperglycemia without ketosis (ketosis-resistant type 2 diabetes), and nine nondiabetic obese control subjects underwent intravenous infusion of 20% intralipid at 40 ml/h for 48 h. beta-Cell function was assessed by changes in insulin and C-peptide concentration during infusions and by changes in acute insulin response to arginine stimulation (AIR(arg)) before and after lipid infusion. RESULTS The mean time to discontinue insulin therapy was 11.0 +/- 8.0 weeks in KPDM and 9.6 +/- 2.2 weeks in ketosis-resistant type 2 diabetes (P = NS). At remission, KPDM and ketosis-resistant type 2 diabetes had similar glucose (94 +/- 14 vs. 109 +/- 20 mg/dl), A1C (5.7 +/- 0.4 vs. 6.3 +/- 1.1%), and baseline AIR(arg) response (34.8 +/- 30 vs. 64 +/- 69 microU/ml). P = NS despite a fourfold increase in free fatty acid (FFA) levels (0.4 +/- 0.3 to 1.8 +/- 1.1 mmol/l, P < 0.01) during the 48-h intralipid infusion; the response to AIR(arg) stimulation, as well as changes in insulin and C-peptide levels, were similar among obese patients with KPDM, patients with ketosis-resistant type 2 diabetes, and nondiabetic control subjects. CONCLUSIONS Near-normoglycemia remission in obese African American patients with KPDM and ketosis-resistant type 2 diabetes is associated with a remarkable recovery in basal and stimulated insulin secretion. A high FFA level by intralipid infusion for 48 h was not associated with beta-cell decompensation (lipotoxicity) in KPDM patients. PMID:20028938

  19. Continuous subcutaneous infusion of glucagon by portable pump in non beta cell tumor hypoglycemia.

    PubMed

    Houlbert, D; Altman, J J; Lageron, A; Capeau, J; Dahan, R; Friedberg, G; Wassef, M; Poffenbarger, P L; Segrestaa, J M

    1985-04-01

    Subcutaneous infusion of glucagon by portable pump appears to give very effective symptomatic relief from non beta cell tumor hypoglycemia when surgery, radiotherapy and chemotherapy are impossible or ineffective. This mode of glucagon administration was proposed in a patient who had severe nocturnal hypoglycemic attacks. The aim of the study was to specify the modes of utilization and to test the efficiency and the tolerance of this treatment. Glucagon was infused at 400 micrograms/h during every 12 hour night. Because of the hepatic action of glucagon it is very important to use this treatment with an adequate diet and to stop the infusion during the day to reconstitute the glycogen overload. This mode of glucagon administration was very effective in over 6 months of use and well tolerated. PMID:4007224

  20. Is Transforming Stem Cells to Pancreatic Beta Cells Still the Holy Grail for Type 2 Diabetes?

    PubMed

    Kahraman, Sevim; Okawa, Erin R; Kulkarni, Rohit N

    2016-08-01

    Diabetes is a progressive disease affecting millions of people worldwide. There are several medications and treatment options to improve the life quality of people with diabetes. One of the strategies for the treatment of diabetes could be the use of human pluripotent stem cells or induced pluripotent stem cells. The recent advances in differentiation of stem cells into insulin-secreting beta-like cells in vitro make the transplantation of the stem cell-derived beta-like cells an attractive approach for treatment of type 1 and type 2 diabetes. While stem cell-derived beta-like cells provide an unlimited cell source for beta cell replacement therapies, these cells can also be used as a platform for drug screening or modeling diseases. PMID:27313072

  1. Transient receptor potential M3 channels are ionotropic steroid receptors in pancreatic beta cells.

    PubMed

    Wagner, Thomas F J; Loch, Sabine; Lambert, Sachar; Straub, Isabelle; Mannebach, Stefanie; Mathar, Ilka; Düfer, Martina; Lis, Annette; Flockerzi, Veit; Philipp, Stephan E; Oberwinkler, Johannes

    2008-12-01

    Transient receptor potential (TRP) cation channels are renowned for their ability to sense diverse chemical stimuli. Still, for many members of this large and heterogeneous protein family it is unclear how their activity is regulated and whether they are influenced by endogenous substances. On the other hand, steroidal compounds are increasingly recognized to have rapid effects on membrane surface receptors that often have not been identified at the molecular level. We show here that TRPM3, a divalent-permeable cation channel, is rapidly and reversibly activated by extracellular pregnenolone sulphate, a neuroactive steroid. We show that pregnenolone sulphate activates endogenous TRPM3 channels in insulin-producing beta cells. Application of pregnenolone sulphate led to a rapid calcium influx and enhanced insulin secretion from pancreatic islets. Our results establish that TRPM3 is an essential component of an ionotropic steroid receptor enabling unanticipated crosstalk between steroidal and insulin-signalling endocrine systems. PMID:18978782

  2. Matrix metalloproteinases, T cell homing and beta-cell mass in type 1 diabetes.

    PubMed

    Savinov, Alexei Y; Strongin, Alex Y

    2009-01-01

    The pathogenesis of type 1 diabetes begins with the activation of autoimmune T killer cells and is followed by their homing into the pancreatic islets. After penetrating the pancreatic islets, T cells directly contact and destroy insulin-producing beta cells. This review provides an overview of the dynamic interactions which link T cell membrane type-1 matrix metalloproteinase (MT1-MMP) and the signaling adhesion CD44 receptor with T cell transendothelial migration and the subsequent homing of the transmigrated cells to the pancreatic islets. MT1-MMP regulates the functionality of CD44 in diabetogenic T cells. By regulating the functionality of T cell CD44, MT1-MMP mediates the transition of T cell adhesion to endothelial cells to the transendothelial migration of T cells, thus, controlling the rate at which T cells home into the pancreatic islets. As a result, the T cell MT1-MMP-CD44 axis controls the severity of the disease. Inhibition of MT1-MMP proteolysis of CD44 using highly specific and potent synthetic inhibitors, which have been clinically tested in cancer patients, reduces the rate of transendothelial migration and the homing of T cells. Result is a decrease in the net diabetogenic efficiency of T cells and a restoration of beta cell mass and insulin production in NOD mice. The latter is a reliable and widely used model of type I diabetes in humans. Overall, existing experimental evidence suggests that there is a sound mechanistic rationale for clinical trials of the inhibitors of T cell MT1-MMP in human type 1 diabetes patients. PMID:19251049

  3. Effects of meal size and composition on incretin, alpha-cell, and beta-cell responses.

    PubMed

    Rijkelijkhuizen, Josina M; McQuarrie, Kelly; Girman, Cynthia J; Stein, Peter P; Mari, Andrea; Holst, Jens J; Nijpels, Giel; Dekker, Jacqueline M

    2010-04-01

    The incretins glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) regulate postprandial insulin release from the beta-cells. We investigated the effects of 3 standardized meals with different caloric and nutritional content in terms of postprandial glucose, insulin, glucagon, and incretin responses. In a randomized crossover study, 18 subjects with type 2 diabetes mellitus and 6 healthy volunteers underwent three 4-hour meal tolerance tests (small carbohydrate [CH]-rich meal, large CH-rich meal, and fat-rich meal). Non-model-based and model-based estimates of beta-cell function and incremental areas under the curve of glucose, insulin, C-peptide, glucagon, GLP-1, and GIP were calculated. Mixed models and Friedman tests were used to test for differences in meal responses. The large CH-rich meal and fat-rich meal resulted in a slightly larger insulin response as compared with the small CH-rich meal and led to a slightly shorter period of hyperglycemia, but only in healthy subjects. Model-based insulin secretion estimates did not show pronounced differences between meals. Both in healthy individuals and in those with diabetes, more CH resulted in higher GLP-1 release. In contrast with the other meals, GIP release was still rising 2 hours after the fat-rich meal. The initial glucagon response was stimulated by the large CH-rich meal, whereas the fat-rich meal induced a late glucagon response. Fat preferentially stimulates GIP secretion, whereas CH stimulates GLP-1 secretion. Differences in meal size and composition led to differences in insulin and incretin responses but not to differences in postprandial glucose levels of the well-controlled patients with diabetes. PMID:19846181

  4. Role of humoral beta-cell autoimmunity in type 1 diabetes.

    PubMed

    Knip, Mikael; Siljander, Heli; Ilonen, Jorma; Simell, Olli; Veijola, Riitta

    2016-07-01

    Islet cell antibodies (ICA) were found for the first time more than 40 yr ago in patients with autoimmune endocrine deficiencies, including type 1 diabetes (T1D). ICA detected by indirect immunofluorescence represent a heterogeneous group of autoantibodies targeting a series of biochemical autoantigens, such as the protein tyrosine phosphatase related islet antigen 2 (IA-2), the 65 kD isoform of glutamic acid decarboxylase (GA65), and zinc transporter 8 (ZnT8) as well as currently unidentified autoantigens. The general view is that the diabetes-associated autoantibodies are not directly involved in beta-cell destruction but function as biomarkers of an ongoing destructive process. The diabetes-associated autoantibodies remain the strongest predictive marker for future development of T1D. Positivity for multiple (≥2) autoantibodies is highly predictive of clinical disease both among first-degree relatives and in the general population. Autoantibody titers are highly variable during the preclinical phase, but in many cases the titers tend to decrease before diagnosis. The first signs of beta-cell autoimmunity may appear early during the first months of life. The majority of those individuals diagnosed with T1D before puberty seroconvert to autoantibody positivity before the age of 3 yr. The natural course and duration of preclinical diabetes vary substantially from one individual to another. The characteristics of the isotype-specific response during preclinical diabetes appear to be antigen-specific. Diabetes-associated autoantibodies may be useful surrogate markers of the subsequent development of T1D in primary prevention trials. T1D may occur, albeit rarely, in the absence of any signs of humoral autoimmunity at diagnosis. PMID:27411432

  5. Serpine1 Mediates Porphyromonas gingivalis Induced Insulin Secretion in the Pancreatic Beta Cell Line MIN6

    PubMed Central

    Bhat, Uppoor G.; Watanabe, Keiko

    2015-01-01

    Periodontitis is an inflammatory disease resulting in destruction of gingiva and alveolar bone caused by an exuberant host immunological response to periodontal pathogens. Results from a number of epidemiological studies indicate a close association between diabetes and periodontitis. Results from cross-sectional studies indicate that subjects with periodontitis have a higher odds ratio of developing insulin resistance (IR). However, the mechanisms by which periodontitis influences the development of diabetes are not known. Results from our previous studies using an animal model of periodontitis suggest that periodontitis accelerates the onset of hyperinsulinemia and IR. In addition, LPS from a periodontal pathogen, Porphyromonas gingivalis (Pg), stimulates Serpine1 expression in the pancreatic beta cell line MIN6. Based on these observations, we hypothesized that a periodontal pathogen induces hyperinsulinemia and Serpine1 may be involved in this process. To test this hypothesis, we co-incubated Pg with the pancreatic beta cell line MIN6 and measured the effect on insulin secretion by MIN6 cells. We further determined the involvement of Serpine1 in insulin secretion by downregulating Serpine1 expression. Our results indicated that Pg stimulated insulin secretion by approximately 3.0 fold under normoglycemic conditions. In a hyperglycemic state, Pg increased insulin secretion by 1.5 fold. Pg significantly upregulated expression of the Serpine1 gene and this was associated with increased secretion of insulin by MIN6 cells. However, cells with downregulated Serpine1 expression were resistant to Pg stimulated insulin secretion under normoglycemic conditions. We conclude that the periodontal pathogen, Pg, induced insulin secretion by MIN6 cells and this induction was, in part, Serpine1 dependent. Thus, Serpine1 may play a pivotal role in insulin secretion during the accelerated development of hyperinsulinemia and the resulting IR in the setting of periodontitis. PMID

  6. Beta-cell function, incretin effect, and incretin hormones in obese youth along the span of glucose tolerance from normal to prediabetes to Type 2 diabetes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Using the hyperglycemic and euglycemic clamp, we demonstrated impaired Beta-cell function in obese youth with increasing dysglycemia. Herein we describe oral glucose tolerance test (OGTT)-modeled Beta-cell function and incretin effect in obese adolescents spanning the range of glucose tolerance. Bet...

  7. Down-regulation of zinc transporter 8 (SLC30A8) in pancreatic beta-cells promotes cell survival

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The pancreatic islet contains high levels of zinc in granular vesicles of beta-cells where insulin is matured, crystallized, and stored before secretion. Zinc is an essential co-factor for insulin crystallization forming dense core in secretory granules. In insulin-containing secretory granules, zin...

  8. A quantitative study of sodium tungstate protective effect on pancreatic beta cells in streptozotocin-induced diabetic rats.

    PubMed

    Heidari, Zahra; Mahmoudzadeh-Sagheb, Hamidreza; Moudi, Bita

    2008-12-01

    Diabetes is a major public health problem. Development of new therapies that are able to improve glycemia management, cure diabetes, and can even protect from it, are of great interest. This study investigated the protective effect of sodium tungstate against STZ-induced beta-cell damages by means of stereological methods. Sixty rats were divided into six groups: control (C), tungstate-treated control (TC), STZ-induced diabetic (D), STZ-induced diabetic rats were treated by sodium tungstate from 1 week before STZ injection (TDB), food-restricted diabetic (FRD), and diabetic rats treated with sodium tungstate 1 week after STZ administration (TDA). Stereological estimation of pancreas volume, islets volume density, volume-weighted mean islets volume and mass of beta cells, islets, and pancreas and total number of islets were done. Islets volume density, volume-weighted mean islets volume, and mass of beta cells, islets, and pancreas of TDB group was significantly higher than D, FRD and TDA groups (P<0.001) and was comparable to controls (C and TC groups). Total number of islets, pancreas wet weight and volume did not show any significant changes between these groups (P>0.05). Results suggested that sodium tungstate preserves pancreatic beta cells from STZ-induced damages and diabetes induction in rats. PMID:18400503

  9. Progressive glucose stimulation of islet beta cells reveals a transition from segregated to integrated modular functional connectivity patterns

    NASA Astrophysics Data System (ADS)

    Markovič, Rene; Stožer, Andraž; Gosak, Marko; Dolenšek, Jurij; Marhl, Marko; Rupnik, Marjan Slak

    2015-01-01

    Collective beta cell activity in islets of Langerhans is critical for the supply of insulin within an organism. Even though individual beta cells are intrinsically heterogeneous, the presence of intercellular coupling mechanisms ensures coordinated activity and a well-regulated exocytosis of insulin. In order to get a detailed insight into the functional organization of the syncytium, we applied advanced analytical tools from the realm of complex network theory to uncover the functional connectivity pattern among cells composing the intact islet. The procedure is based on the determination of correlations between long temporal traces obtained from confocal functional multicellular calcium imaging of beta cells stimulated in a stepwise manner with a range of physiological glucose concentrations. Our results revealed that the extracted connectivity networks are sparse for low glucose concentrations, whereas for higher stimulatory levels they become more densely connected. Most importantly, for all ranges of glucose concentration beta cells within the islets form locally clustered functional sub-compartments, thereby indicating that their collective activity profiles exhibit a modular nature. Moreover, we show that the observed non-linear functional relationship between different network metrics and glucose concentration represents a well-balanced setup that parallels physiological insulin release.

  10. Progressive glucose stimulation of islet beta cells reveals a transition from segregated to integrated modular functional connectivity patterns

    PubMed Central

    Markovič, Rene; Stožer, Andraž; Gosak, Marko; Dolenšek, Jurij; Marhl, Marko; Rupnik, Marjan Slak

    2015-01-01

    Collective beta cell activity in islets of Langerhans is critical for the supply of insulin within an organism. Even though individual beta cells are intrinsically heterogeneous, the presence of intercellular coupling mechanisms ensures coordinated activity and a well-regulated exocytosis of insulin. In order to get a detailed insight into the functional organization of the syncytium, we applied advanced analytical tools from the realm of complex network theory to uncover the functional connectivity pattern among cells composing the intact islet. The procedure is based on the determination of correlations between long temporal traces obtained from confocal functional multicellular calcium imaging of beta cells stimulated in a stepwise manner with a range of physiological glucose concentrations. Our results revealed that the extracted connectivity networks are sparse for low glucose concentrations, whereas for higher stimulatory levels they become more densely connected. Most importantly, for all ranges of glucose concentration beta cells within the islets form locally clustered functional sub-compartments, thereby indicating that their collective activity profiles exhibit a modular nature. Moreover, we show that the observed non-linear functional relationship between different network metrics and glucose concentration represents a well-balanced setup that parallels physiological insulin release. PMID:25598507

  11. Induction of beta-cell resistance to hypoxia and technologies for oxygen delivery to transplanted pancreatic islets.

    PubMed

    Lazard, Daniel; Vardi, Pnina; Bloch, Konstantin

    2012-09-01

    Hypoxia is believed to be a crucial factor involved in cell adaptation to environmental stress. Islet transplantation, especially with immunoisolated islets, interrupts vascular connections, resulting in the substantially decreased delivery of oxygen and nutrients to islet cells. Insulin-producing pancreatic beta cells are known to be highly susceptible to oxygen deficiency. Such susceptibility to hypoxia is believed to be one of the main causes of beta-cell death in the post-transplantation period. Different strategies have been developed for the protection of beta cells against hypoxic injury and for oxygen delivery to transplanted islets. The enhancement of beta-cell defense properties against hypoxia has been achieved using various techniques such as gene transfection, drug supplementation, co-culturing with stem cells and cell selection. Technologies for oxygen delivery to transplanted islets include local neovascularization of subcutaneous sites, electrochemical and photosynthetic oxygen generation, oxygen refuelling of bio-artificial pancreas and whole body oxygenation by using hyperbaric therapy. Progress in the field of oxygen technologies for islet transplantation requires a multidisciplinary approach to explore and optimize the interaction between components of the biological system and different technological processes. This review article focuses mainly on the recently developed strategies for oxygenation and protection from hypoxic injury - to achieve stable and long-term normoglycaemia in diabetic patients with transplanted pancreatic islets. PMID:22389124

  12. Proinsulin maturation disorder is a contributor to the defect of subsequent conversion to insulin in {beta}-cells

    SciTech Connect

    Wang, Jie; Osei, Kwame

    2011-07-22

    Highlights: {yields} Primary proinsulin maturation disorder is inherent in Ins2{sup +/Akita} islets/{beta}-cells. {yields} A consequence is the inefficient conversion of proinsulin to insulin. {yields} Post-translational defects occur as well in the involved PC1/3 and PC2 convertases. {yields} Proinsulin maturation chaos results in defects in the following conversion process. {yields} A link of the proinsulin maturation disorder and hyperproinsulinemia is suggested. -- Abstract: Disproportionate hyperproinsulinemia is an indicator of {beta}-cell dysfunction in diabetes and the basis underlying this abnormality remains obscure. Recently, we have found proinsulin is an aggregation-prone molecule inherent with a low relative folding rate and maintains a homeostatic balance of natively and plentiful non-natively folded states (i.e., proinsulin homeostasis, PIHO) in normal {beta}-cells as a result of the integration of maturation and disposal processes. PIHO is susceptible to environmental and genetic influences. Perturbation of PIHO produces a number of toxic consequences with known association to {beta}-cell failure in diabetes. To explore whether the perturbation of PIHO has a link to disproportionate hyperproinsulinemia, we investigated proinsulin conversion and the involved prohormone convertase 1/3 (PC1/3) and 2 (PC2) in mouse Ins2{sup +/Akita} islets/{beta}-cells that preserve a primary PIHO disorder due to a mutation (C96Y) in the insulin 2 (Ins2) gene. Our metabolic-labeling studies found an increased ratio of proinsulin to insulin in the cellular or released proteins of Ins2{sup +/Akita} islets. Histological, metabolic-labeling, and RT-PCR analyses revealed decreases of the PC1/3 and PC2 immunoreactivities in the {beta}-cells of Ins2{sup +/Akita} islets in spite of no declines of these two convertases at the transcriptional and translational levels. Immunoblot analyses in cloned Ins2{sup +/Akita} {beta}-cells further confirmed the increased ratio of proinsulin

  13. Correlation of insulin resistance, beta cell function and insulin sensitivity with serum sFas and sFasL in newly diagnosed type 2 diabetes.

    PubMed

    Kumar, Hemant; Mishra, Manish; Bajpai, Surabhi; Pokhria, Deepa; Arya, Awadhesh Kumar; Singh, Rakesh Kumar; Tripathi, Kamlakar

    2013-08-01

    Pancreatic beta cell dysfunction and reduced insulin sensitivity are fundamental factors associated with glucotoxicity, lipotoxicity and oxidative stress in type 2 diabetic patients (T2DM). Diabetic milieu can induce apoptosis in several types of cells. The aim of present study was to compare circulating soluble apoptotic markers (sFas and sFas-L) with HOMA-IR, HOMA-%S, HOMA-%B in the serum of newly diagnosed T2DM and healthy subjects. For this study, 94 T2DM and 60 healthy subjects were enroled and evaluated for various parameters. Biochemical quantifications were performed with Syncron CX5 auto-analyzer. The levels of serum sFas-L, TNF-α and IL-6 were estimated by flowcytometry. The fasting serum insulin and sFas quantified by ELISA. HOMA-IR, HOMA-%S and HOMA-%B were calculated with HOMA calculator v2.2.2. The levels of TC, TG, LDL-C, VLDL-C were augmented and HDL declined significantly (P < 0.001) in diabetics. The levels of serum insulin, TNF-α, IL-6, sFas, HOMA-IR were raised (P < 0.001) and sFas-L, HOMA-%S and HOMA-%B were decreased significantly (P < 0.001) in T2DM subjects than healthy. In diabetics, serum sFas was positively correlated with HOMA-IR (r = 0.720, P < 0.001) and negatively with HOMA-%B (r = -0.642, P < 0.001) significantly while serum sFasL was negatively correlated with HOMA-IR (r = -0.483, P < 0.001) and positively with HOMA-%B (r = 0.466, P < 0.001) significantly. Further, the multivariate stepwise regression analysis shows that HOMA-IR contributes significantly to the variance of sFas and sFasL. Our findings suggest that the pancreatic beta cell dysfunction along with increased insulin resistance appears to be associated with apoptotic markers. PMID:21695404

  14. Impairment of Rat Fetal Beta-Cell Development by Maternal Exposure to Dexamethasone during Different Time-Windows

    PubMed Central

    Dumortier, Olivier; Theys, Nicolas; Ahn, Marie-Thérèse; Remacle, Claude; Reusens, Brigitte

    2011-01-01

    Aim Glucocorticoids (GCs) take part in the direct control of cell lineage during the late phase of pancreas development when endocrine and exocrine cell differentiation occurs. However, other tissues such as the vasculature exert a critical role before that phase. This study aims to investigate the consequences of overexposure to exogenous glucocorticoids during different time-windows of gestation for the development of the fetal endocrine pancreas. Methods Pregnant Wistar rats received dexamethasone acetate in their drinking water (1 µg/ml) during the last week or throughout gestation. Fetuses and their pancreases were analyzed at day 15 and 21 of gestation. Morphometrical analysis was performed on pancreatic sections after immunohistochemistry techniques and insulin secretion was evaluated on fetal islets collected in vitro. Results Dexamethasone given the last week or throughout gestation reduced the beta-cell mass in 21-day-old fetuses by respectively 18% or 62%. This was accompanied by a defect in insulin secretion. The alpha-cell mass was reduced similarly. Neither islet vascularization nor beta-cell proliferation was affected when dexamethasone was administered during the last week, which was however the case when given throughout gestation. When given from the beginning of gestation, dexamethasone reduced the number of cells expressing the early marker of endocrine lineage neurogenin-3 when analyzed at 15 days of fetal age. Conclusions GCs reduce the beta- and alpha-cell mass by different mechanisms according to the stage of development during which the treatment was applied. In fetuses exposed to glucocorticoids the last week of gestation only, beta-cell mass is reduced due to impairment of beta-cell commitment, whereas in fetuses exposed throughout gestation, islet vascularization and lower beta-cell proliferation are involved as well, amplifying the reduction of the endocrine mass. PMID:21991320

  15. Adult Human Pancreatic Islet Beta-Cells Display Limited Turnover and Long Lifespan as Determined by In-Vivo Thymidine Analog Incorporation and Radiocarbon Dating

    SciTech Connect

    Perl, S; Kushner, J A; Buchholz, B A; Meeker, A K; Stein, G M; Hsieh, M; Kirby, M; Pechhold, S; Liu, E H; Harlan, D M; Tisdale, J F

    2010-03-15

    Diabetes mellitus results from an absolute or relative deficiency of insulin producing pancreatic beta-cells. The adult human beta-cell's turnover rate remains unknown. We employed novel techniques to examine adult human islet beta-cell turnover and longevity in vivo. Subjects enrolled in NIH clinical trials received thymidine analogues [iododeoxyuridine (IdU) or bromodeoxyuridine (BrdU)] 8-days to 4-years prior to death. Archival autopsy samples from ten patients (aged 17-74 years) were employed to assess beta-cell turnover by scoring nuclear analog labeling within insulin staining cells. Human adult beta-cell longevity was determined by estimating the cells genomic DNA integration of atmospheric carbon-14 ({sup 14}C). DNA was purified from pancreatic islets isolated from cadaveric donors; whole islet prep DNA was obtained from a 15 year old donor, and purified beta-cell DNA was obtained from two donors (age 48 and 80 years). {sup 14}C levels were then determined using accelerator mass spectrometry (AMS). Cellular 'birth date' was determined by comparing the subject's DNA {sup 14}C content relative to a well-established {sup 14}C atmospheric prevalence curve. In the two subjects less than age 20 years, 1-2% of the beta-cell nuclei co-stained for BrdU/IdU. No beta-cell nuclei co-stained in the eight patients more than 30 years old. Consistent with the BrdU/IdU turnover data, beta-cell DNA {sup 14}C content indicated the cells 'birth date' occurred within the subject's first 30 years of life. Under typical circumstances, adult human beta-cells and their cellular precursors are established by young adulthood.

  16. Properties of single potassium channels modulated by glucose in rat pancreatic beta-cells.

    PubMed Central

    Ashcroft, F M; Ashcroft, S J; Harrison, D E

    1988-01-01

    1. The patch clamp method has been used to examine the effect of glucose on single K+ channel currents recorded from cell-attached patches on dissociated rat pancreatic beta-cells. Patch pipettes contained a 140 mM-K+ solution. 2. In glucose-free solution three types of K+ channels were observed. Two of these, having conductances of around 50 pS (G-channel) and 20 pS when the external K+ concentration, [K+]0, was 140 mM, were active at the resting potential of the cell. The G-channel was observed in more patches and showed higher activity; it therefore appears to contribute the major fraction of the resting K+ permeability of the beta-cell. At membrane potentials positive to about +20 mV a third type of K+ channel, having a mean conductance of 120 pS, was activated. The open probability of this channel was strongly voltage dependent and increased with depolarization. 3. The reversal potential of the G-channel current was shifted 59 mV by a 10-fold change in external K+ (Na+ substitution) indicating the channel is highly K+ selective. The single-channel conductance varied with [K+]o as predicted from the Goldman-Hodgkin-Katz equation; at physiological [K+]o (5 mM-K+) an inward conductance of around 10 pS is predicted. The amplitude of the single-channel current showed a tendency to saturate with increasing [K+]o. 4. Single G-channel currents show burst kinetics indicating at least two closed states. The open and closed (gap) times within the bursts were distributed exponentially with time constants of 2.5 ms (tau o) and 0.5 ms (tau c1) respectively at the resting potential of the cell. There was little change in tau c1 over the voltage range -40 to 60 mV (pipette potential) but tau o increased slightly with membrane depolarization. 5. The addition of glucose to the bath solution produced a reversible, dose-dependent decrease in G-channel activity. This decrease results principally from a reduction in the frequency and duration of the bursts of openings with

  17. MicroRNA-29a is up-regulated in beta-cells by glucose and decreases glucose-stimulated insulin secretion

    SciTech Connect

    Bagge, Annika; Clausen, Trine R.; Larsen, Sylvester; Ladefoged, Mette; Rosenstierne, Maiken W.; Larsen, Louise; Vang, Ole; Nielsen, Jens H.; Dalgaard, Louise T.

    2012-09-21

    Highlights: Black-Right-Pointing-Pointer MicroRNA-29a (miR-29a) levels are increased by glucose in human and rat islets and INS-1E cells. Black-Right-Pointing-Pointer miR-29a increases proliferation of INS-1E beta-cells. Black-Right-Pointing-Pointer Forced expression of miR-29a decreases glucose-stimulated insulin secretion (GSIS). Black-Right-Pointing-Pointer Depletion of beta-cell miR-29a improves GSIS. Black-Right-Pointing-Pointer miR-29a may be a mediator of glucose toxicity in beta-cells. -- Abstract: Chronically elevated levels of glucose impair pancreatic beta-cell function while inducing beta-cell proliferation. MicroRNA-29a (miR-29a) levels are increased in several tissues in diabetic animals and mediate decreased insulin-stimulated glucose-transport of adipocytes. The aim was to investigate the impact of glucose on miR-29a levels in INS-1E beta-cells and in human islets of Langerhans and furthermore to evaluate the impact of miR-29a on beta-cell function and proliferation. Increased glucose levels up-regulated miR-29a in beta-cells and human and rat islets of Langerhans. Glucose-stimulated insulin-secretion (GSIS) of INS-1E beta-cells was decreased by forced expression of miR-29a, while depletion of endogenous miR-29a improved GSIS. Over-expression of miR-29a increased INS-1E proliferation. Thus, miR-29a up-regulation is involved in glucose-induced proliferation of beta-cells. Furthermore, as depletion of miR-29a improves beta-cell function, miR-29a is a mediator of glucose-induced beta-cell dysfunction. Glucose-induced up-regulation of miR-29a in beta-cells could be implicated in progression from impaired glucose tolerance to type 2 diabetes.

  18. OSTEOCYTE APOPTOSIS

    PubMed Central

    Jilka, Robert L.; Noble, Brendon; Weinstein, Robert S.

    2012-01-01

    Apoptotic death of osteocytes was recognized over 15 years ago, but its significance for bone homeostasis has remained elusive. A new paradigm has emerged that invokes osteocyte apoptosis as a critical event in the recruitment of osteoclasts to a specific site in response to skeletal unloading, fatigue damage, estrogen deficiency and perhaps in other states where bone must be removed. This is accomplished by yet to be defined signals emanating from dying osteocytes, which stimulate neighboring viable osteocytes to produce osteoclastogenic cytokines. The osteocyte apoptosis caused by chronic glucocorticoid administration does not increase osteoclasts; however, it does negatively impact maintenance of bone hydration, vascularity, and strength. PMID:23238124

  19. Thyrotropin releasing hormone (TRH) affects gene expression in pancreatic beta-cells.

    PubMed

    Luo, LuGuang; Yano, Naohiro

    2005-01-01

    Thyrotropin-releasing hormone (TRH), originally identified as a hypothalamic hormone, is expressed in the pancreas. The peptide has been shown to control glycemia, although the role of TRH in the pancreas has not yet been clarified. In quiescent INS-1 cells (rat immortalized beta-cell line), 200 nM of TRH for 24 hours significantly increased insulin levels in the culture medium and in cell extracts. In studies with gene array technology where about 60% to 75% of the 1081 genes were detected, TRH significantly stimulated multiple groups of gene expressions, including G-protein-coupled receptor and related signaling, such as insulin secretion, endoplasmic reticulum traffic mechanisms, cell-cycle regulators, protein turnover factors, DNA recombination, and growth factors. Noticeably, TRH suppressed the genes of proapoptotic Bcl-2-associated protein X, Bcl-xL/ Bcl-2-associated death promoter, and Fas. The multiple gene expressions in response to TRH in pancreatic cells suggest that the changed microenvironment brought about by TRH may influence beta-cellfunction. PMID:16392621

  20. The Sherman-Rinzel-Keizer model for bursting electrical activity in the pancreatic. beta. -cell

    SciTech Connect

    Pernarowski, M.; Kevorkian, J. . Dept. of Applied Mathematics); Miura, R.M. )

    1990-03-01

    Pancreatic {beta}-cells exhibit periodic bursting electrical activity (BEA) consisting of active and silent phases. The Sherman-Rinzel-Keizer (SRK) model of this phenomenon consists of three coupled first-order nonlinear differential equations which describe the dynamics of the membrane potential, the activation parameter for the voltage-gated potassium channel, and the intracellular calcium concentration. These equations are nondimensionalized and transformed into a Lienard differential equation coupled to a single first-order differential equation for the slowly changing nondimensional calcium concentration. Leading-order perturbation problems are derived for the silent and active phases of the BEA on slow and fast time scales. Numerical solutions of these leading-order problems are compared with those for the exact equation in their respective regions. The leading-order solution in the active phase has a limit cycle behavior with a slowly varying frequency. It is observed that the damping term'' in the Lienard equation is small numerically. 26 refs., 6 figs., 2 tabs.

  1. Measuring phospholipase D activity in insulin-secreting pancreatic beta-cells and insulin-responsive muscle cells and adipocytes.

    PubMed

    Cazzolli, Rosanna; Huang, Ping; Teng, Shuzhi; Hughes, William E

    2009-01-01

    Phospholipase D (PLD) is an enzyme producing phosphatidic acid and choline through hydrolysis of phosphatidylcholine. The enzyme has been identified as a member of a variety of signal transduction cascades and as a key regulator of numerous intracellular vesicle trafficking processes. A role for PLD in regulating glucose homeostasis is emerging as the enzyme has recently been identified in events regulating exocytosis of insulin from pancreatic beta-cells and also in insulin-stimulated glucose uptake through controlling GLUT4 vesicle exocytosis in muscle and adipose tissue. We present methodologies for assessing cellular PLD activity in secretagogue-stimulated insulin-secreting pancreatic beta-cells and also insulin-stimulated adipocyte and muscle cells, two of the principal insulin-responsive cell types controlling blood glucose levels. PMID:19160674

  2. GeneSpeed Beta Cell: An Online Genomics Data Repository and Analysis Resource Tailored for the Islet Cell Biologist

    PubMed Central

    Quayum, Nayeem; Kutchma, Alecksandr; Sarkar, Suparna A.; Juhl, Kirstine; Gradwohl, Gerard; Mellitzer, Georg; Hutton, John C.; Jensen, Jan

    2008-01-01

    Objective. We here describe the development of a freely available online database resource, GeneSpeed Beta Cell, which has been created for the pancreatic islet and pancreatic developmental biology investigator community. Research Design and Methods. We have developed GeneSpeed Beta Cell as a separate component of the GeneSpeed database, providing a genomics-type data repository of pancreas and islet-relevant datasets interlinked with the domain-oriented GeneSpeed database. Results. GeneSpeed Beta Cell allows the query of multiple published and unpublished select genomics datasets in a simultaneous fashion (multiexperiment viewing) and is capable of defining intersection results from precomputed analysis of such datasets (multidimensional querying). Combined with the protein-domain categorization/assembly toolbox provided by the GeneSpeed database, the user is able to define spatial expression constraints of select gene lists in a relatively rigid fashion within the pancreatic expression space. We provide several demonstration case studies of relevance to islet cell biology and development of the pancreas that provide novel insight into islet biology. Conclusions. The combination of an exhaustive domain-based compilation of the transcriptome with gene array data of interest to the islet biologist affords novel methods for multidimensional querying between individual datasets in a rapid fashion, presently not available elsewhere. PMID:18795106

  3. Induced ICER I{gamma} down-regulates cyclin A expression and cell proliferation in insulin-producing {beta} cells

    SciTech Connect

    Inada, Akari; Weir, Gordon C.; Bonner-Weir, Susan . E-mail: susan.bonner-weir@joslin.harvard.edu

    2005-04-15

    We have previously found that cyclin A expression is markedly reduced in pancreatic {beta}-cells by cell-specific overexpression of repressor inducible cyclic AMP early repressor (ICER I{gamma}) in transgenic mice. Here we further examined regulatory effects of ICER I{gamma} on cyclin A gene expression using Min6 cells, an insulin-producing cell line. The cyclin A promoter luciferase assay showed that ICER I{gamma} directly repressed cyclin A gene transcription. In addition, upon ICER I{gamma} overexpression, cyclin A mRNA levels markedly decreased, thereby confirming an inhibitory effect of ICER I{gamma} on cyclin A expression. Suppression of cyclin A results in inhibition of BrdU incorporation. Under normal culture conditions endogenous cyclin A is abundant in these cells, whereas ICER is hardly detectable. However, serum starvation of Min6 cells induces ICER I{gamma} expression with a concomitant very low expression level of cyclin A. Cyclin A protein is not expressed unless the cells are in active DNA replication. These results indicate a potentially important anti-proliferative effect of ICER I{gamma} in pancreatic {beta} cells. Since ICER I{gamma} is greatly increased in diabetes as well as in FFA- or high glucose-treated islets, this effect may in part exacerbate diabetes by limiting {beta}-cell proliferation.

  4. Usurping the mitochondrial supremacy: extramitochondrial sources of reactive oxygen intermediates and their role in beta cell metabolism and insulin secretion.

    PubMed

    Gray, Joshua P; Heart, Emma

    2010-05-01

    Insulin secretion from pancreatic beta cells is a process dependent on metabolism. While oxidative stress is a well-known inducer of beta cell toxicity and impairs insulin secretion, recent studies suggest that low levels of metabolically-derived reactive oxygen intermediates (ROI) also play a positive role in insulin secretion. Glucose metabolism is directly correlated with ROI production, particularly in beta cells in which glucose uptake is proportional to the extracellular concentration of glucose. Low levels of exogenously added ROI or quinones, which stimulate ROI production, positively affect insulin secretion, while antioxidants block insulin secretion, suggesting that ROI activate unidentified redox-sensitive signal transduction components within these cells. The mitochondria are one source of ROI: increased metabolic flux increases mitochondrial membrane potential resulting in electron leakage and adventitious ROI production. A second source of ROI are cytosolic and plasma membrane oxidoreductases which oxidize NAD(P)H and directly produce ROI through the reduction of molecular oxygen. The mechanism of ROI-mediated potentiation of insulin secretion remains an important topic for future study. PMID:20397883

  5. Glucagon-Like Peptide-1 Triggers Protective Pathways in Pancreatic Beta-Cells Exposed to Glycated Serum

    PubMed Central

    Puddu, Alessandra; Sanguineti, Roberta; Durante, Arianna; Nencioni, Alessio; Mach, François; Montecucco, Fabrizio; Viviani, Giorgio L.

    2013-01-01

    Advanced glycation end products (AGEs) might play a pathophysiological role in the development of diabetes and its complications. AGEs negatively affect pancreatic beta-cell function and the expression of transcriptional factors regulating insulin gene. Glucagon-like peptide-1 (GLP-1), an incretin hormone that regulates glucose homeostasis, might counteract the harmful effects of AGEs on the beta cells in culture. The aim of this study was to identify the intracellular mechanisms underlying GLP-1-mediated protection from AGE-induced detrimental activities in pancreatic beta cells. HIT-T15 cells were cultured for 5 days with glycated serum (GS, consisting in a pool of AGEs), in the presence or absence of 10 nmol/L GLP-1. After evaluation of oxidative stress, we determined the expression and subcellular localization of proteins involved in maintaining redox balance and insulin gene expression, such as nuclear factor erythroid-derived 2 (Nrf2), glutathione reductase, PDX-1, and MafA. Then, we investigated proinsulin production. The results showed that GS increased oxidative stress, reduced protein expression of all investigated factors through proteasome activation, and decreased proinsulin content. Furthermore, GS reduced ability of PDX-1 and MafA to bind DNA. Coincubation with GLP-1 reversed these GS-mediated detrimental effects. In conclusion, GLP-1, protecting cells against oxidants, triggers protective intercellular pathways in HIT-T15 cells exposed to GS. PMID:23737644

  6. Inter-domain tagging implicates caveolin-1 in insulin receptor trafficking and Erk signaling bias in pancreatic beta-cells

    PubMed Central

    Boothe, Tobias; Lim, Gareth E.; Cen, Haoning; Skovsø, Søs; Piske, Micah; Li, Shu Nan; Nabi, Ivan R.; Gilon, Patrick; Johnson, James D.

    2016-01-01

    Objective The role and mechanisms of insulin receptor internalization remain incompletely understood. Previous trafficking studies of insulin receptors involved fluorescent protein tagging at their termini, manipulations that may be expected to result in dysfunctional receptors. Our objective was to determine the trafficking route and molecular mechanisms of functional tagged insulin receptors and endogenous insulin receptors in pancreatic beta-cells. Methods We generated functional insulin receptors tagged with pH-resistant fluorescent proteins between domains. Confocal, TIRF and STED imaging revealed a trafficking pattern of inter-domain tagged insulin receptors and endogenous insulin receptors detected with antibodies. Results Surprisingly, interdomain-tagged and endogenous insulin receptors in beta-cells bypassed classical Rab5a- or Rab7-mediated endocytic routes. Instead, we found that removal of insulin receptors from the plasma membrane involved tyrosine-phosphorylated caveolin-1, prior to trafficking within flotillin-1-positive structures to lysosomes. Multiple methods of inhibiting caveolin-1 significantly reduced Erk activation in vitro or in vivo, while leaving Akt signaling mostly intact. Conclusions We conclude that phosphorylated caveolin-1 plays a role in insulin receptor internalization towards lysosomes through flotillin-1-positive structures and that caveolin-1 helps bias physiological beta-cell insulin signaling towards Erk activation. PMID:27110488

  7. Incretin Receptor Null Mice Reveal Key Role of GLP-1 but Not GIP in Pancreatic Beta Cell Adaptation to Pregnancy

    PubMed Central

    Moffett, R. Charlotte; Vasu, Srividya; Thorens, Bernard; Drucker, Daniel J.; Flatt, Peter R.

    2014-01-01

    Islet adaptations to pregnancy were explored in C57BL6/J mice lacking functional receptors for glucagon-like peptide 1 (GLP-1) and gastric inhibitory polypeptide (GIP). Pregnant wild type mice and GIPRKO mice exhibited marked increases in islet and beta cell area, numbers of medium/large sized islets, with positive effects on Ki67/Tunel ratio favouring beta cell growth and enhanced pancreatic insulin content. Alpha cell area and glucagon content were unchanged but prohormone convertases PC2 and PC1/3 together with significant amounts of GLP-1 and GIP were detected in alpha cells. Knockout of GLP-1R abolished these islet adaptations and paradoxically decreased pancreatic insulin, GLP-1 and GIP. This was associated with abolition of normal pregnancy-induced increases in plasma GIP, L-cell numbers, and intestinal GIP and GLP-1 stores. These data indicate that GLP-1 but not GIP is a key mediator of beta cell mass expansion and related adaptations in pregnancy, triggered in part by generation of intra-islet GLP-1. PMID:24927416

  8. Furosemide reduces insulin release by inhibition of Cl sup minus and Ca sup 2+ fluxes in. beta. -cells

    SciTech Connect

    Sandstroem, P.E.; Sehlin, J. )

    1988-11-01

    The effect of furosemide on insulin release, glucose oxidation, {sup 36}Cl{sup {minus}} fluxes, and {sup 45}Ca{sup 2+} uptake was studied in isolated, {beta}-cell-rich pancreatic islets from ob/ob mice. Low concentrations of furosemide (0.01-0.1 mM) reduced the glucose-induced insulin release, whereas high doses (1-10 mM) increased basal and glucose-induced release. Furosemide at concentrations that reduced glucose-induced insulin release did not affect the islet production of {sup 14}CO{sub 2} from D-(U-{sup 14}C)glucose. The influx rate and equilibrium content of {sup 36}Cl{sup {minus}} were reduced by furosemide, whereas the basal and glucose-stimulated {sup 36}Cl{sup {minus}} efflux rates were unaffected. The glucose-induced uptake of {sup 45}Ca{sup 2+} was inhibited by furosemide. It is suggested that the diabetogenic action of furosemide may be due, at least in part, to direct inhibition of insulin release from the pancreatic {beta}-cells. This may be caused primarily by inhibition of an inwardly directed Cl{sup {minus}} pump, leading to a reduced transmembrane electrochemical gradient for chloride in the {beta}-cells. This reduced gradient in combination with unaltered Cl{sup {minus}} permeability may lead to decreased total outward Cl{sup {minus}} transport, a factor associated with stimulated calcium uptake and insulin release.

  9. Canine Fibroblast Growth Factor 21 Ameliorates Hyperglycemia Associated with Inhibiting Hepatic Gluconeogenesis and Improving Pancreatic Beta-Cell Survival in Diabetic Mice and Dogs

    PubMed Central

    Xu, Pengfei; Zhang, Yingjie; Jiang, Xinghao; Li, Junyan; Song, Liying; Khoso, Mir Hasson; Liu, Yunye; Wu, Qiang; Ren, Guiping; Li, Deshan

    2016-01-01

    Diabetes mellitus is a common endocrinopathy in dog. Fibroblast growth factor 21 (FGF-21) is a secreted protein, which is involved in glucose homeostasis. We speculate that the recombinant canine FGF-21 (cFGF-21) has the potential to become a powerful therapeutics to treat canine diabetes. The cFGF-21 gene was cloned and expressed in E. coli Rosetta (DE3). After purification, a cFGF-21 protein with the purity exceeding 95% was obtained. Mouse 3T3-L1 adipocytes and type 1 diabetic mice/dogs induced by STZ were used to examine the biological activity of cFGF-21 in vitro and in vivo, respectively. Results showed that cFGF-21 stimulated glucose uptake in adipocytes significantly in a dose-dependent manner, and reduced plasma glucose significantly in diabetic mice/dogs. After treatment with cFGF-21, the serum insulin level, glycosylated hemoglobin (HbA1c) level and the expressions of the hepatic gluconeogenesis genes (glucose-6-phosphatase, G6Pase and phosphoenolpyruvate carboxykinase, PCK) of the diabetic mice/dogs were attenuated significantly. In the mouse experiment, we also found that the phosphorylation of signal transducer and activator of transcription 3 (STAT3) and the expression of suppressor of cytokine signaling 3 (SOCS3) were up-regulated significantly in the livers after treatment. Histopathological and immunohistochemical results showed that treatment with cFGF-21 promoted recovery of pancreatic islets from STZ-induced apoptosis. Besides, we also found that treatment with cFGF-21 protected liver against STZ or hyperglycemia induced damage and the mechanism of this action associated with inhibiting oxidative stress. In conclusion, cFGF-21 represents a promising candidate for canine diabetes therapeutics. The mechanism of cFGF-21 ameliorates hyperglycemia associated with inhibiting hepatic gluconeogenesis by regulation of STAT3 signal pathway and improving pancreatic beta-cell survival. PMID:27203422

  10. Canine Fibroblast Growth Factor 21 Ameliorates Hyperglycemia Associated with Inhibiting Hepatic Gluconeogenesis and Improving Pancreatic Beta-Cell Survival in Diabetic Mice and Dogs.

    PubMed

    Xu, Pengfei; Zhang, Yingjie; Jiang, Xinghao; Li, Junyan; Song, Liying; Khoso, Mir Hasson; Liu, Yunye; Wu, Qiang; Ren, Guiping; Li, Deshan

    2016-01-01

    Diabetes mellitus is a common endocrinopathy in dog. Fibroblast growth factor 21 (FGF-21) is a secreted protein, which is involved in glucose homeostasis. We speculate that the recombinant canine FGF-21 (cFGF-21) has the potential to become a powerful therapeutics to treat canine diabetes. The cFGF-21 gene was cloned and expressed in E. coli Rosetta (DE3). After purification, a cFGF-21 protein with the purity exceeding 95% was obtained. Mouse 3T3-L1 adipocytes and type 1 diabetic mice/dogs induced by STZ were used to examine the biological activity of cFGF-21 in vitro and in vivo, respectively. Results showed that cFGF-21 stimulated glucose uptake in adipocytes significantly in a dose-dependent manner, and reduced plasma glucose significantly in diabetic mice/dogs. After treatment with cFGF-21, the serum insulin level, glycosylated hemoglobin (HbA1c) level and the expressions of the hepatic gluconeogenesis genes (glucose-6-phosphatase, G6Pase and phosphoenolpyruvate carboxykinase, PCK) of the diabetic mice/dogs were attenuated significantly. In the mouse experiment, we also found that the phosphorylation of signal transducer and activator of transcription 3 (STAT3) and the expression of suppressor of cytokine signaling 3 (SOCS3) were up-regulated significantly in the livers after treatment. Histopathological and immunohistochemical results showed that treatment with cFGF-21 promoted recovery of pancreatic islets from STZ-induced apoptosis. Besides, we also found that treatment with cFGF-21 protected liver against STZ or hyperglycemia induced damage and the mechanism of this action associated with inhibiting oxidative stress. In conclusion, cFGF-21 represents a promising candidate for canine diabetes therapeutics. The mechanism of cFGF-21 ameliorates hyperglycemia associated with inhibiting hepatic gluconeogenesis by regulation of STAT3 signal pathway and improving pancreatic beta-cell survival. PMID:27203422

  11. Hormetic and regulatory effects of lipid peroxidation mediators in pancreatic beta cells.

    PubMed

    Maulucci, Giuseppe; Daniel, Bareket; Cohen, Ofir; Avrahami, Yossef; Sasson, Shlomo

    2016-06-01

    proliferator-activated receptor δ (PPARδ) in vascular endothelial cells and insulin secreting beta cells, promote such adaptive responses to ameliorate detrimental effects of high glucose and diabetes-like conditions. In addition, due to the electrophilic nature of these reactive aldehydes they form covalent adducts with electronegative moieties in proteins, phosphatidylethanolamine and nucleotides. Normally these non-enzymatic modifications are maintained below the cytotoxic range due to efficient cellular neutralization processes of 4-hydroxyalkenals. The major neutralizing enzymes include fatty aldehyde dehydrogenase (FALDH), aldose reductase (AR) and alcohol dehydrogenase (ADH), which transform the aldehyde to the corresponding carboxylic acid or alcohols, respectively, or by biding to the thiol group in glutathione (GSH) by the action of glutathione-S-transferase (GST). This review describes the hormetic and cytotoxic roles of oxygen free radicals and 4-hydroxyalkenals in beta cells exposed to nutritional challenges and the cellular mechanisms they employ to maintain their level at functional range below the cytotoxic threshold. PMID:27012748

  12. The Glucotoxicity Protecting Effect of Ezetimibe in Pancreatic Beta Cells via Inhibition of CD36

    PubMed Central

    2016-01-01

    Inhibition of CD36, a fatty acid transporter, has been reported to prevent glucotoxicity and ameliorate high glucose induced beta cell dysfunction. Ezetimibe is a selective cholesterol absorption inhibitor that blocks Niemann Pick C1-like 1 protein, but may exert its effect through suppression of CD36. We attempted to clarify the beneficial effect of ezetimibe on insulin secreting cells and to determine whether this effect is related to change of CD36 expression. mRNA expression of insulin and CD36, intracellular peroxide level and glucose stimulated insulin secretion (GSIS) under normal (5.6 mM) or high glucose (30 mM) condition in INS-1 cells and primary rat islet cells were compared. Changes of the aforementioned factors with treatment with ezetimibe (20 μM) under normal or high glucose condition were also assessed. mRNA expression of insulin was decreased with high glucose, which was reversed by ezetimibe in both INS-1 cells and primary rat islets. CD36 mRNA expression was increased with high glucose, but decreased by ezetimibe in INS-1 cells and primary rat islets. Three-day treatment with high glucose resulted in an increase in intracellular peroxide level; however, it was decreased by treatment with ezetimibe. Decrease in GSIS by three-day treatment with high glucose was reversed by ezetimibe. Palmitate uptake following exposure to high glucose conditions for three days was significantly elevated, which was reversed by ezetimibe in INS-1 cells. Ezetimibe may prevent glucotoxicity in pancreatic β-cells through a decrease in fatty acid influx via inhibition of CD36. PMID:27051238

  13. The Glucotoxicity Protecting Effect of Ezetimibe in Pancreatic Beta Cells via Inhibition of CD36.

    PubMed

    Yoon, Ji Sung; Moon, Jun Sung; Kim, Yong-Woon; Won, Kyu Chang; Lee, Hyoung Woo

    2016-04-01

    Inhibition of CD36, a fatty acid transporter, has been reported to prevent glucotoxicity and ameliorate high glucose induced beta cell dysfunction. Ezetimibe is a selective cholesterol absorption inhibitor that blocks Niemann Pick C1-like 1 protein, but may exert its effect through suppression of CD36. We attempted to clarify the beneficial effect of ezetimibe on insulin secreting cells and to determine whether this effect is related to change of CD36 expression. mRNA expression of insulin and CD36, intracellular peroxide level and glucose stimulated insulin secretion (GSIS) under normal (5.6 mM) or high glucose (30 mM) condition in INS-1 cells and primary rat islet cells were compared. Changes of the aforementioned factors with treatment with ezetimibe (20 μM) under normal or high glucose condition were also assessed. mRNA expression of insulin was decreased with high glucose, which was reversed by ezetimibe in both INS-1 cells and primary rat islets. CD36 mRNA expression was increased with high glucose, but decreased by ezetimibe in INS-1 cells and primary rat islets. Three-day treatment with high glucose resulted in an increase in intracellular peroxide level; however, it was decreased by treatment with ezetimibe. Decrease in GSIS by three-day treatment with high glucose was reversed by ezetimibe. Palmitate uptake following exposure to high glucose conditions for three days was significantly elevated, which was reversed by ezetimibe in INS-1 cells. Ezetimibe may prevent glucotoxicity in pancreatic β-cells through a decrease in fatty acid influx via inhibition of CD36. PMID:27051238

  14. Nicotinamide-functionalized multiwalled carbon nanotubes increase insulin production in pancreatic beta cells via MIF pathway

    PubMed Central

    Ilie, Ioana; Ilie, Razvan; Mocan, Teodora; Tabaran, Flaviu; Iancu, Cornel; Mocan, Lucian

    2013-01-01

    Recent data in the literature support the role of nicotinamide (NA) as a pharmacologic agent that stimulates pancreatic beta-cells to produce insulin in vitro. There are data showing that carbon nanotubes may be useful in initiating and maintaining cellular metabolic responses. This study shows that administration of multiwalled carbon nanotubes (MWCNTs) functionalized with nicotinamide (NA-MWCNTs) leads to significant insulin production compared with individual administration of NA, MWCNTs, and a control solution. Treatment of 1.4E7 cells for 30 minutes with NA-MWCNTs at concentrations ranging from 1 mg/L to 20 mg/L resulted in significantly increased insulin release (0.18 ± 0.026 ng/mL for 1 mg/L, 0.21 ± 0.024 ng/mL for 5 mg/L, and 0.27 ± 0.028 ng/mL for 20 mg/L). Thus, compared with cells treated with NA only (0.1 ± 0.01 ng/mL for 1 mg/L, 0.12 ± 0.017 ng/mL for 5 mg/L, and 0.17 ± 0.01 ng/mL for 20 mg/L) we observed a significant positive effect on insulin release in cells treated with NA-MWCNTs. The results were confirmed using flow cytometry, epifluorescence microscopy combined with immunochemistry staining, and enzyme-linked immunosorbent assay techniques. In addition, using immunofluorescence microscopy techniques, we were able to demonstrate that MWCNTs enhance insulin production via the macrophage migration inhibitory factor pathway. The application and potential of NA combined with MWCNTs as an antidiabetic agent may represent the beginning of a new chapter in the nanomediated treatment of diabetes mellitus. PMID:24039418

  15. Distinct glucose lowering and beta cell protective effects of vanadium and food restriction in streptozotocin-diabetes.

    PubMed

    Cam, M C; Rodrigues, B; McNeill, J H

    1999-11-01

    Vanadium is an oral insulin-mimetic agent that diminishes hyperglycemia, improves beta-cell insulin store and secretory function, and can reverse the diabetic state chronically after withdrawal from treatment. As food restriction has been reported to enhance insulin sensitivity and reduce insulin demand, we assessed the contribution of a reduced food intake to the glucose lowering and beta-cell protective effects of vanadium. Streptozotocin (STZ)-diabetic rats were untreated (D) or administered vanadyl sulfate in the drinking water (DT) at one week prior to and for 5 weeks following the administration of STZ. An additional group was pair-fed (DP) with an equal amount of food as that consumed by the DT group. Shortly after the induction of diabetes, hyperglycemic D rats demonstrated a significant rise in plasma insulin to levels that initially exceeded that of the controls. This was followed by a steady reduction over several weeks, suggesting a gradual depletion of functional beta-cells. Both vanadium treatment and pair-feeding abolished the insulin hypersecretory response following STZ administration. Glucose lowering was enhanced in DT animals when administered higher concentrations of vanadium, despite no further reduction in food intake, and all DT animals (10/10) were normoglycemic by 5 weeks. Mean pancreatic insulin content in DT rats was improved fourfold and was associated with a greater number of granulated beta-cells. Conversely, food restriction only modestly improved glycemia and the pancreatic insulin store and, unlike DT, DP rats remained highly glucose-intolerant. At 5 weeks of diabetes, fed circulating glucose and insulin levels were strongly correlated (P=0.0002) in the D and DP groups, supporting the notion that glucose lowering with food restriction is dependent on improved plasma insulin levels. A separate correlation was observed in DT animals within a lower range of plasma insulin, suggesting that vanadium, unlike food restriction, reduced

  16. Increased secretion of insulin and proliferation of islet {beta}-cells in rats with mesenteric lymph duct ligation

    SciTech Connect

    Nagino, Ko; Yokozawa, Junji; Sasaki, Yu; Matsuda, Akiko; Takeda, Hiroaki; Kawata, Sumio

    2012-08-24

    Highlights: Black-Right-Pointing-Pointer Insulin secretion was increased during the OGTT or IVGTT in mesenteric lymph duct-ligated rats. Black-Right-Pointing-Pointer Proliferation of islet {beta}-cells was upregulated in lymph duct-ligated rats. Black-Right-Pointing-Pointer Mesenteric lymph duct flow has a role in glucose metabolism. -- Abstract: Background and aims: It has been suggested that intestinal lymph flow plays an important role in insulin secretion and glucose metabolism after meals. In this study, we investigated the influence of ligation of the mesenteric lymph duct on glucose metabolism and islet {beta}-cells in rats. Methods: Male Sprague-Dawley rats (10 weeks old) were divided into two groups: one underwent ligation of the mesenteric lymph duct above the cistern (ligation group), and the other underwent a sham operation (sham group). After 1 and 2 weeks, fasting plasma concentrations of glucose, insulin, triglyceride, glucose-dependent insulinotropic polypeptide (GIP), and the active form of glucagon-like peptide-1 (GLP-1) were measured. At 2 weeks after the operation, the oral glucose tolerance test (OGTT) and intravenous glucose tolerance test (IVGTT) were performed. After the rats had been sacrificed, the insulin content of the pancreas was measured and the proliferation of {beta}-cells was assessed immunohistochemically using antibodies against insulin and Ki-67. Results: During the OGTT, the ligation group showed a significant decrease in the plasma glucose concentration at 120 min (p < 0.05) and a significant increase in the plasma insulin concentration by more than 2-fold at 15 min (p < 0.01). On the other hand, the plasma GIP concentration was significantly decreased at 60 min (p < 0.01) in the ligated group, while the active form of GLP-1 showed a significantly higher level at 90 min (1.7-fold; p < 0.05) and 120 min (2.5-fold; p < 0.01). During the IVGTT, the plasma insulin concentration in the ligation group was significantly higher at 2

  17. Evidence that down-regulation of. beta. -cell glucose transporters in non-insulin-dependent diabetes may be the cause of diabetic hyperglycemia

    SciTech Connect

    Orci, L.; Ravazzola, M.; Baetens, D.; Amherdt, M. ); Inman, L.; Johnson, J.H.; Unger, R.H. Dept. of Veterans Affairs Medical Center, Dallas, TX ); Peterson, R.G. ); Newgard, C.B. )

    1990-12-01

    Non-insulin-dependent diabetes mellitus (NIDDM) is attributed to a failure of pancreatic {beta} cells to maintain insulin secretion at a level sufficient to compensate for underlying insulin resistance. In the ZDF rat, a model of NIDDM that closely resembles the human syndrome, the authors have previously reported profound underexpression of GLUT-2, the high-K{sub m} facilitative glucose transporter expressed by {beta} cells of normal animals. Here they report that islets of diabetic rats exhibit a marked decrease in the volume density of GLUT-2-positive {beta} cells and a reduction at the electron-microscopic level in the number of GLUT-2-immunoreactive sites per unit of {beta}-cell plasma membrane. The deficiency of GLUT-2 cannot be induced in normal {beta} cells by in vivo or in vitro exposure to high levels of glucose nor can it be prevented in {beta} cells of prediabetic ZDF rats by elimination of hyperglycemia. They conclude that this dearth of immunodetectable GLUT-2 in NIDDM is not secondary to hyperglycemia and therefore that it may well play a causal role in the development of hyperglycemia.

  18. Neogenesis and proliferation of {beta}-cells induced by human betacellulin gene transduction via retrograde pancreatic duct injection of an adenovirus vector

    SciTech Connect

    Tokui, Yae . E-mail: ytokui@imed2.med.osaka-u.ac.jp; Kozawa, Junji; Yamagata, Kazuya; Zhang, Jun; Ohmoto, Hiroshi; Tochino, Yoshihiro; Okita, Kohei; Iwahashi, Hiromi; Namba, Mitsuyoshi; Shimomura, Iichiro; Miyagawa, Jun-ichiro |

    2006-12-01

    Betacellulin (BTC) has been shown to have a role in the differentiation and proliferation of {beta}-cells both in vitro and in vivo. We administered a human betacellulin (hBTC) adenovirus vector to male ICR mice via retrograde pancreatic duct injection. As a control, we administered a {beta}-galactosidase adenovirus vector. In the mice, hBTC protein was mainly overexpressed by pancreatic duct cells. On immunohistochemical analysis, we observed features of {beta}-cell neogenesis as newly formed insulin-positive cells in the duct cell lining or islet-like cell clusters (ICCs) closely associated with the ducts. The BrdU labeling index of {beta}-cells was also increased by the betacellulin vector compared with that of control mice. These results indicate that hBTC gene transduction into adult pancreatic duct cells promoted {beta}-cell differentiation (mainly from duct cells) and proliferation of pre-existing {beta}-cells, resulting in an increase of the {beta}-cell mass that improved glucose tolerance in diabetic mice.

  19. Proteomic analysis of conditioned media from glucose responsive and glucose non-responsive phenotypes reveals a panel of secreted proteins associated with beta cell dysfunction.

    PubMed

    Dowling, Paul; Shields, William; Rani, Sweta; Meleady, Paula; Henry, Michael; Jeppesen, Per; O'Driscoll, Lorraine; Clynes, Martin

    2008-11-01

    Media conditioned by dysfunctioning pancreatic beta cells offer an excellent source of potential protein markers associated with this phenotype. Proteins identified from cell culture model systems are often found to be of importance clinically. Previous work by us and others have shown that low-passage MIN-6 cells (MIN-6(L)) respond to changes in glucose concentrations, producing an approximately 5.5-fold glucose-stimulated insulin secretion (GSIS) in response to 26.7 mmol/L, compared with 3.3 mmol/L, glucose. After continuous culture or high-passage (MIN-(H)), this GSIS was no longer present and thus represents an excellent model system for investigating beta cell dysfunction. Employing 2-D difference gel electrophoresis and mass spectrometry a panel of protein markers were identified in conditioned media (CM) from MIN-6(L) and MIN-6(H) beta cells. These proteins, including secretogranin II, secretogranin III and transthyretin, are associated with secretory granule biogenesis and were found to have substantially increased levels in the CM from the non-responsive high-passage MIN-6 beta cells. A panel of protein markers found to have increased abundance levels in CM from MIN-6(H) compared with MIN-6(L) beta cells may have the potential to be used clinically for assessing beta cell function and to monitor the effects of specific therapeutics. PMID:18924105

  20. Drp1 guarding of the mitochondrial network is important for glucose-stimulated insulin secretion in pancreatic beta cells.

    PubMed

    Reinhardt, Florian; Schultz, Julia; Waterstradt, Rica; Baltrusch, Simone

    2016-06-10

    Mitochondria form a tubular network in mammalian cells, and the mitochondrial life cycle is determined by fission, fusion and autophagy. Dynamin-related protein 1 (Drp1) has a pivotal role in these processes because it alone is able to constrict mitochondria. However, the regulation and function of Drp1 have been shown to vary between cell types. Mitochondrial morphology affects mitochondrial metabolism and function. In pancreatic beta cells mitochondrial metabolism is a key component of the glucose-induced cascade of insulin secretion. The goal of the present study was to investigate the action of Drp1 in pancreatic beta cells. For this purpose Drp1 was down-regulated by means of shDrp1 in insulin-secreting INS1 cells and mouse pancreatic islets. In INS1 cells reduced Drp1 expression resulted in diminished expression of proteins regulating mitochondrial fusion, namely mitofusin 1 and 2, and optic atrophy protein 1. Diminished mitochondrial dynamics can therefore be assumed. After down-regulation of Drp1 in INS1 cells and spread mouse islets the initially homogenous mitochondrial network characterised by a moderate level of interconnections shifted towards high heterogeneity with elongated, clustered and looped mitochondria. These morphological changes were found to correlate directly with functional alterations. Mitochondrial membrane potential and ATP generation were significantly reduced in INS1 cells after Drp1down-regulation. Finally, a significant loss of glucose-stimulated insulin secretion was demonstrated in INS1 cells and mouse pancreatic islets. In conclusion, Drp1 expression is important in pancreatic beta cells to maintain the regulation of insulin secretion. PMID:27154223

  1. A Novel GLP1 Receptor Interacting Protein ATP6ap2 Regulates Insulin Secretion in Pancreatic Beta Cells.

    PubMed

    Dai, Feihan F; Bhattacharjee, Alpana; Liu, Ying; Batchuluun, Battsetseg; Zhang, Ming; Wang, Xinye Serena; Huang, Xinyi; Luu, Lemieux; Zhu, Dan; Gaisano, Herbert; Wheeler, Michael B

    2015-10-01

    GLP1 activates its receptor, GLP1R, to enhance insulin secretion. The activation and transduction of GLP1R requires complex interactions with a host of accessory proteins, most of which remain largely unknown. In this study, we used membrane-based split ubiquitin yeast two-hybrid assays to identify novel GLP1R interactors in both mouse and human islets. Among these, ATP6ap2 (ATPase H(+)-transporting lysosomal accessory protein 2) was identified in both mouse and human islet screens. ATP6ap2 was shown to be abundant in islets including both alpha and beta cells. When GLP1R and ATP6ap2 were co-expressed in beta cells, GLP1R was shown to directly interact with ATP6ap2, as assessed by co-immunoprecipitation. In INS-1 cells, overexpression of ATP6ap2 did not affect insulin secretion; however, siRNA knockdown decreased both glucose-stimulated and GLP1-induced insulin secretion. Decreases in GLP1-induced insulin secretion were accompanied by attenuated GLP1 stimulated cAMP accumulation. Because ATP6ap2 is a subunit required for V-ATPase assembly of insulin granules, it has been reported to be involved in granule acidification. In accordance with this, we observed impaired insulin granule acidification upon ATP6ap2 knockdown but paradoxically increased proinsulin secretion. Importantly, as a GLP1R interactor, ATP6ap2 was required for GLP1-induced Ca(2+) influx, in part explaining decreased insulin secretion in ATP6ap2 knockdown cells. Taken together, our findings identify a group of proteins that interact with the GLP1R. We further show that one interactor, ATP6ap2, plays a novel dual role in beta cells, modulating both GLP1R signaling and insulin processing to affect insulin secretion. PMID:26272612

  2. The reprogrammed pancreatic progenitor-like intermediate state of hepatic cells is more susceptible to pancreatic beta cell differentiation.

    PubMed

    Wang, Qiwei; Wang, Hai; Sun, Yu; Li, Shi-Wu; Donelan, William; Chang, Lung-Ji; Jin, Shouguang; Terada, Naohiro; Cheng, Henrique; Reeves, Westley H; Yang, Li-Jun

    2013-08-15

    Induced pluripotent stem cells (iPSCs) hold great promise for cell therapy. However, their low efficiency of lineage-specific differentiation and tumorigenesis severely hinder clinical translation. We hypothesized that reprogramming of somatic cells into lineage-specific progenitor cells might allow for large-scale expansion, avoiding the tumorigenesis inherent with iPSCs and simultaneously facilitating lineage-specific differentiation. Here we aimed at reprogramming rat hepatic WB cells, using four Yamanaka factors, into pancreatic progenitor cells (PPCs) or intermediate (IM) cells that have characteristics of PPCs. IM clones were selected based on their specific morphology and alkaline phosphatase activity and stably passaged under defined culture conditions. IM cells did not have iPSC properties, could be stably expanded in large quantity, and expressed all 14 genes that are used to define the PPC developmental stage. Directed differentiation of IM and WB cells by Pdx1-Ngn3-MafA (PNM) into pancreatic beta-like cells revealed that the IM cells are more susceptible to directed beta cell differentiation because of their open chromatin configuration, as demonstrated by expression of key pancreatic beta cell genes, secretion of insulin in response to glucose stimulation, and easy access to exogenous PNM proteins at the rat insulin 1 and Pdx1 promoters. This notion that IM cells are superior to their parental cells is further supported by the epigenetic demonstration of accessibility of Pdx1 and insulin 1 promoters. In conclusion, we have developed a strategy to derive and expand PPC cells from hepatic WB cells using conventional cell reprogramming. This proof-of-principal study may offer a novel, safe and effective way to generate autologous pancreatic beta cells for cell therapy of diabetes. PMID:23750005

  3. Membrane Potential and Calcium Dynamics in Beta Cells from Mouse Pancreas Tissue Slices: Theory, Experimentation, and Analysis.

    PubMed

    Dolenšek, Jurij; Špelič, Denis; Klemen, Maša Skelin; Žalik, Borut; Gosak, Marko; Rupnik, Marjan Slak; Stožer, Andraž

    2015-01-01

    Beta cells in the pancreatic islets of Langerhans are precise biological sensors for glucose and play a central role in balancing the organism between catabolic and anabolic needs. A hallmark of the beta cell response to glucose are oscillatory changes of membrane potential that are tightly coupled with oscillatory changes in intracellular calcium concentration which, in turn, elicit oscillations of insulin secretion. Both membrane potential and calcium changes spread from one beta cell to the other in a wave-like manner. In order to assess the properties of the abovementioned responses to physiological and pathological stimuli, the main challenge remains how to effectively measure membrane potential and calcium changes at the same time with high spatial and temporal resolution, and also in as many cells as possible. To date, the most wide-spread approach has employed the electrophysiological patch-clamp method to monitor membrane potential changes. Inherently, this technique has many advantages, such as a direct contact with the cell and a high temporal resolution. However, it allows one to assess information from a single cell only. In some instances, this technique has been used in conjunction with CCD camera-based imaging, offering the opportunity to simultaneously monitor membrane potential and calcium changes, but not in the same cells and not with a reliable cellular or subcellular spatial resolution. Recently, a novel family of highly-sensitive membrane potential reporter dyes in combination with high temporal and spatial confocal calcium imaging allows for simultaneously detecting membrane potential and calcium changes in many cells at a time. Since the signals yielded from both types of reporter dyes are inherently noisy, we have developed complex methods of data denoising that permit for visualization and pixel-wise analysis of signals. Combining the experimental approach of high-resolution imaging with the advanced analysis of noisy data enables novel

  4. Maternal obesity accelerates fetal pancreatic beta-cell but not alpha-cell development in sheep: prenatal consequences.

    PubMed

    Ford, Stephen P; Zhang, Liren; Zhu, Meijun; Miller, Myrna M; Smith, Derek T; Hess, Bret W; Moss, Gary E; Nathanielsz, Peter W; Nijland, Mark J

    2009-09-01

    Maternal obesity affects offspring weight, body composition, and organ function, increasing diabetes and metabolic syndrome risk. We determined effects of maternal obesity and a high-energy diet on fetal pancreatic development. Sixty days prior to breeding, ewes were assigned to control [100% of National Research Council (NRC) recommendations] or obesogenic (OB; 150% NRC) diets. At 75 days gestation, OB ewes exhibited elevated insulin-to-glucose ratios at rest and during a glucose tolerance test, demonstrating insulin resistance compared with control ewes. In fetal studies, ewes ate their respective diets from 60 days before to 75 days after conception when animals were euthanized under general anesthesia. OB and control ewes increased in body weight by approximately 43% and approximately 6%, respectively, from diet initiation until necropsy. Although all organs were heavier in fetuses from OB ewes, only pancreatic weight increased as a percentage of fetal weight. Blood glucose, insulin, and cortisol were elevated in OB ewes and fetuses on day 75. Insulin-positive cells per unit pancreatic area were 50% greater in fetuses from OB ewes as a result of increased beta-cell mitoses rather than decreased programmed cell death. Lambs of OB ewes were born earlier but weighed the same as control lambs; however, their crown-to-rump length was reduced, and their fat mass was increased. We conclude that increased systemic insulin in fetuses from OB ewes results from increased glucose exposure and/or cortisol-induced accelerated fetal beta-cell maturation and may contribute to premature beta-cell function loss and predisposition to obesity and metabolic disease in offspring. PMID:19605766

  5. Membrane Potential and Calcium Dynamics in Beta Cells from Mouse Pancreas Tissue Slices: Theory, Experimentation, and Analysis

    PubMed Central

    Dolenšek, Jurij; Špelič, Denis; Skelin Klemen, Maša; Žalik, Borut; Gosak, Marko; Slak Rupnik, Marjan; Stožer, Andraž

    2015-01-01

    Beta cells in the pancreatic islets of Langerhans are precise biological sensors for glucose and play a central role in balancing the organism between catabolic and anabolic needs. A hallmark of the beta cell response to glucose are oscillatory changes of membrane potential that are tightly coupled with oscillatory changes in intracellular calcium concentration which, in turn, elicit oscillations of insulin secretion. Both membrane potential and calcium changes spread from one beta cell to the other in a wave-like manner. In order to assess the properties of the abovementioned responses to physiological and pathological stimuli, the main challenge remains how to effectively measure membrane potential and calcium changes at the same time with high spatial and temporal resolution, and also in as many cells as possible. To date, the most wide-spread approach has employed the electrophysiological patch-clamp method to monitor membrane potential changes. Inherently, this technique has many advantages, such as a direct contact with the cell and a high temporal resolution. However, it allows one to assess information from a single cell only. In some instances, this technique has been used in conjunction with CCD camera-based imaging, offering the opportunity to simultaneously monitor membrane potential and calcium changes, but not in the same cells and not with a reliable cellular or subcellular spatial resolution. Recently, a novel family of highly-sensitive membrane potential reporter dyes in combination with high temporal and spatial confocal calcium imaging allows for simultaneously detecting membrane potential and calcium changes in many cells at a time. Since the signals yielded from both types of reporter dyes are inherently noisy, we have developed complex methods of data denoising that permit for visualization and pixel-wise analysis of signals. Combining the experimental approach of high-resolution imaging with the advanced analysis of noisy data enables novel

  6. The transcription factors Nkx6.1 and Nkx6.2 possess equivalent activities in promoting beta-cell fate specification in Pdx1+ pancreatic progenitor cells.

    PubMed

    Nelson, Shelley B; Schaffer, Ashleigh E; Sander, Maike

    2007-07-01

    Despite much progress in identifying transcriptional regulators that control the specification of the different pancreatic endocrine cell types, the spatiotemporal aspects of endocrine subtype specification have remained largely elusive. Here, we address the mechanism by which the transcription factors Nkx6.1 (Nkx6-1) and Nkx6.2 (Nkx6-2) orchestrate development of the endocrine alpha- and beta-cell lineages. Specifically, we assayed for the rescue of insulin-producing beta-cells in Nkx6.1 mutant mice upon restoring Nkx6 activity in select progenitor cell populations with different Nkx6-expressing transgenes. Beta-cell formation and maturation was restored when Nkx6.1 was expressed in multipotential Pdx1(+) pancreatic progenitors, whereas no rescue was observed upon expression in committed Ngn3(+) (Neurog3(+)) endocrine progenitors. Although not excluding additional roles downstream of Ngn3, this finding suggests a first requirement for Nkx6.1 in specifying beta-cell progenitors prior to Ngn3 activation. Surprisingly, although Nkx6.2 only compensates for Nkx6.1 in alpha-but not in beta-cell development in Nkx6.1(-/-) mice, a Pdx1-promoter-driven Nkx6.2 transgene had the same ability to rescue beta-cells as the Pdx1-Nkx6.1 transgene. This demonstrates that the distinct requirements for Nkx6.1 and Nkx6.2 in endocrine differentiation are a consequence of their divergent spatiotemporal expression domains rather than their biochemical activities and implies that both Nkx6.1 and Nkx6.2 possess alpha- and beta-cell-specifying activities. PMID:17537793

  7. Cytotoxic T-cells from T-cell receptor transgenic NOD8.3 mice destroy beta-cells via the perforin and Fas pathways.

    PubMed

    Dudek, Nadine L; Thomas, Helen E; Mariana, Lina; Sutherland, Robyn M; Allison, Janette; Estella, Eugene; Angstetra, Eveline; Trapani, Joseph A; Santamaria, Pere; Lew, Andrew M; Kay, Thomas W H

    2006-09-01

    Cytotoxic T-cells are the major mediators of beta-cell destruction in type 1 diabetes, but the molecular mechanisms are not definitively established. We have examined the contribution of perforin and Fas ligand to beta-cell destruction using islet-specific CD8(+) T-cells from T-cell receptor transgenic NOD8.3 mice. NOD8.3 T-cells killed Fas-deficient islets in vitro and in vivo. Perforin-deficient NOD8.3 T-cells were able to destroy wild-type but not Fas-deficient islets in vitro. These results imply that NOD8.3 T-cells use both pathways and that Fas is required for beta-cell killing only when perforin is missing. Consistent with this theory, transgenic NOD8.3 mice with beta-cells that do not respond to Fas ligation were not protected from diabetes. We next investigated the mechanism of protection provided by overexpression of suppressor of cytokine signaling-1 (SOCS-1) in beta-cells of NOD8.3 mice. SOCS-1 islets remained intact when grafted into NOD8.3 mice and were less efficiently killed in vitro. However, addition of exogenous peptide rendered SOCS-1 islets susceptible to 8.3 T-cell-mediated lysis. Therefore, NOD8.3 T-cells use both perforin and Fas pathways to kill beta-cells and the surprising blockade of NOD8.3 T-cell-mediated beta-cell death by SOCS-1 overexpression may be due in part to reduced target cell recognition. PMID:16936188

  8. Perforin facilitates beta cell killing and regulates autoreactive CD8+ T-cell responses to antigen in mouse models of type 1 diabetes.

    PubMed

    Trivedi, Prerak; Graham, Kate L; Krishnamurthy, Balasubramaninan; Fynch, Stacey; Slattery, Robyn M; Kay, Thomas W H; Thomas, Helen E

    2016-04-01

    In type 1 diabetes, cytotoxic CD8(+) T lymphocytes (CTLs) directly interact with pancreatic beta cells through major histocompatibility complex class I. An immune synapse facilitates delivery of cytotoxic granules, comprised mainly of granzymes and perforin. Perforin deficiency protects the majority of non-obese diabetic (NOD) mice from autoimmune diabetes. Intriguingly perforin deficiency does not prevent diabetes in CD8(+) T-cell receptor transgenic NOD8.3 mice. We therefore investigated the importance of perforin-dependent killing via CTL-beta cell contact in autoimmune diabetes. Perforin-deficient CTL from NOD mice or from NOD8.3 mice were significantly less efficient at adoptive transfer of autoimmune diabetes into NODRag1(-/-) mice, confirming that perforin is essential to facilitate beta cell destruction. However, increasing the number of transferred in vitro-activated perforin-deficient 8.3 T cells reversed the phenotype and resulted in diabetes. Perforin-deficient NOD8.3 T cells were present in increased proportion in islets, and proliferated more in response to antigen in vivo indicating that perforin may regulate the activation of CTLs, possibly by controlling cytokine production. This was confirmed when we examined the requirement for direct interaction between beta cells and CD8(+) T cells in NOD8.3 mice, in which beta cells specifically lack major histocompatibility complex (MHC) class I through conditional deletion of β2-microglobulin. Although diabetes was significantly reduced, 40% of these mice developed diabetes, indicating that NOD8.3 T cells can kill beta cells in the absence of direct interaction. Our data indicate that although perforin delivery is the main mechanism that CTL use to destroy beta cells, they can employ alternative mechanisms to induce diabetes in a perforin-independent manner. PMID:26446877

  9. Islet-selectivity of G-protein coupled receptor ligands evaluated for PET imaging of pancreatic {beta}-cell mass

    SciTech Connect

    Cline, Gary W.; Zhao, Xiaojian; Jakowski, Amy B.; Soeller, Walter C.; Treadway, Judith L.

    2011-09-02

    Highlights: {yields} We screened G-protein coupled receptors for imaging pancreatic. {yields} Database mining and immunohistochemistry identified GPCRs enriched in {beta}-cells. {yields} In vitro and in vivo assays were used to determine exocrine vs endocrine specificity. {yields} GPCR candidates for imaging of {beta}-cell mass are Prokineticin-1R, mGluR5, and GLP-1R. -- Abstract: A critical unmet need exists for methods to quantitatively measure endogenous pancreatic {beta}-cell mass (BCM) for the clinical evaluation of therapies to prevent or reverse loss of BCM and diabetes progression. Our objective was to identify G-protein coupled receptors (GPCRs) that are expressed with a high degree of specificity to islet {beta}-cells for receptor-targeted imaging of BCM. GPCRs enriched in pancreatic islets relative to pancreas acinar and hepatic tissue were identified using a database screen. Islet-specific expression was confirmed by human pancreas immunohistochemistry (IHC). In vitro selectivity assessment was determined from the binding and uptake of radiolabeled ligands to the rat insulinoma INS-1 832/13 cell line and isolated rat islets relative to the exocrine pancreas cell-type, PANC-1. Tail-vein injections of radioligands into rats were used to determine favorable image criteria of in vivo biodistribution to the pancreas relative to other internal organs (i.e., liver, spleen, stomach, and lungs). Database and IHC screening identified four candidate receptors for further in vitro and in vivo evaluation for PET imaging of BCM: prokineticin-1 receptor (PK-1R), metabotropic glutamate receptor type-5 (mGluR5), neuropeptide Y-2 receptor (NPY-2R), and glucagon-like peptide 1 receptor (GLP-1R). In vitro specificity ratios gave the following receptor rank order: PK-1R > GLP-1R > NPY-2R > mGluR5. The biodistribution rank order of selectivity to the pancreas was found to be PK-1R > VMAT2 {approx} GLP-1R > mGluR5. Favorable islet selectivity and biodistribution

  10. Relationship of Soluble RAGE with Insulin Resistance and Beta Cell Function during Development of Type 2 Diabetes Mellitus.

    PubMed

    Biswas, Subrata Kumar; Mohtarin, Sabreena; Mudi, Sonchita Rani; Anwar, Taznuva; Banu, Laila Anjuman; Alam, Sheikh Md Khorshed; Fariduddin, Md; Arslan, M Iqbal

    2015-01-01

    This study examined whether circulating levels of soluble receptor for advanced glycation end products (sRAGE) alter in prediabetes and correlate with insulin resistance (IR) and beta cell function in prediabetes and newly diagnosed type 2 diabetes mellitus (T2DM). Subjects without previous history of diabetes were recruited and grouped as control, prediabetes, and newly diagnosed T2DM. The control subjects (n = 40) and people with prediabetes (n = 52) and diabetes (n = 66) were similar in terms of age, sex, BMI, systolic and diastolic BP, and fasting insulin level. HOMA-IR was found significantly higher in people with diabetes than control subjects (p < 0.001) and people with prediabetes (p = 0.005); and HOMA-%B was found significantly deteriorated in people with diabetes (p < 0.001) compared to control subjects and people with prediabetes. However, serum sRAGE levels did not show any significant alteration in people with prediabetes compared to control subjects. Moreover, univariate and multivariate analyses did not identify any significant correlation and statistical association of sRAGE with HOMA-IR and HOMA-%B in people with prediabetes and newly diagnosed T2DM. Our data suggest that serum sRAGE levels do not alter in people with prediabetes compared to control subjects and do not correlate or associate with IR and beta cell function during development of T2DM. PMID:26078977

  11. Relationship of Soluble RAGE with Insulin Resistance and Beta Cell Function during Development of Type 2 Diabetes Mellitus

    PubMed Central

    Biswas, Subrata Kumar; Mohtarin, Sabreena; Mudi, Sonchita Rani; Anwar, Taznuva; Banu, Laila Anjuman; Alam, Sheikh Md. Khorshed; Fariduddin, Md.; Arslan, M. Iqbal

    2015-01-01

    This study examined whether circulating levels of soluble receptor for advanced glycation end products (sRAGE) alter in prediabetes and correlate with insulin resistance (IR) and beta cell function in prediabetes and newly diagnosed type 2 diabetes mellitus (T2DM). Subjects without previous history of diabetes were recruited and grouped as control, prediabetes, and newly diagnosed T2DM. The control subjects (n = 40) and people with prediabetes (n = 52) and diabetes (n = 66) were similar in terms of age, sex, BMI, systolic and diastolic BP, and fasting insulin level. HOMA-IR was found significantly higher in people with diabetes than control subjects (p < 0.001) and people with prediabetes (p = 0.005); and HOMA-%B was found significantly deteriorated in people with diabetes (p < 0.001) compared to control subjects and people with prediabetes. However, serum sRAGE levels did not show any significant alteration in people with prediabetes compared to control subjects. Moreover, univariate and multivariate analyses did not identify any significant correlation and statistical association of sRAGE with HOMA-IR and HOMA-%B in people with prediabetes and newly diagnosed T2DM. Our data suggest that serum sRAGE levels do not alter in people with prediabetes compared to control subjects and do not correlate or associate with IR and beta cell function during development of T2DM. PMID:26078977

  12. Combined Optical Coherence and Fluorescence Microscopy to assess dynamics and specificity of pancreatic beta-cell tracers

    PubMed Central

    Berclaz, Corinne; Pache, Christophe; Bouwens, Arno; Szlag, Daniel; Lopez, Antonio; Joosten, Lieke; Ekim, Selen; Brom, Maarten; Gotthardt, Martin; Grapin-Botton, Anne; Lasser, Theo

    2015-01-01

    The identification of a beta-cell tracer is a major quest in diabetes research. However, since MRI, PET and SPECT cannot resolve individual islets, optical techniques are required to assess the specificity of these tracers. We propose to combine Optical Coherence Microscopy (OCM) with fluorescence detection in a single optical platform to facilitate these initial screening steps from cell culture up to living rodents. OCM can image islets and vascularization without any labeling. Thereby, it alleviates the need of both genetically modified mice to detect islets and injection of external dye to reveal vascularization. We characterized Cy5.5-exendin-3, an agonist of glucagon-like peptide 1 receptor (GLP1R), for which other imaging modalities have been used and can serve as a reference. Cultured cells transfected with GLP1R and incubated with Cy5.5-exendin-3 show full tracer internalization. We determined that a dose of 1 μg of Cy5.5-exendin-3 is sufficient to optically detect in vivo the tracer in islets with a high specificity. In a next step, time-lapse OCM imaging was used to monitor the rapid and specific tracer accumulation in murine islets and its persistence over hours. This optical platform represents a versatile toolbox for selecting beta-cell specific markers for diabetes research and future clinical diagnosis. PMID:25988507

  13. Sulforaphane protects against cytokine- and streptozotocin-induced {beta}-cell damage by suppressing the NF-{kappa}B pathway

    SciTech Connect

    Song, Mi-Young; Kim, Eun-Kyung; Moon, Woo-Sung; Park, Jin-Woo; Kim, Hyung-Jin; So, Hong-Seob; Park, Raekil; Kwon, Kang-Beom Park, Byung-Hyun

    2009-02-15

    Sulforaphane (SFN) is an indirect antioxidant that protects animal tissues from chemical or biological insults by stimulating the expression of several NF-E2-related factor-2 (Nrf2)-regulated phase 2 enzymes. Treatment of RINm5F insulinoma cells with SFN increases Nrf2 nuclear translocation and expression of phase 2 enzymes. In this study, we investigated whether the activation of Nrf2 by SFN treatment or ectopic overexpression of Nrf2 inhibited cytokine-induced {beta}-cell damage. Treatment of RIN cells with IL-1{beta} and IFN-{gamma} induced {beta}-cell damage through a NF-{kappa}B-dependent signaling pathway. Activation of Nrf2 by treatment with SFN and induction of Nrf2 overexpression by transfection with Nrf2 prevented cytokine toxicity. The mechanism by which Nrf2 activation inhibited NF-{kappa}B-dependent cell death signals appeared to involve the reduction of oxidative stress, as demonstrated by the inhibition of cytokine-induced H{sub 2}O{sub 2} production. The protective effect of SFN was further demonstrated by the restoration of normal insulin secreting responses to glucose in cytokine-treated rat pancreatic islets. Furthermore, pretreatment with SFN blocked the development of type 1 diabetes in streptozotocin-treated mice.

  14. Combined Optical Coherence and Fluorescence Microscopy to assess dynamics and specificity of pancreatic beta-cell tracers.

    PubMed

    Berclaz, Corinne; Pache, Christophe; Bouwens, Arno; Szlag, Daniel; Lopez, Antonio; Joosten, Lieke; Ekim, Selen; Brom, Maarten; Gotthardt, Martin; Grapin-Botton, Anne; Lasser, Theo

    2015-01-01

    The identification of a beta-cell tracer is a major quest in diabetes research. However, since MRI, PET and SPECT cannot resolve individual islets, optical techniques are required to assess the specificity of these tracers. We propose to combine Optical Coherence Microscopy (OCM) with fluorescence detection in a single optical platform to facilitate these initial screening steps from cell culture up to living rodents. OCM can image islets and vascularization without any labeling. Thereby, it alleviates the need of both genetically modified mice to detect islets and injection of external dye to reveal vascularization. We characterized Cy5.5-exendin-3, an agonist of glucagon-like peptide 1 receptor (GLP1R), for which other imaging modalities have been used and can serve as a reference. Cultured cells transfected with GLP1R and incubated with Cy5.5-exendin-3 show full tracer internalization. We determined that a dose of 1 μg of Cy5.5-exendin-3 is sufficient to optically detect in vivo the tracer in islets with a high specificity. In a next step, time-lapse OCM imaging was used to monitor the rapid and specific tracer accumulation in murine islets and its persistence over hours. This optical platform represents a versatile toolbox for selecting beta-cell specific markers for diabetes research and future clinical diagnosis. PMID:25988507

  15. Relationship Between Beta Cell Dysfunction and Severity of Disease Among Critically Ill Children: A STROBE-Compliant Prospective Observational Study.

    PubMed

    Liu, Ping-Ping; Lu, Xiu-Lan; Xiao, Zheng-Hui; Qiu, Jun; Zhu, Yi-Min

    2016-05-01

    Although beta cell dysfunction has been proved to predict prognosis among humans and animals, its prediction on severity of disease remains unclear among children. The present study was aimed to examine the relationship between beta cell dysfunction and severity of disease among critically ill children.This prospective study included 1146 critically ill children, who were admitted to Pediatric Intensive Care Unit (PICU) of Hunan Children's Hospital from November 2011 to August 2013. Information on characteristics, laboratory tests, and prognostic outcomes was collected. Homeostasis model assessment (HOMA)-β, evaluating beta cell function, was used to divide all participants into 4 groups: HOMA-β = 100% (group I, n = 339), 80% ≤ HOMA-β < 100% (group II, n = 71), 40% ≤ HOMA-β < 80% (group III, n = 293), and HOMA-β < 40% (group IV, n = 443). Severity of disease was assessed using the worst Sequential Organ Failure Assessment (SOFA) score, Pediatric Risk of Mortality (PRISM) III score, incidence of organ damage, septic shock, multiple organ dysfunction syndrome (MODS), mechanical ventilation (MV) and mortality. Logistic regression analysis was used to evaluate the risk of developing poor outcomes among patients in different HOMA-β groups, with group I as the reference group.Among 1146 children, incidence of HOMA-β < 100% was 70.41%. C-peptide and insulin declined with the decrement of HOMA-β (P < 0.01). C-reactive protein and procalcitonin levels, rather than white blood cell, were significantly different among 4 groups (P < 0.01). In addition, the worst SOFA score and the worst PRISMIII score increased with declined HOMA-β. For example, the worst SOFA score in group I, II, III, and IV was 1.55 ± 1.85, 1.71 ± 1.93, 1.92 ± 1.63, and 2.18 ± 1.77, respectively. Furthermore, patients with declined HOMA-β had higher risk of developing septic shock, MODS, MV, and mortality, even after adjusting age, gender

  16. Metformin prevents endoplasmic reticulum stress-induced apoptosis through AMPK-PI3K-c-Jun NH2 pathway

    USGS Publications Warehouse

    Jung, T.W.; Lee, M.W.; Lee, Y.-J.; Kim, S.M.

    2012-01-01

    Type 2 diabetes mellitus is thought to be partially associated with endoplasmic reticulum (ER) stress toxicity on pancreatic beta cells and the result of decreased insulin synthesis and secretion. In this study, we showed that a well-known insulin sensitizer, metformin, directly protects against dysfunction and death of ER stress-induced NIT-1 cells (a mouse pancreatic beta cell line) via AMP-activated protein kinase (AMPK) and phosphatidylinositol-3 (PI3) kinase activation. We also showed that exposure of NIT-1 cells to metformin (5mM) increases cellular resistance against ER stress-induced NIT-1 cell dysfunction and death. AMPK and PI3 kinase inhibitors abolished the effect of metformin on cell function and death. Metformin-mediated protective effects on ER stress-induced apoptosis were not a result of an unfolded protein response or the induced inhibitors of apoptotic proteins. In addition, we showed that exposure of ER stressed-induced NIT-1 cells to metformin decreases the phosphorylation of c-Jun NH(2) terminal kinase (JNK). These data suggest that metformin is an important determinant of ER stress-induced apoptosis in NIT-1 cells and may have implications for ER stress-mediated pancreatic beta cell destruction via regulation of the AMPK-PI3 kinase-JNK pathway.

  17. GLUT2 (SLC2A2) is not the principal glucose transporter in human pancreatic beta cells: implications for understanding genetic association signals at this locus.

    PubMed

    McCulloch, Laura J; van de Bunt, Martijn; Braun, Matthias; Frayn, Keith N; Clark, Anne; Gloyn, Anna L

    2011-12-01

    SLC2A2 encoding glucose transporter -2 (GLUT2) acts as the primary glucose transporter and sensor in rodent pancreatic islets and is widely assumed to play a similar role in humans. In healthy adults SLC2A2 variants are associated with elevated fasting plasma glucose (fpg) concentrations but physiological characterisation does not support a defect in pancreatic beta-cell function. Interspecies differences can create barriers for the follow up of disease association signals. We hypothesised that GLUT2 is not the principal glucose transporter in human beta-cells and that SLC2A2 variants exert their effect on fpg levels through defects in other tissues. SLC2A1-4 (GLUT 1-4) mRNA expression levels were determined in human and mouse islets, beta-cells, liver, muscle and adipose tissue by qRT-PCR whilst GLUT1-3 protein levels were examined by immunohistochemistry. The presence of all three glucose transporters was demonstrated in human and mouse islets and purified beta-cells. Quantitative expression profiling demonstrated that Slc2a2 is the predominant glucose transporter (expression >10 fold higher that Slc2a1) in mouse islets whilst SLC2A1 and SLC2A3 predominate in both human islets and beta-cells (expression 2.8 and 2.7 fold higher than SLC2A2 respectively). Our data therefore suggest that GLUT2 is unlikely to be the principal glucose transporter in human beta-cells and that SLC2A2 defects in other metabolic tissues drive the observed differences in glucose levels between carriers of SLC2A2 variants. Direct extrapolation from rodent to human islet glucose transporter activity is unlikely to be appropriate. PMID:21920790

  18. Selective destruction of mouse islet beta cells by human T lymphocytes in a newly-established humanized type 1 diabetic model

    SciTech Connect

    Zhao, Yong; Guo, Chengshan; Hwang, David; Lin, Brian; Dingeldein, Michael; Mihailescu, Dan; Sam, Susan; Sidhwani, Seema; Zhang, Yongkang; Jain, Sumit; Skidgel, Randal A.; Prabhakar, Bellur S.; Mazzone, Theodore; Holterman, Mark J.

    2010-09-03

    Research highlights: {yields} Establish a human immune-mediated type 1 diabetic model in NOD-scid IL2r{gamma}{sup null} mice. {yields} Using the irradiated diabetic NOD mouse spleen mononuclear cells as trigger. {yields} The islet {beta} cells were selectively destroyed by infiltrated human T cells. {yields} The model can facilitate translational research to find a cure for type 1 diabetes. -- Abstract: Type 1 diabetes (T1D) is caused by a T cell-mediated autoimmune response that leads to the loss of insulin-producing {beta} cells. The optimal preclinical testing of promising therapies would be aided by a humanized immune-mediated T1D model. We develop this model in NOD-scid IL2r{gamma}{sup null} mice. The selective destruction of pancreatic islet {beta} cells was mediated by human T lymphocytes after an initial trigger was supplied by the injection of irradiated spleen mononuclear cells (SMC) from diabetic nonobese diabetic (NOD) mice. This resulted in severe insulitis, a marked loss of total {beta}-cell mass, and other related phenotypes of T1D. The migration of human T cells to pancreatic islets was controlled by the {beta} cell-produced highly conserved chemokine stromal cell-derived factor 1 (SDF-1) and its receptor C-X-C chemokine receptor (CXCR) 4, as demonstrated by in vivo blocking experiments using antibody to CXCR4. The specificity of humanized T cell-mediated immune responses against islet {beta} cells was generated by the local inflammatory microenvironment in pancreatic islets including human CD4{sup +} T cell infiltration and clonal expansion, and the mouse islet {beta}-cell-derived CD1d-mediated human iNKT activation. The selective destruction of mouse islet {beta} cells by a human T cell-mediated immune response in this humanized T1D model can mimic those observed in T1D patients. This model can provide a valuable tool for translational research into T1D.

  19. Markers of beta cell failure predict poor glycemic response to GLP-1 receptor agonist therapy in type 2 diabetes

    PubMed Central

    Jones, Angus G; McDonald, Timothy J; Shields, Beverley M; Hill, Anita V; Hyde, Christopher J; Knight, Bridget A; Hattersley, Andrew T

    2016-01-01

    Objective To assess whether clinical characteristics and simple biomarkers of beta cell failure are associated with individual variation in glycemic response to GLP-1 receptor agonist therapy in patients with type 2 diabetes. Research Design and Methods We prospectively studied 620 participants with type 2 diabetes and HbA1c ≥58mmol/mol (7.5%) commencing GLP-1 receptor agonist therapy as part of their usual diabetes care and assessed response to therapy over 6 months. We assessed the association between baseline clinical measurements associated with beta cell failure and glycemic response (HbA1c change 0 to 6 months, primary outcome) with change in weight (0 to 6 months) as a secondary outcome using linear regression and ANOVA with adjustment for baseline HbA1c and co-treatment change. Results Reduced glycemic response to GLP-1R agonists was associated with longer duration diabetes, insulin co-treatment, lower fasting C-peptide, lower post meal urine C-peptide creatinine ratio and positive GAD or IA2 islet autoantibodies (p≤0.01 for all). Participants with positive autoantibodies or severe insulin deficiency (fasting C-peptide ≤0.25nmol/L) had markedly reduced glycemic response to GLP-1RA therapy (autoantibodies: mean HbA1c change -5.2 vs -15.2 mmol/mol (-0.5 vs -1.4%), p=0.005 C-peptide <0.25nmol/L: mean change -2.1 vs -15.3mmol/mol (-0.2 vs -1.4%), p=0.002). These markers were predominantly present in insulin treated participants and were not associated with weight change. Conclusions Clinical markers of low beta cell function are associated with reduced glycemic response to GLP-1R agonist therapy. C-peptide and islet autoantibodies represent potential biomarkers for the stratification of GLP-1R agonist therapy in insulin treated diabetes. PMID:26242184

  20. Analysis of the noise-induced bursting-spiking transition in a pancreatic beta-cell model.

    PubMed

    Aguirre, Jacobo; Mosekilde, Erik; Sanjuán, Miguel A F

    2004-04-01

    A stochastic model of the electrophysiological behavior of the pancreatic beta cell is studied, as a paradigmatic example of a bursting biological cell embedded in a noisy environment. The analysis is focused on the distortion that a growing noise causes to the basic properties of the membrane potential signals, such as their periodic or chaotic nature, and their bursting or spiking behavior. We present effective computational tools to obtain as much information as possible from these signals, and we suggest that the methods could be applied to real time series. Finally, a universal dependence of the main characteristics of the membrane potential on the size of the considered cell cluster is presented. PMID:15169046

  1. Endocytosis of secretory granules in mouse pancreatic beta-cells evoked by transient elevation of cytosolic calcium.

    PubMed Central

    Eliasson, L; Proks, P; Ammälä, C; Ashcroft, F M; Bokvist, K; Renström, E; Rorsman, P; Smith, P A

    1996-01-01

    1. To investigate the mechanisms regulating the reuptake of secretory granule membranes following regulated exocytosis, we have monitored changes in cell capacitance in single pancreatic beta-cells. 2. Membrane retrieval (endocytosis) occurred both in a continuous manner and in abrupt steps, corresponding to the simultaneous retrieval of 50-100 granules. The large endocytotic steps were associated with a conductance change of about 1 nS which we attribute to the formation of a fission pore with a pore radius of approximately 1 nm. 3. In some cells, we observed large amplitude capacitance fluctuations, suggesting that aggregates of granules are connected to the plasma membrane by a single pore and are subsequently retrieved as a single unit. 4. Endocytosis was evoked by elevation of cytosolic [Ca2+]i, but once initiated, a sustained increase in [Ca2+]i was not required for endocytosis to continue. 5. The [Ca2+]i dependence of exo- and endocytosis was studied by photorelease of Ca2+ from the 'caged' precursor Ca(2+)-nitrophenyl-EGTA (Ca(2+)-NP-EGTA). Both exo- and endocytosis were initiated at between 0.5 and 2 microM Cai(2+). The rate of endocytosis saturated above 2 microM Cai(2+), whereas exocytosis continued to increase up to 4 microM Cai(2+). The maximum rate of endocytosis was < 25% of that of exocytosis. 6. Unlike exocytosis, endocytosis proceeded equally well in the presence or absence of Mg-ATP. 7. Our data indicate that in the pancreatic beta-cell, exocytosis and endocytosis are regulated by different mechanisms. Images Figure 6 Figure 8 PMID:8799897

  2. Non-steroidal anti-inflammatory drugs increase insulin release from beta cells by inhibiting ATP-sensitive potassium channels

    PubMed Central

    Li, J; Zhang, N; Ye, B; Ju, W; Orser, B; Fox, J E M; Wheeler, M B; Wang, Q; Lu, W-Y

    2007-01-01

    Background and purpose: Some non-steroidal anti-inflammatory drugs (NSAIDs) incidentally induce hypoglycemia, which is often seen in diabetic patients receiving sulphonylureas. NSAIDs influence various ion channel activities, thus they may cause hypoglycemia by affecting ion channel functions in insulin secreting beta cells. This study investigated the effects of the NSAID meclofenamic acid (MFA) on the electrical excitability and the secretion of insulin from pancreatic beta cells. Experimental approach: Using patch clamp techniques and insulin secretion assays, the effects of MFA on the membrane potential and transmembrane current of INS-1 cells, and insulin secretion were studied. Key results: Under perforated patch recordings, MFA induced a rapid depolarization in INS-1 cells bathed in low (2.8mM), but not high (28mM) glucose solutions. MFA, as well as acetylsalicylic acid (ASA) and flufenamic acid (FFA), excited the cells by inhibiting ATP-sensitive potassium channels (KATP). In whole cell recordings, KATP conductance consistently appeared when intracellular ATP was diluted. Intracellular glibenclamide prevented the development of KATP activity, whereas intracellular MFA had no effect. At low glibenclamide concentrations, MFA induced additional inhibition of the KATP current. Live cell Ca2+ imaging displayed that MFA elevated intracellular Ca2+ at low glucose concentrations. Furthermore, MFA dose-dependently increased insulin release under low, but not high, glucose conditions. Conclusions and Implications: MFA blocked KATP through an extracellular mechanism and thus increased insulin secretion. As some NSAIDs synergistically inhibit KATP activity together with sulphonylureas, the risk of NSAID-induced hypoglycemia should be considered when glucose-lowering compounds are administered. PMID:17435793

  3. Pancreatic Beta Cell G-Protein Coupled Receptors and Second Messenger Interactions: A Systems Biology Computational Analysis

    PubMed Central

    Fridlyand, Leonid E.; Philipson, Louis H.

    2016-01-01

    Insulin secretory in pancreatic beta-cells responses to nutrient stimuli and hormonal modulators include multiple messengers and signaling pathways with complex interdependencies. Here we present a computational model that incorporates recent data on glucose metabolism, plasma membrane potential, G-protein-coupled-receptors (GPCR), cytoplasmic and endoplasmic reticulum calcium dynamics, cAMP and phospholipase C pathways that regulate interactions between second messengers in pancreatic beta-cells. The values of key model parameters were inferred from published experimental data. The model gives a reasonable fit to important aspects of experimentally measured metabolic and second messenger concentrations and provides a framework for analyzing the role of metabolic, hormones and neurotransmitters changes on insulin secretion. Our analysis of the dynamic data provides support for the hypothesis that activation of Ca2+-dependent adenylyl cyclases play a critical role in modulating the effects of glucagon-like peptide 1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP) and catecholamines. The regulatory properties of adenylyl cyclase isoforms determine fluctuations in cytoplasmic cAMP concentration and reveal a synergistic action of glucose, GLP-1 and GIP on insulin secretion. On the other hand, the regulatory properties of phospholipase C isoforms determine the interaction of glucose, acetylcholine and free fatty acids (FFA) (that act through the FFA receptors) on insulin secretion. We found that a combination of GPCR agonists activating different messenger pathways can stimulate insulin secretion more effectively than a combination of GPCR agonists for a single pathway. This analysis also suggests that the activators of GLP-1, GIP and FFA receptors may have a relatively low risk of hypoglycemia in fasting conditions whereas an activator of muscarinic receptors can increase this risk. This computational analysis demonstrates that study of second messenger

  4. Insulin resistance and beta-cell function in different ethnic groups in Kenya: the role of abdominal fat distribution.

    PubMed

    Christensen, D L; Faurholt-Jepsen, D; Faerch, K; Mwaniki, D L; Boit, M K; Kilonzo, B; Tetens, I; Friis, H; Borch-Johnsen, K

    2014-02-01

    Little is known about the pathophysiology of diabetes in Africans. Thus, we assessed whether insulin resistance and beta-cell function differed by ethnicity in Kenya and whether differences were modified by abdominal fat distribution. A cross-sectional study in 1,087 rural Luo (n = 361), Kamba (n = 378), and Maasai (n = 348) was conducted. All participants had a standard 75-g oral glucose tolerance test (OGTT). Venous blood samples were collected at 0, 30, and 120 min. Serum insulin was analysed at 0 and 30 min. From the OGTT, we assessed the homoeostasis model assessment of insulin resistance by computer model, early phase insulin secretion, and disposition index (DI) dividing insulin secretion by insulin resistance. Abdominal subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) thickness were carried out by ultrasonography. Linear regression analyses were done to assess ethnic differences in insulin indices. The Maasai had 32 and 17% higher insulin resistance than the Luo and Kamba, respectively (p < 0.001). Early phase insulin secretion was 16% higher in the Maasai compared to the Luo (p < 0.001). DI was 12% (p = 0.002) and 10% (p = 0.015) lower in the Maasai compared to the Luo and Kamba, respectively. Adjustments of SAT (range 0.1-7.1 cm) and VAT (range 1.5-14.2 cm) largely explained these inter-group differences with the Maasai having the highest combined abdominal fat accumulation. The Maasai had the highest insulin resistance and secretion, but the lowest relative beta-cell function compared to the Luo and Kamba. These differences were primarily explained by abdominal fat distribution. PMID:23563691

  5. Partial regeneration of beta-cells in the islets of Langerhans by Nymphayol a sterol isolated from Nymphaea stellata (Willd.) flowers.

    PubMed

    Subash-Babu, P; Ignacimuthu, S; Agastian, P; Varghese, Babu

    2009-04-01

    Reduction of the beta-cell mass is critical in the pathogenesis of diabetes mellitus. The discovery of agents which induce regeneration of pancreatic beta-cells would be useful to develop new therapeutic approaches to treat diabetes. The present study was aimed at identifying a new agent for the control of diabetes through regeneration of pancreatic beta cells and insulin secretory potential. Nymphaea stellata flower chloroform extract (NSFCExt) showed significant plasma glucose lowering effect. Further NSFCExt was utilized to isolate and identify the lead compound based on bioassay guided fractionation; we found Nymphayol (25,26-dinorcholest-5-en-3beta-ol) a new crystal [space group P2(1) (No. 4), a=9.618(5), b=7.518(5), c=37.491(5)]. It was purified by repeat column. The structure was determined on the basis of X-ray crystallography and spectral data. Oral administration of Nymphayol for 45 days significantly (p<0.05) lowered the blood glucose level and more importantly it effectively increased the insulin content in diabetic rats. In addition, Nymphayol increased the number of beta cell mass enormously. Islet-like cell clusters in the islets of Langerhans were clearly observed based on histochemical and immunohistochemical study. PMID:19272781

  6. Differences in beta-cell function and insulin secretion in Black vs. White obese adolescents: Do incretin hormones play a role?

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Black youth are at higher risk for type 2 diabetes (T2D) than their White peers. Previously we demonstrated that for the same degree of insulin sensitivity, Black youth have an upregulated beta-cell function and insulin hypersecretion, in response to intravenous (IV) glucose, compared with Whites. T...

  7. Beta-cell-specific production of IL6 in conjunction with a mainly intracellular but not mainly surface viral protein causes diabetes

    PubMed Central

    Van Belle, Tom L.; Pagni, Philippe P.; Liao, Jeanette; Sachithanantham, Sowbarnika; Dave, Amy; Hani, Amira Bel; Manenkova, Yulia; Amirian, Natalie; Yang, Cheng; Morin, Bret; Zhang, Haiqing; Campbell, Iain L.; von Herrath, Matthias G.

    2014-01-01

    Inflammatory mechanisms play a key role in the pathogenesis of type 1 and type 2 diabetes. IL6, a pleiotropic cytokine with impact on immune and non-immune cell types, has been proposed to be involved in the events causing both forms of diabetes and to play a key role in experimental insulin-dependent diabetes development. The aim of this study was to investigate how beta-cell specific overexpression of IL-6 influences diabetes development. We developed two lines of rat insulin promoter (RIP)-lymphocytic choriomeningitis virus (LCMV) mice that also co-express IL6 in their beta-cells. Expression of the viral nucleoprotein (NP), which has a predominantly intracellular localization, together with IL6 led to hyperglycemia, which was associated with a loss of GLUT-2 expression in the pancreatic beta-cells and infiltration of CD11b+ cells, but not T cells, in the pancreas. In contrast, over-expression of the LCMV glycoprotein (GP), which can localize to the surface, with IL-6 did not lead to spontaneous diabetes, but accelerated virus-induced diabetes by increasing autoantigen-specific CD8+ T cell responses and reducing the regulatory T cell fraction, leading to increased pancreatic infiltration by CD4+ and CD8+ T cells as well as CD11b+ and CD11c+ cells. The production of IL-6 in beta-cells acts prodiabetic, underscoring the potential benefit of targeting IL6 in diabetes. PMID:24582317

  8. Restoring Mitochondrial Function: A Small Molecule-mediated Approach to Enhance Glucose Stimulated Insulin Secretion in Cholesterol Accumulated Pancreatic beta cells.

    PubMed

    Asalla, Suman; Girada, Shravan Babu; Kuna, Ramya S; Chowdhury, Debabrata; Kandagatla, Bhaskar; Oruganti, Srinivas; Bhadra, Utpal; Bhadra, Manika Pal; Kalivendi, Shasi Vardhan; Rao, Swetha Pavani; Row, Anupama; Ibrahim, A; Ghosh, Partha Pratim; Mitra, Prasenjit

    2016-01-01

    Dyslipidemia, particularly the elevated serum cholesterol levels, aggravate the pathophysiology of type 2 diabetes. In the present study we explored the relationship between fasting blood sugar and serum lipid parameters in human volunteers which revealed a significant linear effect of serum cholesterol on fasting blood glucose. Short term feeding of cholesterol enriched diet to rodent model resulted in elevated serum cholesterol levels, cholesterol accumulation in pancreatic islets and hyperinsulinemia with modest increase in plasma glucose level. To explore the mechanism, we treated cultured BRIN-BD11 pancreatic beta cells with soluble cholesterol. Our data shows that cholesterol treatment of cultured pancreatic beta cells enhances total cellular cholesterol. While one hour cholesterol exposure enhances insulin exocytosis, overnight cholesterol accumulation in cultured pancreatic beta cells affects cellular respiration, and inhibits Glucose stimulated insulin secretion. We further report that (E)-4-Chloro-2-(1-(2-(2,4,6-trichlorophenyl) hydrazono) ethyl) phenol (small molecule M1) prevents the cholesterol mediated blunting of cellular respiration and potentiates Glucose stimulated insulin secretion which was abolished in pancreatic beta cells on cholesterol accumulation. PMID:27282931

  9. Restoring Mitochondrial Function: A Small Molecule-mediated Approach to Enhance Glucose Stimulated Insulin Secretion in Cholesterol Accumulated Pancreatic beta cells

    PubMed Central

    Asalla, Suman; Girada, Shravan Babu; Kuna, Ramya S.; Chowdhury, Debabrata; Kandagatla, Bhaskar; Oruganti, Srinivas; Bhadra, Utpal; Bhadra, Manika Pal; Kalivendi, Shasi Vardhan; Rao, Swetha Pavani; Row, Anupama; Ibrahim, A; Ghosh, Partha Pratim; Mitra, Prasenjit

    2016-01-01

    Dyslipidemia, particularly the elevated serum cholesterol levels, aggravate the pathophysiology of type 2 diabetes. In the present study we explored the relationship between fasting blood sugar and serum lipid parameters in human volunteers which revealed a significant linear effect of serum cholesterol on fasting blood glucose. Short term feeding of cholesterol enriched diet to rodent model resulted in elevated serum cholesterol levels, cholesterol accumulation in pancreatic islets and hyperinsulinemia with modest increase in plasma glucose level. To explore the mechanism, we treated cultured BRIN-BD11 pancreatic beta cells with soluble cholesterol. Our data shows that cholesterol treatment of cultured pancreatic beta cells enhances total cellular cholesterol. While one hour cholesterol exposure enhances insulin exocytosis, overnight cholesterol accumulation in cultured pancreatic beta cells affects cellular respiration, and inhibits Glucose stimulated insulin secretion. We further report that (E)-4-Chloro-2-(1-(2-(2,4,6-trichlorophenyl) hydrazono) ethyl) phenol (small molecule M1) prevents the cholesterol mediated blunting of cellular respiration and potentiates Glucose stimulated insulin secretion which was abolished in pancreatic beta cells on cholesterol accumulation. PMID:27282931

  10. Primary prevention of beta-cell autoimmunity and type 1 diabetes – The Global Platform for the Prevention of Autoimmune Diabetes (GPPAD) perspectives

    PubMed Central

    Ziegler, A.G.; Danne, T.; Dunger, D.B.; Berner, R.; Puff, R.; Kiess, W.; Agiostratidou, G.; Todd, J.A.; Bonifacio, E.

    2016-01-01

    Objective Type 1 diabetes can be identified by the presence of beta-cell autoantibodies that often arise in the first few years of life. The purpose of this perspective is to present the case for primary prevention of beta-cell autoimmunity and to provide a study design for its implementation in Europe. Methods We examined and summarized recruitment strategies, enrollment rates, and outcomes in published TRIGR, FINDIA and BABYDIET primary prevention trials, and the TEDDY intensive observational study. A proposal for a recruitment and implementation strategy to perform a phase II/III primary prevention randomized controlled trial in infants with genetic risk for developing beta-cell autoimmunity is outlined. Results Infants with a family history of type 1 diabetes (TRIGR, BABYDIET, TEDDY) and infants younger than age 3 months from the general population (FINDIA, TEDDY) were enrolled into these studies. All studies used HLA genotyping as part of their eligibility criteria. Predicted beta-cell autoimmunity risk in the eligible infants ranged from 3% (FINDIA, TEDDY general population) up to 12% (TRIGR, BABYDIET). Amongst eligible infants, participation was between 38% (TEDDY general population) and 97% (FINDIA). Outcomes, defined as multiple beta-cell autoantibodies, were consistent with predicted risks. We subsequently modeled recruitment into a randomized controlled trial (RCT) that could assess the efficacy of oral insulin treatment as adapted from the Pre-POINT pilot trial. The RCT would recruit infants with and without a first-degree family history of type 1 diabetes and be based on general population genetic risk testing. HLA genotyping and, for the general population, genotyping at additional type 1 diabetes susceptibility SNPs would be used to identify children with around 10% risk of beta-cell autoimmunity. The proposed RCT would have 80% power to detect a 50% reduction in multiple beta-cell autoantibodies by age 4 years at a two-tailed alpha of 0.05, and

  11. Sphingosine in apoptosis signaling.

    PubMed

    Cuvillier, Olivier

    2002-12-30

    The sphingolipid metabolites ceramide, sphingosine, and sphingosine 1-phosphate contribute to controlling cell proliferation and apoptosis. Ceramide and its catabolite sphingosine act as negative regulators of cell proliferation and promote apoptosis. Conversely, sphingosine 1-phosphate, formed by phosphorylation of sphingosine by a sphingosine kinase, has been involved in stimulating cell growth and inhibiting apoptosis. As the phosphorylation of sphingosine diminishes apoptosis, while dephosphorylation of sphingosine 1-phosphate potentiates it, the role of sphingosine as a messenger of apoptosis is of importance. Herein, the effects of sphingosine on diverse signaling pathways implicated in the apoptotic process are reviewed. PMID:12531549

  12. Determinants of glucose toxicity and its reversibility in the pancreatic islet beta-cell line, HIT-T15.

    PubMed

    Gleason, C E; Gonzalez, M; Harmon, J S; Robertson, R P

    2000-11-01

    HIT-T15 cells, a clonal beta-cell line, were cultured and passaged weekly for 6 mo in RPMI 1640 media containing various concentrations of glucose. Insulin content decreased in the intermediate- and late-passage cells as a continuous rather than a threshold glucose concentration effect. In a second series of experiments, cells were grown in media containing either 0.8 or 16.0 mM glucose from passages 76 through 105. Subcultures of passages 86, 92, and 99 that had been grown in media containing 16.0 mM glucose were switched to media containing 0.8 mM glucose and also carried forward to passage 105. Dramatic increases in insulin content and secretion and insulin gene expression were observed when the switches were made at passages 86 and 92 but not when the switch was made at passage 99. These findings suggest that glucose toxicity of insulin-secreting cells is a continuous rather than a threshold function of glucose concentration and that the shorter the period of antecedent glucose toxicity, the more likely that full recovery of cell function will occur. PMID:11052953

  13. DPP-4 inhibitor des-F-sitagliptin treatment increased insulin exocytosis from db/db mice {beta} cells

    SciTech Connect

    Nagamatsu, Shinya; Ohara-Imaizumi, Mica; Nakamichi, Yoko; Aoyagi, Kyota; Nishiwaki, Chiyono

    2011-09-09

    Highlights: {yields} Anti-diabetic new drug, DPP-4 inhibitor, can affect the insulin exocytosis. {yields} DPP-4 inhibitor treatment altered syntaxin 1 expression. {yields} Treatment of db/db mice with DPP-4 inhibitor increased insulin release. -- Abstract: Incretin promotes insulin secretion acutely. Recently, orally-administered DPP-4 inhibitors represent a new class of anti-hyperglycemic agents. Indeed, inhibitors of dipeptidyl peptidase-IV (DPP-4), sitagliptin, has just begun to be widely used as therapeutics for type 2 diabetes. However, the effects of sitagliptin-treatment on insulin exocytosis from single {beta}-cells are yet unknown. We therefore investigated how sitagliptin-treatment in db/db mice affects insulin exocytosis by treating db/db mice with des-F-sitagliptin for 2 weeks. Perfusion studies showed that 2 weeks-sitagliptin treatment potentiated insulin secretion. We then analyzed insulin granule motion and SNARE protein, syntaxin 1, by TIRF imaging system. TIRF imaging of insulin exocytosis showed the increased number of docked insulin granules and increased fusion events from them during first-phase release. In accord with insulin exocytosis data, des-F-sitagliptin-treatment increased the number of syntaxin 1 clusters on the plasma membrane. Thus, our data demonstrated that 2-weeks des-F-sitagliptin-treatment increased the fusion events of insulin granules, probably via increased number of docked insulin granules and that of syntaxin 1 clusters.

  14. Autoantibodies to the GLUT-2 glucose transporter of beta cells in insulin-dependent diabetes mellitus of recent onset.

    PubMed Central

    Inman, L R; McAllister, C T; Chen, L; Hughes, S; Newgard, C B; Kettman, J R; Unger, R H; Johnson, J H

    1993-01-01

    Purified immunoglobulin G (IgG) from the serum of patients with insulin-dependent diabetes mellitus (IDDM) of recent onset inhibits high-Km uptake of 3-O-methyl-beta-D-glucose by rat pancreatic islets. To determine if the inhibition is the result of antibodies against GLUT-2, the high-Km glucose transporter of beta cells, we incubated IDDM sera with rat islet cells and with AtT-20ins cells transfected to express GLUT-2. IDDM sera inhibited glucose uptake in islet cells and in GLUT-2-expressing AtT-20ins cells but not in AtT-20ins cells transfected to express the low-Km isoform, GLUT-1. In 24 of 30 (77%) patients with newly diagnosed IDDM, IgG binding as measured by immunofluorescence and flow cytometry of the cells transfected to express GLUT-2 was > 2 standard deviations from the mean of the nondiabetic population; 29 of 31 (96%) of nondiabetic children were negative (P < 0.0001). Increased IgG binding could be removed by absorption with GLUT-2-expressing cells but not with GLUT-1-expressing cells. We conclude that most patients with IDDM of recent onset have autoantibodies to GLUT-2. PMID:8433987

  15. Gene networks modified by sulphonylureas in beta cells: a pathway-based analysis of insulin secretion and cell death.

    PubMed

    Magnusson, Nils E; Dyrskjøt, Lars; Grimm, Daniela; Wehland, Markus; Pietsch, Jessica; Rungby, Jørgen

    2012-10-01

    Sulphonylureas (SUs) used in the treatment for type 2 diabetes have been shown to result in different clinical outcome. This study hypothesized that three widely used SUs, glibenclamide, glimepiride and gliclazide, may affect function and survival of insulin-producing cells differently. To evaluate differences between SUs, insulin secretion and cell death were measured, and genome-wide gene expression patterns were compared using a bioinformatics approach focusing on functional relationships between molecules. Insulin-producing INS-1E cells exposed to SUs for 6 and 24 hr were assayed using GeneChip. Cluster and pathway analyses were used to identify differentially expressed genes and patterns of potential biological functions associated with SU treatment. Cell death was measured using acridine orange/Hoechst 33342 staining. Short-term treatment (6 hr) yielded up-regulation of insulin secretion and genes associated with insulin secretion for all three SUs applied. While long-term treatment (24-72 hr) with gliclazide did not change gene expression or cell survival, treatment with glibenclamide or glimepiride up-regulated genes associated with oxidative stress and hypoxia, but did not induce cell death. Short-term treatment with SUs initiates gene regulation that can be attributed to insulin secretion with few differences between individual SUs. This regulation was temporal and returned to baseline after 24 hr. Individual differences observed after 24-72 hr indicate that glibenclamide and glimepiride induce potentially harmful cell signalling insufficient for triggering beta cell death. PMID:22642398

  16. Protective effect of Withania somnifera against oxidative stress and pancreatic beta-cell damage in type 2 diabetic rats.

    PubMed

    Anwer, Tarique; Sharma, Manju; Pillai, Krishna Kolappa; Khan, Gyas

    2012-01-01

    The aim of the present study was to investigate the effects of Withania somnifera (WS) on lipid peroxidation (LPO), activities of both non-enzymatic and enzymatic antioxidants and histopathological examination of pancreas in type 2 diabetic rats. Type 2 diabetes was induced by single intraperitoneal injection of STZ (100 mg/kg) to 2 days old rat pups. Oxidative stress was measured by tissue LPO levels, reduced glutathione (GSH) contents and by enzymatic activities of glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-S-transferase (GST), superoxide dismutase (SOD) and catalase (CAT). Administration of WS to type 2 diabetic rats caused a significant decrease in blood glucose and tissue LPO levels with significant increase in GSH contents when compared with the type 2 diabetic control rats. In addition, WS treated rats also showed a significant increase in the activities of antioxidant enzymes namely GPx, GR, GST, SOD and CAT when compared with type 2 diabetic control rats. Significant reduction in the number and size of pancreatic beta-cells were preserved to near normal morphology by the administration of WS in type 2 diabetic rats as evident from histopathological examination. The results obtained clearly indicate that WS has shown strong free radical scavenging activity and helped in improving the non-enzymatic and enzymatic antioxidants in type 2 diabetic rats. PMID:23285670

  17. Characterization of Phospholipids in Insulin Secretory Granules and Mitochondria in Pancreatic Beta Cells and Their Changes with Glucose Stimulation*

    PubMed Central

    MacDonald, Michael J.; Ade, Lacmbouh; Ntambi, James M.; Ansari, Israr-Ul H.; Stoker, Scott W.

    2015-01-01

    The lipid composition of insulin secretory granules (ISG) has never previously been thoroughly characterized. We characterized the phospholipid composition of ISG and mitochondria in pancreatic beta cells without and with glucose stimulation. The phospholipid/protein ratios of most phospholipids containing unsaturated fatty acids were higher in ISG than in whole cells and in mitochondria. The concentrations of negatively charged phospholipids, phosphatidylserine, and phosphatidylinositol in ISG were 5-fold higher than in the whole cell. In ISG phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin, fatty acids 12:0 and 14:0 were high, as were phosphatidylserine and phosphatidylinositol containing 18-carbon unsaturated FA. With glucose stimulation, the concentration of many ISG phosphatidylserines and phosphatidylinositols increased; unsaturated fatty acids in phosphatidylserine increased; and most phosphatidylethanolamines, phosphatidylcholines, sphingomyelins, and lysophosphatidylcholines were unchanged. Unsaturation and shorter fatty acid length in phospholipids facilitate curvature and fluidity of membranes, which favors fusion of membranes. Recent evidence suggests that negatively charged phospholipids, such as phosphatidylserine, act as coupling factors enhancing the interaction of positively charged regions in SNARE proteins in synaptic or secretory vesicle membrane lipid bilayers with positively charged regions in SNARE proteins in the plasma membrane lipid bilayer to facilitate docking of vesicles to the plasma membrane during exocytosis. The results indicate that ISG phospholipids are in a dynamic state and are consistent with the idea that changes in ISG phospholipids facilitate fusion of ISG with the plasma membrane-enhancing glucose-stimulated insulin exocytosis. PMID:25762724

  18. Extracellular ATP stimulates exocytosis via localized Ca(2+) release from acidic stores in rat pancreatic beta cells.

    PubMed

    Xie, Li; Zhang, Ming; Zhou, Wei; Wu, Zhengxing; Ding, Jiuping; Chen, Liangyi; Xu, Tao

    2006-04-01

    Three different methods, membrane capacitance (C(m)) measurement, amperometry and FM dye labeling were used to investigate the role of extracellular ATP in insulin secretion from rat pancreatic beta cells. We found that extracellular application of ATP mobilized intracellular Ca(2+) stores and synchronously triggered vigorous exocytosis. No influence of ATP on the readily releasable pool of vesicles was observed, which argues against a direct modulation of the secretory machinery at a level downstream of Ca(2+) elevation. The stimulatory effects of ATP were greatly reduced by intracellular perfusion of BAPTA but not EGTA, suggesting a close spatial association of fusion sites with intracellular Ca(2+) releasing sites. ATP-induced Ca(2+) transients and exocytosis were not blocked by thapsigargin (TG), by a ryanodine receptor antagonist or by dissipation of pH in acidic stores by monensin alone, but they were greatly attenuated by IP(3) receptor inhibition as well as ionomycin plus monensin, suggesting involvement of IP(3)-sensitive acidic Ca(2+) stores. Taken together, our data suggest that extracellular ATP triggers exocytosis by mobilizing spatially limited acidic Ca(2+) stores through IP(3) receptors. This mechanism may explain how insulin secretion from the pancreas is coordinated through diffusible ATP that is co-released with insulin. PMID:16536741

  19. AMP-activated protein kinase mediates apoptosis in response to bioenergetic stress through activation of the pro-apoptotic Bcl-2 homology domain-3-only protein BMF.

    PubMed

    Kilbride, Seán M; Farrelly, Angela M; Bonner, Caroline; Ward, Manus W; Nyhan, Kristine C; Concannon, Caoimhín G; Wollheim, Claes B; Byrne, Maria M; Prehn, Jochen H M

    2010-11-12

    Heterozygous loss-of-function mutations in the hepatocyte nuclear factor 1A (HNF1A) gene result in the pathogenesis of maturity-onset diabetes-of-the-young type 3, (HNF1A-MODY). This disorder is characterized by a primary defect in metabolism-secretion coupling and decreased beta cell mass, attributed to excessive beta cell apoptosis. Here, we investigated the link between energy stress and apoptosis activation following HNF1A inactivation. This study employed single cell fluorescent microscopy, flow cytometry, gene expression analysis, and gene silencing to study the effects of overexpression of dominant-negative (DN)-HNF1A expression on cellular bioenergetics and apoptosis in INS-1 cells. Induction of DN-HNF1A expression led to reduced ATP levels and diminished the bioenergetic response to glucose. This was coupled with activation of the bioenergetic stress sensor AMP-activated protein kinase (AMPK), which preceded the onset of apoptosis. Pharmacological activation of AMPK using aminoimidazole carboxamide ribonucleotide (AICAR) was sufficient to induce apoptosis in naive cells. Conversely, inhibition of AMPK with compound C or AMPKα gene silencing protected against DN-HNF1A-induced apoptosis. Interestingly, AMPK mediated the induction of the pro-apoptotic Bcl-2 homology domain-3-only protein Bmf (Bcl-2-modifying factor). Bmf expression was also elevated in islets of DN-HNF1A transgenic mice. Furthermore, knockdown of Bmf expression in INS-1 cells using siRNA was sufficient to protect against DN-HNF1A-induced apoptosis. Our study suggests that overexpression of DN-HNF1A induces bioenergetic stress and activation of AMPK. This in turn mediates the transcriptional activation of the pro-apoptotic Bcl-2-homology protein BMF, coupling prolonged energy stress to apoptosis activation. PMID:20841353

  20. The Influence of Exposure to Maternal Diabetes In Utero on the Rate Of Decline in Beta-Cell Function Among Youth with Diabetes

    PubMed Central

    Crume, T.L.; Andrews, J.S.; D’Agostino, R.; Pettitt, D.J.; Mayer-Davis, E.J.; Law, J.R.; Dolan, L.; Lawrence, J.M.; Saydah, S.; Greenbaum, C.; Rodriguez, B.L.; Dabelea, D.

    2014-01-01

    A relationship between exposure to maternal diabetes in utero and a younger age at diagnosis of type 2 diabetes was detected in SEARCH for Diabetes in Youth Study, while no significant association was detected with paternal diabetes status, suggesting an independent effect of the intrauterine exposure to hyperglycemia. We assessed the influence of exposure to maternal diabetes in utero on beta cell decline measured using fasting C-peptide (FCP) among 1079 youth with diabetes, including 941 with type 1 and 138 with type 2, who were followed post-diagnosis for an average of 58 months. No significant relationship was detected between exposure to maternal diabetes in utero and change in FCP levels in youth with type 1 or type 2 diabetes. These findings suggest that exposure to maternal diabetes in utero may not be an important determinant of short-term beta-cell function decline in youth with type 1 or type 2 diabetes. PMID:23645121

  1. Extracellular ATP-induced nuclear Ca{sup 2+} transient is mediated by inositol 1,4,5-trisphosphate receptors in mouse pancreatic {beta}-cells

    SciTech Connect

    Chen, Zheng; Li, Zhengzheng; Peng, Gong; Chen, Xiaoli; Yin, Wenxuan; Kotlikoff, Michael I.; Yuan, Zeng-qiang; Ji, Guangju

    2009-05-01

    Extracellular ATP (eATP) induces an intracellular Ca{sup 2+} transient by activating phospholipase C (PLC)-associated P2X4 purinergic receptors, leading to production of inositol 1,4,5-trisphosphate (IP3) and subsequent Ca{sup 2+} release from intracellular stores in mouse pancreatic {beta}-cells. Using laser scanning confocal microscopy, Ca{sup 2+} indicator fluo-4 AM, and the cell permeable nuclear indicator Hoechst 33342, we examined the properties of eATP-induced Ca{sup 2+} release in pancreatic {beta}-cell nuclei. eATP induced a higher nuclear Ca{sup 2+} transient in pancreatic {beta}-cell nuclei than in the cytosol. After pretreatment with thapsigargin (TG), an inhibitor of sarco-endoplasmic reticulum Ca{sup 2+}-ATPase (SERCA) pumps, the amplitude of eATP-induced Ca{sup 2+} transients in the nucleus was still much higher than those in the cytosol. This effect of eATP was not altered by inhibition of either the plasma membrane Ca{sup 2+}-ATPase (PMCA) or the plasma membrane Na{sup +}/Ca{sup 2+} exchanger (NCX) by LaCl{sub 3} or by replacement of Na{sup +} with N-Methyl-Glucosamine. eATP-induced nuclear Ca{sup 2+} transients were abolished by a cell-permeable IP3R inhibitor, 2-aminoethoxydiphenyl borate (2-APB), but were not blocked by the ryanodine receptor (RyR) antagonist ryanodine. Immunofluorescence studies showed that IP3Rs are expressed on the nuclear envelope of pancreatic {beta}-cells. These results indicate that eATP triggers nuclear Ca{sup 2+} transients by mobilizing a nuclear Ca{sup 2+} store via nuclear IP3Rs.

  2. Beneficial effect of 17{beta}-estradiol on hyperglycemia and islet {beta}-cell functions in a streptozotocin-induced diabetic rat model

    SciTech Connect

    Yamabe, Noriko; Kang, Ki Sung; Zhu Baoting

    2010-11-15

    The modulating effect of estrogen on glucose homeostasis remains a controversial issue at present. In this study, we sought to determine the beneficial effect of 17{beta}-estradiol (E{sub 2}) on hyperglycemia and islet {beta}-cell functions in streptozotocin (STZ)-induced diabetic rats. Male Sprague-Dawley rats were injected i.p. with STZ to induce a relatively mild diabetic condition. The rats were then treated with E{sub 2} orally at 500 {mu}g/kg body weight/day for 15 days to evaluate the modulating effect on hyperglycemia, insulin secretion, and islet {beta}-cell proliferation. E{sub 2} administration for 10 days significantly lowered plasma glucose levels, increased plasma insulin levels, and improved glucose tolerance by attenuating insulin response to oral glucose loading. These beneficial effects of E{sub 2} were accompanied by increases in islet number and volume, rate of islet cell proliferation, and the amount of insulin secreted. The growth-stimulatory effect of E{sub 2} on islet cells was linked to the functions of the estrogen receptor {alpha}. Notably, these protective effects of E{sub 2} on diabetic conditions were basically not observed when the STZ-treated rats had a more severe degree of islet damage and hyperglycemia. Taken together, we conclude that E{sub 2} can promote the regeneration of damaged pancreatic islets by stimulating {beta}-cell proliferation in diabetic rats, and this effect is accompanied by improvements in glucose tolerance and a decrease in plasma glucose levels. These findings suggest that oral administration of E{sub 2} may be beneficial in diabetic patients with an accelerated loss of islet {beta}-cells.

  3. Beneficial effects of candesartan, an angiotensin II type 1 receptor blocker, on beta-cell function and morphology in db/db mice.

    PubMed

    Shao, Jiaqing; Iwashita, Noseki; Ikeda, Fuki; Ogihara, Takeshi; Uchida, Toyoyoshi; Shimizu, Tomoaki; Uchino, Hiroshi; Hirose, Takahisa; Kawamori, Ryuzo; Watada, Hirotaka

    2006-06-16

    Several epidemiological studies suggested that treatment with angiotensin II type 1 receptor blocker (ARB) provided a risk reduction of developing type 2 diabetes. In this study, we investigated whether and how ARB treatment can improve abnormalities of pancreatic islets in diabetes state. We randomized db/db mice, a model of type 2 diabetes with obesity, at the age of 8 weeks to receive candesartan, an ARB, for 6 weeks. We also studied age-matched db/misty mice as control. Glucose tolerance test revealed that candesartan treatment improved glucose tolerance with the modest increase in serum insulin level in db/db mice. Concurrently, candesartan increased beta-cell mass, increased staining intensity of insulin, and decreased staining intensity of components of NAD(P)H oxidase, p22phox and gp91phox, and those of oxidative stress markers in beta-cells. These changes were accompanied by reduction of mitochondrial volume. Treatment with candesartan also reduced fibrosis in and around the islets and prevented the loss of endothelial cells in islets. Our results showed that candesartan partially prevented deterioration of glucose tolerance by providing protection against progressive beta-cell damage in diabetes. PMID:16650382

  4. O-Linked β-N-acetylglucosamine (O-GlcNAc) Acts as a Glucose Sensor to Epigenetically Regulate the Insulin Gene in Pancreatic Beta Cells.

    PubMed

    Durning, Sean P; Flanagan-Steet, Heather; Prasad, Nripesh; Wells, Lance

    2016-01-29

    The post-translational protein modification O-linked β-N-acetylglucosamine (O-GlcNAc) is a proposed nutrient sensor that has been shown to regulate multiple biological pathways. This dynamic and inducible enzymatic modification to intracellular proteins utilizes the end product of the nutrient sensing hexosamine biosynthetic pathway, UDP-GlcNAc, as its substrate donor. Type II diabetic patients have elevated O-GlcNAc-modified proteins within pancreatic beta cells due to chronic hyperglycemia-induced glucose overload, but a molecular role for O-GlcNAc within beta cells remains unclear. Using directed pharmacological approaches in the mouse insulinoma-6 (Min6) cell line, we demonstrate that elevating nuclear O-GlcNAc increases intracellular insulin levels and preserves glucose-stimulated insulin secretion during chronic hyperglycemia. The molecular mechanism for these observed changes appears to be, at least in part, due to elevated O-GlcNAc-dependent increases in Ins1 and Ins2 mRNA levels via elevations in histone H3 transcriptional activation marks. Furthermore, RNA deep sequencing reveals that this mechanism of altered gene transcription is restricted and that the majority of genes regulated by elevated O-GlcNAc levels are similarly regulated by a shift from euglycemic to hyperglycemic conditions. These findings implicate the O-GlcNAc modification as a potential mechanism for hyperglycemic-regulated gene expression in the beta cell. PMID:26598517

  5. Beneficial effects of Murraya koenigii leaves on antioxidant defense system and ultra structural changes of pancreatic beta-cells in experimental diabetes in rats.

    PubMed

    Arulselvan, Palanisamy; Subramanian, Sorimuthu Pillai

    2007-01-30

    Oxidative stress and oxidative damage to tissues are common end points of chronic diseases such as atherosclerosis, diabetes, and rheumatoid arthritis. Oxidative stress in diabetes coexists with a reduction in the antioxidant status, which can further increase the deleterious effects of free radicals. The aim of the present study was to evaluate the possible protective effects of Murraya koenigii leaves extract against beta-cell damage and antioxidant defense systems of plasma and pancreas in streptozotocin induced diabetes in rats. The levels of glucose and glycosylated hemoglobin in blood and insulin, Vitamin C, Vitamin E, ceruloplasmin, reduced glutathione and TBARS were estimated in plasma of control and experimental groups of rats. To assess the changes in the cellular antioxidant defense system such as the level of reduced glutathione and activities of superoxide dismutase, catalase and glutathione peroxidase were assayed in pancreatic tissue homogenate. The levels of glucose, glycosylated hemoglobin, insulin, TBARS, enzymatic and non-enzymatic antioxidants were altered in diabetic rats. These alterations were reverted back to near control levels after the treatment of M. koenigii leaves extract. Transmission electron microscopic studies also revealed the protective nature of M. koenigii leaves on pancreatic beta-cells. These findings suggest that M. koenigii treatment exerts a therapeutic protective nature in diabetes by decreasing oxidative stress and pancreatic beta-cell damage. The antioxidant effect of the M. koenigii extract was compared with glibenclamide, a well-known hypoglycemic drug. PMID:17188670

  6. Direct visualisation of peptide hormones in cultured pancreatic islet alpha- and beta-cells by intact-cell mass spectrometry.

    PubMed

    Buchanan, Christina M; Malik, Arpita S; Cooper, Garth J S

    2007-01-01

    The application of intact-cell mass spectrometry (ICM) by matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry to achieve direct protein-profiling of bacterial species is now well established. However, this methodology has not to our knowledge been applied to the analysis of mammalian cells in routine culture. Here, we describe a novel application of ICM by which we have identified proteins in intact cells from two lines representative of pancreatic islet alpha- and beta-cells. Adherent alphaTC1 clone 9 and betaTC6 F7 cells were harvested into phosphate-buffered saline (PBS) using enzyme-free dissociation buffer before 1 microL of cell suspension was spotted onto MALDI plates. Cells were overlaid with sinapinic acid then washed with pure water before application of a final coat of sinapinic acid. Data in the 2000-20,000 m/z range were acquired in linear mode on a Voyager DE-Pro mass spectrometer. The proteins which ionised were composed in large part of peptide hormones (e.g. insulin and glucagon) known to be packaged into the secretory granules of the beta- and alpha-cells respectively. However, in addition to visualising the peptides expected to be associated with these cells, a mass consistent with oxyntomodulin was identified in the cultured alpha-cells, a finding not previously reported to our knowledge. In summary, this paper describes, for the first time, a rapid and direct method useful for identifying secretory products in intact endocrine cells. PMID:17918213

  7. βIV-Spectrin and CaMKII facilitate Kir6.2 regulation in pancreatic beta cells

    PubMed Central

    Kline, Crystal F.; Wright, Patrick J.; Koval, Olha M.; Zmuda, Erik J.; Johnson, Benjamin L.; Anderson, Mark E.; Hai, Tsonwin; Hund, Thomas J.; Mohler, Peter J.

    2013-01-01

    Identified over a dozen years ago in the brain and pancreatic islet, βIV-spectrin is critical for the local organization of protein complexes throughout the nervous system. βIV-Spectrin targets ion channels and adapter proteins to axon initial segments and nodes of Ranvier in neurons, and βIV-spectrin dysfunction underlies ataxia and early death in mice. Despite advances in βIV-spectrin research in the nervous system, its role in pancreatic islet biology is unknown. Here, we report that βIV-spectrin serves as a multifunctional structural and signaling platform in the pancreatic islet. We report that βIV-spectrin directly associates with and targets the calcium/calmodulin-dependent protein kinase II (CaMKII) in pancreatic islets. In parallel, βIV-spectrin targets ankyrin-B and the ATP-sensitive potassium channel. Consistent with these findings, βIV-spectrin mutant mice lacking CaMKII- or ankyrin-binding motifs display selective loss of expression and targeting of key protein components, including CaMKIIδ. βIV-Spectrin–targeted CaMKII directly phosphorylates the inwardly-rectifying potassium channel, Kir6.2 (alpha subunit of KATP channel complex), and we identify the specific residue, Kir6.2 T224, responsible for CaMKII-dependent regulation of KATP channel function. CaMKII-dependent phosphorylation alters channel regulation resulting in KATP channel inhibition, a cellular phenotype consistent with aberrant insulin regulation. Finally, we demonstrate aberrant KATP channel phosphorylation in βIV-spectrin mutant mice. In summary, our findings establish a broader role for βIV-spectrin in regulation of cell membrane excitability in the pancreatic islet, define the pathway for CaMKII local control in pancreatic beta cells, and identify the mechanism for CaMKII-dependent regulation of KATP channels. PMID:24101510

  8. Lipodystrophy in human immunodeficiency virus patients impairs insulin action and induces defects in beta-cell function.

    PubMed

    Andersen, Ove; Haugaard, Steen B; Andersen, Ulrik B; Friis-Møller, Nina; Storgaard, Heidi; Vølund, Aage; Nielsen, Jens Ole; Iversen, Johan; Madsbad, Sten

    2003-10-01

    The pathophysiology of insulin resistance in human immunodeficiency virus (HIV)-associated lipodystrophy syndrome (HALS) is not fully clarified. We investigated 18 men with HALS and 18 HIV-positive males without lipodystrophy (control subjects). Duration and modality of antiretroviral therapy were similar between study groups. A hyperinsulinemic euglycemic clamp showed an impaired glucose disposal rate (GDR) in HALS patients (5.6 v 8.3 mg glucose/min. kg(FFM), P =.0006). As demonstrated by indirect calorimetry, HALS patients showed an impaired nonoxidative glucose metabolism (NOGM, 2.2 v 4.2, P =.006), whereas levels of basal and insulin-stimulated oxidative glucose metabolism (OGM) (2.4 v 2.3, P =.55, and 3.3 v 4.0, P =.064, respectively) were not significantly different between groups. Despite comparable total fat masses, dual energy x-ray absorptiometry (DEXA) scans showed that the percentage of limb fat (ie, peripheral-fat-mass/[peripheral-fat-mass + trunk-fat-mass]. 100%) was reduced in HALS patients (36% v 46%, P =.0002). Multiple linear regression analysis indicated that percentage of limb fat explained 53% of the variability of GDR and 45% of the variability of NOGM in HALS patients. In HALS patients, leg fat mass correlated positively with NOGM (r =.51, P <.05), whereas abdominal fat mass and NOGM did not correlate (P =.91). Analyzing the relationship between first phase insulin secretion and insulin sensitivity, 6 HALS patients compared with none of the control subjects exhibited impaired insulin secretion (P <.05). Our data suggest that fat redistribution independently of antiretroviral therapy is highly related to insulin resistance in HALS patients. Furthermore, in HALS patients, impaired glucose metabolism most likely relates to decreased NOGM and to defects in beta-cell function. PMID:14564688

  9. βIV-Spectrin and CaMKII facilitate Kir6.2 regulation in pancreatic beta cells.

    PubMed

    Kline, Crystal F; Wright, Patrick J; Koval, Olha M; Zmuda, Erik J; Johnson, Benjamin L; Anderson, Mark E; Hai, Tsonwin; Hund, Thomas J; Mohler, Peter J

    2013-10-22

    Identified over a dozen years ago in the brain and pancreatic islet, βIV-spectrin is critical for the local organization of protein complexes throughout the nervous system. βIV-Spectrin targets ion channels and adapter proteins to axon initial segments and nodes of Ranvier in neurons, and βIV-spectrin dysfunction underlies ataxia and early death in mice. Despite advances in βIV-spectrin research in the nervous system, its role in pancreatic islet biology is unknown. Here, we report that βIV-spectrin serves as a multifunctional structural and signaling platform in the pancreatic islet. We report that βIV-spectrin directly associates with and targets the calcium/calmodulin-dependent protein kinase II (CaMKII) in pancreatic islets. In parallel, βIV-spectrin targets ankyrin-B and the ATP-sensitive potassium channel. Consistent with these findings, βIV-spectrin mutant mice lacking CaMKII- or ankyrin-binding motifs display selective loss of expression and targeting of key protein components, including CaMKIIδ. βIV-Spectrin-targeted CaMKII directly phosphorylates the inwardly-rectifying potassium channel, Kir6.2 (alpha subunit of KATP channel complex), and we identify the specific residue, Kir6.2 T224, responsible for CaMKII-dependent regulation of KATP channel function. CaMKII-dependent phosphorylation alters channel regulation resulting in KATP channel inhibition, a cellular phenotype consistent with aberrant insulin regulation. Finally, we demonstrate aberrant KATP channel phosphorylation in βIV-spectrin mutant mice. In summary, our findings establish a broader role for βIV-spectrin in regulation of cell membrane excitability in the pancreatic islet, define the pathway for CaMKII local control in pancreatic beta cells, and identify the mechanism for CaMKII-dependent regulation of KATP channels. PMID:24101510

  10. Calpains, mitochondria, and apoptosis

    PubMed Central

    Smith, Matthew A.; Schnellmann, Rick G.

    2012-01-01

    Mitochondrial activity is critical for efficient function of the cardiovascular system. In response to cardiovascular injury, mitochondrial dysfunction occurs and can lead to apoptosis and necrosis. Calpains are a 15-member family of Ca2+-activated cysteine proteases localized to the cytosol and mitochondria, and several have been shown to regulate apoptosis and necrosis. For example, in endothelial cells, Ca2+ overload causes mitochondrial calpain 1 cleavage of the Na+/Ca2+ exchanger leading to mitochondrial Ca2+ accumulation. Also, activated calpain 1 cleaves Bid, inducing cytochrome c release and apoptosis. In renal cells, calpains 1 and 2 promote apoptosis and necrosis by cleaving cytoskeletal proteins, which increases plasma membrane permeability and cleavage of caspases. Calpain 10 cleaves electron transport chain proteins, causing decreased mitochondrial respiration and excessive activation, or inhibition of calpain 10 activity induces mitochondrial dysfunction and apoptosis. In cardiomyocytes, calpain 1 activates caspase 3 and poly-ADP ribose polymerase during tumour necrosis factor-α-induced apoptosis, and calpain 1 cleaves apoptosis-inducing factor after Ca2+ overload. Many of these observations have been elucidated with calpain inhibitors, but most calpain inhibitors are not specific for calpains or a specific calpain family member, creating more questions. The following review will discuss how calpains affect mitochondrial function and apoptosis within the cardiovascular system. PMID:22581845

  11. Delayed-rectifier (KV2.1) regulation of pancreatic beta-cell calcium responses to glucose: inhibitor specificity and modeling.

    PubMed

    Tamarina, Natalia A; Kuznetsov, Andrey; Fridlyand, Leonid E; Philipson, Louis H

    2005-10-01

    The delayed-rectifier (voltage-activated) K(+) conductance (K(V)) in pancreatic islet beta-cells has been proposed to regulate plasma membrane repolarization during responses to glucose, thereby determining bursting and Ca(2+) oscillations. Here, we verified the expression of K(V)2.1 channel protein in mouse and human islets of Langerhans. We then probed the function of K(V)2.1 channels in islet glucose responses by comparing the effect of hanatoxin (HaTx), a specific blocker of K(V)2.1 channels, with a nonspecific K(+) channel blocker, tetraethylammonium (TEA). Application of HaTx (1 microM) blocked delayed-rectifier currents in mouse beta-cells, resulting in a 40-mV rightward shift in threshold of activation of the voltage-dependent outward current. In the presence of HaTx, there was negligible voltage-activated outward current below 0 mV, suggesting that K(V)2.1 channels form the predominant part of this current in the physiologically relevant range. We then employed HaTx to study the role of K(V)2.1 in the beta-cell Ca(2+) responses to elevated glucose in comparison with TEA. Only HaTx was able to induce slow intracellular Ca(2+) concentration ([Ca(2+)](i)) oscillations in cells stimulated with 20 mM glucose, whereas TEA induced an immediate rise in [Ca(2+)](i) followed by rapid oscillations. In human islets, HaTx acted in a similar fashion. The data were analyzed using a detailed mathematical model of ionic flux and Ca(2+) regulation in beta-cells. The results can be explained by a specific HaTx effect on the K(V) current, whereas TEA affects multiple K(+) conductances. The results underscore the importance of K(V)2.1 channel in repolarization of the pancreatic beta-cell plasma membrane and its role in regulating insulin secretion. PMID:16014354

  12. Fluorescence microscopy studies with a fluorescent glibenclamide derivative, a high-affinity blocker of pancreatic beta-cell ATP-sensitive K+ currents.

    PubMed

    Zünkler, Bernd J; Wos-Maganga, Maria; Panten, Uwe

    2004-04-15

    Hypoglycemic sulfonylureas (e.g. tolbutamide, glibenclamide) exert their stimulatory effects on pancreatic beta-cells by closure of ATP-sensitive K(+) (K(ATP)) channels. Pancreatic K(ATP) channels are composed of two subunits, a pore-forming inwardly rectifying K(+) channel (Kir6.2) subunit and a regulatory subunit (the sulfonylurea receptor of subtype 1 (SUR1)) in a (SUR1/Kir6.2)(4) stoichiometry. The aim of the present study was to characterize the interaction of green-fluorescent 3-[3-(4,4 difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-S-indacen-3-yl)propanamido] glibenclamide (Bodipy-glibenclamide) with pancreatic beta-cell K(ATP) channels using patch-clamp and fluorescence microscopy techniques. Bodipy-glibenclamide inhibited K(ATP) currents from the clonal insulinoma cell line RINm5F half-maximally at a concentration of 0.6nM. Using laser-scanning confocal microscopy Bodipy-glibenclamide was shown to induce a diffuse fluorescence across the RINm5F cell, but only about 17% of total Bodipy-glibenclamide-induced fluorescence intensity in RINm5F cells was due to specific binding to SUR1. Using fluorescence correlation spectroscopy, it could be demonstrated that the fluorescence label contributes to the protein binding and, therefore, possibly also to the non-specific binding of Bodipy-glibenclamide observed in RINm5F cells. Specific binding of Bodipy-glibenclamide to SUR1 in RINm5F cells might be localized to different intracellular structures (nuclear envelope, endoplasmic reticulum, Golgi compartment, insulin secretory granules) as well as to the plasma membrane. In conclusion, Bodipy-glibenclamide is a high-affinity blocker of pancreatic beta-cell K(ATP) currents and can be used for visualizing SUR1 in intact pancreatic beta-cells, although non-specific binding must be taken into account in confocal microscopy experiments on intact beta-cells. PMID:15041461

  13. Recent advances in the Okamoto model: the CD38-cyclic ADP-ribose signal system and the regenerating gene protein (Reg)-Reg receptor system in beta-cells.

    PubMed

    Okamoto, Hiroshi; Takasawa, Shin

    2002-12-01

    Twenty years ago, we first proposed our hypothesis on beta-cell damage and its prevention (the Okamoto model), according to which poly(ADP-ribose) synthetase/polymerase (PARP) activation is critically involved in the consumption of NAD(+), leading to energy depletion and cell death by necrosis. Recently, the model was reconfirmed by results using PARP knockout mice and has been recognized as providing the basis for necrotic death of various cells and tissues. Based on the model, we proposed two signal systems in beta-cells: one is the CD38-cyclic ADP-ribose (cADPR) signal system for insulin secretion, and the other is the regenerating gene protein (Reg)-Reg receptor system for beta-cell regeneration. The physiological and pathological significance of the two signal systems in a variety of cells and tissues as well as in pancreatic beta-cells has recently been recognized. Here, we describe the Okamoto model and its descendents, the CD38-cADPR signal system and the Reg-Reg receptor system, focusing on recent advances and how their significance came to light. Because PARP is involved in Reg gene transcription to induce beta-cell regeneration, and the PARP activation reduces the cellular NAD(+) to decrease the formation of cADPR (a second messenger for insulin secretion) and further to cause necrotic beta-cell death, PARP and its inhibitors have key roles in the induction of beta-cell regeneration, the maintenance of insulin secretion, and the prevention of beta-cell death. PMID:12475791

  14. The transcription factor C/EBP delta has anti-apoptotic and anti-inflammatory roles in pancreatic beta cells.

    PubMed

    Moore, Fabrice; Santin, Izortze; Nogueira, Tatiane C; Gurzov, Esteban N; Marselli, Lorella; Marchetti, Piero; Eizirik, Decio L

    2012-01-01

    In the course of Type 1 diabetes pro-inflammatory cytokines (e.g., IL-1β, IFN-γ and TNF-α) produced by islet-infiltrating immune cells modify expression of key gene networks in β-cells, leading to local inflammation and β-cell apoptosis. Most known cytokine-induced transcription factors have pro-apoptotic effects, and little is known regarding "protective" transcription factors. To this end, we presently evaluated the role of the transcription factor CCAAT/enhancer binding protein delta (C/EBPδ) on β-cell apoptosis and production of inflammatory mediators in the rat insulinoma INS-1E cells, in purified primary rat β-cells and in human islets. C/EBPδ is expressed and up-regulated in response to the cytokines IL-1β and IFN-γ in rat β-cells and human islets. Small interfering RNA-mediated C/EBPδ silencing exacerbated IL-1β+IFN-γ-induced caspase 9 and 3 cleavage and apoptosis in these cells. C/EBPδ deficiency increased the up-regulation of the transcription factor CHOP in response to cytokines, enhancing expression of the pro-apoptotic Bcl-2 family member BIM. Interfering with C/EBPδ and CHOP or C/EBPδ and BIM in double knockdown approaches abrogated the exacerbating effects of C/EBPδ deficiency on cytokine-induced β-cell apoptosis, while C/EBPδ overexpression inhibited BIM expression and partially protected β-cells against IL-1β+IFN-γ-induced apoptosis. Furthermore, C/EBPδ silencing boosted cytokine-induced production of the chemokines CXCL1, 9, 10 and CCL20 in β-cells by hampering IRF-1 up-regulation and increasing STAT1 activation in response to cytokines. These observations identify a novel function of C/EBPδ as a modulatory transcription factor that inhibits the pro-apoptotic and pro-inflammatory gene networks activated by cytokines in pancreatic β-cells. PMID:22347430

  15. The Rab-binding protein Noc2 is associated with insulin-containing secretory granules and is essential for pancreatic beta-cell exocytosis.

    PubMed

    Cheviet, Séverine; Coppola, Thierry; Haynes, Lee P; Burgoyne, Robert D; Regazzi, Romano

    2004-01-01

    The small GTPases Rab3 and Rab27 are associated with secretory granules of pancreatic beta-cells and regulate insulin exocytosis. In this study, we investigated the role of Noc2, a potential partner of these two GTPases, in insulin secretion. In the beta-cell line INS-1E wild-type Noc2, Noc265E, and Noc258A, a mutant capable of interacting with Rab27 but not Rab3, colocalized with insulin-containing vesicles. In contrast, two mutants (Noc2138S,141S and Noc2154A,155A,156A) that bind neither Rab3 nor Rab27 did not associate with secretory granules and were uniformly distributed throughout the cell cytoplasm. Overexpression of wild-type Noc2, Noc265E, or Noc258A inhibited hormone secretion elicited by insulin secretagogues. In contrast, overexpression of the mutants not targeted to secretory granules was without effect. Silencing of the Noc2 gene by RNA interference led to a strong impairment in the capacity of INS-1E cells to respond to insulin secretagogues, indicating that appropriate levels of Noc2 are essential for pancreatic beta-cell exocytosis. The defect was already detectable in the early secretory phase (0-10 min) but was particularly evident during the sustained release phase (10-45 min). Protein-protein binding studies revealed that Noc2 is a potential partner of Munc13, a component of the machinery that controls vesicle priming and insulin exocytosis. These data suggest that Noc2 is involved in the recruitment of secretory granules at the plasma membrane possibly via the interaction with Munc13. PMID:14593078

  16. Differentiation and transplantation of functional pancreatic beta cells generated from induced pluripotent stem cells derived from a type 1 diabetes mouse model.

    PubMed

    Jeon, Kilsoo; Lim, Hyejin; Kim, Jung-Hyun; Thuan, Nguyen Van; Park, Seung Hwa; Lim, Yu-Mi; Choi, Hye-Yeon; Lee, Eung-Ryoung; Kim, Jin-Hoi; Lee, Myung-Shik; Cho, Ssang-Goo

    2012-09-20

    The nonobese diabetic (NOD) mouse is a classical animal model for autoimmune type 1 diabetes (T1D), closely mimicking features of human T1D. Thus, the NOD mouse presents an opportunity to test the effectiveness of induced pluripotent stem cells (iPSCs) as a therapeutic modality for T1D. Here, we demonstrate a proof of concept for cellular therapy using NOD mouse-derived iPSCs (NOD-iPSCs). We generated iPSCs from NOD mouse embryonic fibroblasts or NOD mouse pancreas-derived epithelial cells (NPEs), and applied directed differentiation protocols to differentiate the NOD-iPSCs toward functional pancreatic beta cells. Finally, we investigated whether the NPE-iPSC-derived insulin-producing cells could normalize hyperglycemia in transplanted diabetic mice. The NOD-iPSCs showed typical embryonic stem cell-like characteristics such as expression of markers for pluripotency, in vitro differentiation, teratoma formation, and generation of chimeric mice. We developed a method for stepwise differentiation of NOD-iPSCs into insulin-producing cells, and found that NPE-iPSCs differentiate more readily into insulin-producing cells. The differentiated NPE-iPSCs expressed diverse pancreatic beta cell markers and released insulin in response to glucose and KCl stimulation. Transplantation of the differentiated NPE-iPSCs into diabetic mice resulted in kidney engraftment. The engrafted cells responded to glucose by secreting insulin, thereby normalizing blood glucose levels. We propose that NOD-iPSCs will provide a useful tool for investigating genetic susceptibility to autoimmune diseases and generating a cellular interaction model of T1D, paving the way for the potential application of patient-derived iPSCs in autologous beta cell transplantation for treating diabetes. PMID:22512788

  17. PTBP1 is required for glucose-stimulated cap-independent translation of insulin granule proteins and Coxsackieviruses in beta cells.

    PubMed

    Knoch, Klaus-Peter; Nath-Sain, Suchita; Petzold, Antje; Schneider, Hendryk; Beck, Mike; Wegbrod, Carolin; Sönmez, Anke; Münster, Carla; Friedrich, Anne; Roivainen, Merja; Solimena, Michele

    2014-08-01

    Glucose and GLP-1 stimulate not only insulin secretion, but also the post-transcriptional induction of insulin granule biogenesis. This process involves the nucleocytoplasmic translocation of the RNA binding protein PTBP1. Binding of PTBP1 to the 3'-UTRs of mRNAs for insulin and other cargoes of beta cell granules increases their stability. Here we show that glucose enhances also the binding of PTBP1 to the 5'-UTRs of these transcripts, which display IRES activity, and their translation exclusively in a cap-independent fashion. Accordingly, glucose-induced biosynthesis of granule cargoes was unaffected by pharmacological, genetic or Coxsackievirus-mediated inhibition of cap-dependent translation. Infection with Coxsackieviruses, which also depend on PTBP1 for their own cap-independent translation, reduced instead granule stores and insulin release. These findings provide insight into the mechanism for glucose-induction of insulin granule production and on how Coxsackieviruses, which have been implicated in the pathogenesis of type 1 diabetes, can foster beta cell failure. PMID:25061557

  18. The relationship between node degree and dissipation rate in networks of diffusively coupled oscillators and its significance for pancreatic beta cells

    NASA Astrophysics Data System (ADS)

    Gosak, Marko; Stožer, Andraž; Markovič, Rene; Dolenšek, Jurij; Marhl, Marko; Slak Rupnik, Marjan; Perc, Matjaž

    2015-07-01

    Self-sustained oscillatory dynamics is a motion along a stable limit cycle in the phase space, and it arises in a wide variety of mechanical, electrical, and biological systems. Typically, oscillations are due to a balance between energy dissipation and generation. Their stability depends on the properties of the attractor, in particular, its dissipative characteristics, which in turn determine the flexibility of a given dynamical system. In a network of oscillators, the coupling additionally contributes to the dissipation, and hence affects the robustness of the oscillatory solution. Here, we therefore investigate how a heterogeneous network structure affects the dissipation rate of individual oscillators. First, we show that in a network of diffusively coupled oscillators, the dissipation is a linearly decreasing function of the node degree, and we demonstrate this numerically by calculating the average divergence of coupled Hopf oscillators. Subsequently, we use recordings of intracellular calcium dynamics in pancreatic beta cells in mouse acute tissue slices and the corresponding functional connectivity networks for an experimental verification of the presented theory. We use methods of nonlinear time series analysis to reconstruct the phase space and calculate the sum of Lyapunov exponents. Our analysis reveals a clear tendency of cells with a higher degree, that is, more interconnected cells, having more negative values of divergence, thus confirming our theoretical predictions. We discuss these findings in the context of energetic aspects of signaling in beta cells and potential risks for pathological changes in the tissue.

  19. Free radicals and the pathogenesis of type 1 diabetes: beta-cell cytokine-mediated free radical generation via cyclooxygenase-2.

    PubMed

    Tabatabaie, Tahereh; Vasquez-Weldon, Angelica; Moore, Danny R; Kotake, Yashige

    2003-08-01

    Free radical formation evoked by proinflammatory cytokines has been suggested to be involved in the destruction of beta-cells in the course of type 1 diabetes development. However, there is no direct evidence to support this hypothesis. In this study, we used electron paramagnetic resonance spectroscopy in conjunction with spin-trapping methodology to directly determine whether cytokines give rise to free radical formation in the islets. Our results demonstrate that direct, in vivo administration of tumor necrosis factor-alpha (1,000 units), interleukin-1beta (1,000 units), and interferon-gamma (2,000 units) into the rat pancreas through a bile duct cannula leads to the formation of lipid-derived free radicals in this tissue. These free radicals most likely are generated by the beta-cells because previous depletion of these cells by streptozotocin abolished the cytokine-induced free radical formation. Furthermore, macrophage depletion was found to decrease the production of free radicals. Inhibition of the enzyme inducible cyclooxygenase (COX-2) and the transcription factor nuclear factor-kappaB (NF-kappaB) significantly diminished the free radicals' signal intensity, implicating these factors in the formation of free radicals. We have also demonstrated that cytokine treatment leads to the activation of NF-kappaB in the pancreatic islets of the rats. PMID:12882915

  20. Improved glucose regulation in type 2 diabetic patients with DPP-4 inhibitors: focus on alpha and beta cell function and lipid metabolism.

    PubMed

    Ahrén, Bo; Foley, James E

    2016-05-01

    Inhibition of dipeptidyl peptidase-4 (DPP-4) is an established glucose-lowering strategy for the management of type 2 diabetes mellitus. DPP-4 inhibitors reduce both fasting and postprandial plasma glucose levels, resulting in reduced HbA1c with low risk for hypoglycaemia and weight gain. They act primarily by preventing inactivation of the incretin hormones glucose-dependent insulinotropic polypeptide and glucagon-like peptide-1, thereby prolonging the enhanced endogenous levels of these hormones after meal ingestion. This in turn causes islet and extrapancreatic effects, including increased glucose sensing in islet alpha and beta cells. These effects result in increased insulin secretion and decreased glucagon secretion being more effective in hyperglycaemic states and reduced insulin secretion and increased glucagon secretion being more effective during hypoglycaemia. Other secondary pharmacological actions of DPP-4 inhibitors include mobilisation and burning of fat during meals, decrease in fat extraction from the gut, reduction of fasting lipolysis and liver fat and increase in LDL particle size. These actions contribute to the clinical effects of DPP-4 inhibition, and the reduced demand for insulin could also lead to a durability benefit. This review summarises the current knowledge of the secondary pharmacological actions of DPP-4 inhibitors that lead to improved glucose regulation in patients with type 2 diabetes, focusing on alpha and beta cell function and lipid metabolism. PMID:26894277

  1. Glucose enhances insulin promoter activity in MIN6 beta-cells independently of changes in intracellular Ca2+ concentration and insulin secretion.

    PubMed Central

    Kennedy, H J; Rafiq, I; Pouli, A E; Rutter, G A

    1999-01-01

    Recent studies have suggested that glucose may activate insulin gene transcription through increases in intracellular Ca(2+) concentration, possibly acting via the release of stored insulin. We have investigated this question by dynamic photon-counting imaging of insulin- and c-fos-promoter-firefly luciferase reporter construct activity. Normalized to constitutive viral promoter activity, insulin promoter activity in MIN6 beta-cells was increased 1.6-fold after incubation at 30 mM compared with 3 mM glucose, but was unaltered at either glucose concentration by the presence of insulin (100 nM) or the Ca(2+) channel inhibitor, verapamil (100 microM). Increases in intracellular [Ca(2+)] achieved by plasma membrane depolarization with KCl failed to enhance either insulin or c-fos promoter activity in MIN6 cells, but increased c-fos promoter activity 5-fold in AtT20 cells. Together, these results demonstrate that glucose can exert a direct effect on insulin promoter activity in islet beta-cells, via a signalling pathway which does not require increases in intracellular [Ca(2+)] nor insulin release and insulin receptor activation. PMID:10455011

  2. The relationship between node degree and dissipation rate in networks of diffusively coupled oscillators and its significance for pancreatic beta cells.

    PubMed

    Gosak, Marko; Stožer, Andraž; Markovič, Rene; Dolenšek, Jurij; Marhl, Marko; Rupnik, Marjan Slak; Perc, Matjaž

    2015-07-01

    Self-sustained oscillatory dynamics is a motion along a stable limit cycle in the phase space, and it arises in a wide variety of mechanical, electrical, and biological systems. Typically, oscillations are due to a balance between energy dissipation and generation. Their stability depends on the properties of the attractor, in particular, its dissipative characteristics, which in turn determine the flexibility of a given dynamical system. In a network of oscillators, the coupling additionally contributes to the dissipation, and hence affects the robustness of the oscillatory solution. Here, we therefore investigate how a heterogeneous network structure affects the dissipation rate of individual oscillators. First, we show that in a network of diffusively coupled oscillators, the dissipation is a linearly decreasing function of the node degree, and we demonstrate this numerically by calculating the average divergence of coupled Hopf oscillators. Subsequently, we use recordings of intracellular calcium dynamics in pancreatic beta cells in mouse acute tissue slices and the corresponding functional connectivity networks for an experimental verification of the presented theory. We use methods of nonlinear time series analysis to reconstruct the phase space and calculate the sum of Lyapunov exponents. Our analysis reveals a clear tendency of cells with a higher degree, that is, more interconnected cells, having more negative values of divergence, thus confirming our theoretical predictions. We discuss these findings in the context of energetic aspects of signaling in beta cells and potential risks for pathological changes in the tissue. PMID:26232966

  3. Apoptosis in Anthracycline Cardiomyopathy

    PubMed Central

    Shi, Jianjian; Abdelwahid, Eltyeb; Wei, Lei

    2011-01-01

    Apoptosis is a tightly regulated physiologic process of programmed cell death that occurs in both normal and pathologic tissues. Numerous in vitro or in vivo studies have indicated that cardiomyocyte death through apoptosis and necrosis is a primary contributor to the progression of anthracycline-induced cardiomyopathy. There are now several pieces of evidence to suggest that activation of intrinsic and extrinsic apoptotic pathways contribute to anthracycline-induced apoptosis in the heart. Novel strategies were developed to address a wide variety of cardiotoxic mechanisms and apoptotic pathways by which anthracycline influences cardiac structure and function. Anthracycline-induced apoptosis provides a very valid representation of cardiotoxicity in the heart, an argument which has implications for the most appropriate animal models of damaged heart plus diverse pharmacological effects. In this review we describe various aspects of the current understanding of apoptotic cell death triggered by anthracycline. Differences in the sensitivity to anthracycline-induced apoptosis between young and adult hearts are also discussed. PMID:22212952

  4. Spaceflight Associated Apoptosis

    NASA Technical Reports Server (NTRS)

    Ichiki, Albert T.; Gibson, Linda A.; Allebban, Zuhair

    1996-01-01

    Lymphoid tissues have been shown to atrophy in rats flown on Russian spaceflights. Histological examination indicated evidence for cell degradation. Lymphoid tissues from rats flown on Spacelab Life Sciences-2 mission were analyzed for apoptosis by evidence of fragmented lymphocytes, which could be engulfed by macrophages, or DNA strand breaks using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. Apoptosis was not detected in the thymus and spleen collected inflight or from the synchronous ground rats but was detected in the thymus, spleen and inguinal lymph node of the flight animals on recovery. These results indicate that the apoptosis observed in the lymphatic tissues of the rats on recovery could have been induced by the gravitational stress of reentry, corroborating the findings from the early space-flight observations.

  5. [Sphingolipid and apoptosis].

    PubMed

    Wang, Jing; Hu, Xiao-Song; Shi, Jie-Ping

    2003-07-01

    Over the last decade, considerable progress has been made in the study of sphingolipids with the development of biological techniques. Sphingolipids play important roles in diverse physiological process, including cytoskeleton migration, angiogenesis, embryonic development and signal transduction. Except for this, the lastest evidence has suggested that sphingolipids and their metabolite (ceramide, sphingosine, sphingosine 1-phosphate) can induce apoptosis in a wide variety of tumor cell lines such as LoVo HT29, Bel7402, A549, CNE2 cells. This paper is attempted to review the recent advances of investigation into the relationship between sphingolipids and apoptosis. PMID:14628466

  6. [Apoptosis during embryo development].

    PubMed

    Jezek, Davor; Kozina, Viviana

    2009-10-01

    The development of human embryo includes two essential processes, i.e., rapid mitotic activity of cells and gradual differentiation of tissues and organs. The latter process is very often characterized by extensive migration of cells from their site of origin to the site of definitive location, inductive action of the neighboring germ layers and programmed cell death (apoptosis). This paper describes examples of proliferative and apoptotic processes during the development of human embryo. The development of trilaminar germ disk, skin, gonads, central and peripheral nerve system as well as limbs provides instructive examples of how apoptosis regulates the development and differentiation of cells. PMID:19999545

  7. Apoptosis in colorectal cancer.

    PubMed

    Stoian, M; State, N; Stoica, V; Radulian, G

    2014-06-15

    Apoptosis is an inborn process that has been preserved during evolution; it allows the cells to systematically inactivate, destroy and dispose of their own components thus leading to their death. This programme can be activated by both intra and extracellular mechanisms. The intracellular components involve a genetically defined development programme while the extracellular aspects regard endogenous proteins, cytokines and hormones as well as xenobiotics, radiations, oxidative stress and hypoxia. The ability of a cell to enter apoptosis as a response to a "death" signal depends on its proliferative status, the position in the cell cycle and also on the controlled expression of those genes that have the capacity of promoting and inhibiting cell death. The fine regulation of these parameters needs to be maintained in order to ensure the physiological environment required for the induction of apoptosis. Any malfunction in any of the steps of controlled cellular death can lead to dysfunctions and, as a consequence, to different pathological conditions. The importance of apoptosis lies in its active nature and in the potential of controlling biological systems. PMID:25408720

  8. Oxidative stress-mediated, post-translational loss of MafA protein as a contributing mechanism to loss of insulin gene expression in glucotoxic beta cells.

    PubMed

    Harmon, Jamie S; Stein, Roland; Robertson, R Paul

    2005-03-25

    Glucose toxicity in pancreatic islet beta cells causes loss of insulin gene expression, content, and secretion due to loss of binding of transcription factors, most notably PDX-1 and RIPE-3b1 activator, to the promoter region of the insulin gene. Recently, RIPE-3b1 activator was cloned and identified as the mammalian homologue of avian MafA/Maf-L (MafA). This enabled us to carry out more extensive studies of the role of MafA in glucotoxicity than were hitherto possible. Northern analysis of glucotoxic HIT-T15 cells revealed normal amounts of MafA mRNA, but Western analysis demonstrated a 97 +/- 1% reduction in MafA protein (p < 0.0001). The proteasome is a likely site for MafA degradation as lactacystin, an irreversible proteasome inhibitor, caused an accumulation of MafA protein. Antioxidants have previously been shown to prevent the adverse effects of glucose toxicity on beta cell function both in vivo and in vitro. In the current study, chronic culturing of HIT-T15 cells with the antioxidant N-acetylcysteine (NAC) prevented loss of MafA protein (late passage = 18.9 +/- 10.4% of early passage, p < 0.001; late passage with NAC = 68.7 +/- 19.7% of early passage, p = not significant) and loss of DNA binding (late passage = 63.7 +/- 9% of early passage, p < 0.02; late passage with NAC = 116 +/- 10% of early passage, p = not significant). Additionally, transient transfection of PDX-1 or MafA cDNA into glucotoxic cells increased PDX-1 and MafA protein levels and individually increased insulin promoter activity (untreated = 34%, PDX-1 = 70%, MafA = 78%; percentage of activity of early passage cells), whereas the combined transfection of MafA and PDX-1 completely restored insulin promoter activity. This recovery of promoter activity following transient transfection had no effect on endogenous insulin mRNA. However, adenoviral infection of MafA and PDX-1 significantly increased endogenous insulin mRNA levels by 93% (121 +/- 9 versus 233 +/- 18 density light units; n = 5

  9. Calbindin-D(28k) controls [Ca(2+)](i) and insulin release. Evidence obtained from calbindin-d(28k) knockout mice and beta cell lines

    NASA Technical Reports Server (NTRS)

    Sooy, K.; Schermerhorn, T.; Noda, M.; Surana, M.; Rhoten, W. B.; Meyer, M.; Fleischer, N.; Sharp, G. W.; Christakos, S.

    1999-01-01

    The role of the calcium-binding protein, calbindin-D(28k) in potassium/depolarization-stimulated increases in the cytosolic free Ca(2+) concentration ([Ca(2+)](i)) and insulin release was investigated in pancreatic islets from calbindin-D(28k) nullmutant mice (knockouts; KO) or wild type mice and beta cell lines stably transfected and overexpressing calbindin. Using single islets from KO mice and stimulation with 45 mM KCl, the peak of [Ca(2+)](i) was 3.5-fold greater in islets from KO mice compared with wild type islets (p < 0.01) and [Ca(2+)](i) remained higher during the plateau phase. In addition to the increase in [Ca(2+)](i) in response to KCl there was also a significant increase in insulin release in islets isolated from KO mice. Evidence for modulation by calbindin of [Ca(2+)](i) and insulin release was also noted using beta cell lines. Rat calbindin was stably expressed in betaTC-3 and betaHC-13 cells. In response to depolarizing concentrations of K(+), insulin release was decreased by 45-47% in calbindin expressing betaTC cells and was decreased by 70-80% in calbindin expressing betaHC cells compared with insulin release from vector transfected betaTC or betaHC cells (p < 0.01). In addition, the K(+)-stimulated intracellular calcium peak was markedly inhibited in calbindin expressing betaHC cells compared with vector transfected cells (225 nM versus 1,100 nM, respectively). Buffering of the depolarization-induced rise in [Ca(2+)](i) was also observed in calbindin expressing betaTC cells. In summary, our findings, using both isolated islets from calbindin-D(28k) KO mice and beta cell lines, establish a role for calbindin in the modulation of depolarization-stimulated insulin release and suggest that calbindin can control the rate of insulin release via regulation of [Ca(2+)](i).

  10. Multiple low-dose streptozotocin-induced diabetes in the mouse. Evidence for stimulation of a cytotoxic cellular immune response against an insulin-producing beta cell line.

    PubMed Central

    McEvoy, R C; Andersson, J; Sandler, S; Hellerström, C

    1984-01-01

    Mice were examined for the presence of splenocytes specifically cytotoxic for a rat insulinoma cell line (RIN) during the induction of diabetes by streptozotocin (SZ) in multiple low doses (Multi-Strep). Cytotoxicity was quantitated by the release of 51Cr from damaged cells. A low but statistically significant level of cytolysis (5%) by splenocytes was first detectable on day 8 after the first dose of SZ. The cytotoxicity reached a maximum of approximately 9% on day 10 and slowly decreased thereafter, becoming undetectable 42 d after SZ was first given. The time course of the in vitro cytotoxic response correlated with the degree of insulitis demonstrable in the pancreata of the Multi-Strep mice. The degree of cytotoxicity after Multi-Strep was related to the number of effector splenocytes to which the target RIN cells were exposed and was comparable to that detectable after immunization by intraperitoneal injection of RIN cells in normal mice. The cytotoxicity was specific for insulin-producing cells; syngeneic, allogeneic, and xenogeneic lymphocytes and lymphoblasts, 3T3 cells, and a human keratinocyte cell line were not specifically lysed by the splenocytes of the Multi-Strep mice. This phenomenon was limited to the Multi-Strep mice. Splenocytes from mice made diabetic by a single, high dose of SZ exhibited a very low level of cytotoxicity against the RIN cells. The cytotoxic response was also quantitated in splenocytes from control and Multi-Strep mice (10 d after the first dose of SZ) before and after culture with mitomycin-treated RIN cells in the presence of T cell growth factor (TCGF). The cytotoxicity of the Multi-Strep splenocytes was enhanced more than fivefold after such culture, suggesting the proliferation of an effector cell that could be stimulated and supported in vitro by TCGF. These results support the hypothesis that cell-mediated anti-beta cell autoimmunity may play a role in the destruction of the beta cells in this animal model. The

  11. Dynamic magnetic fields remote-control apoptosis via nanoparticle rotation.

    PubMed

    Zhang, Enming; Kircher, Moritz F; Koch, Martin; Eliasson, Lena; Goldberg, S Nahum; Renström, Erik

    2014-04-22

    The ability to control the movement of nanoparticles remotely and with high precision would have far-reaching implications in many areas of nanotechnology. We have designed a unique dynamic magnetic field (DMF) generator that can induce rotational movements of superparamagnetic iron oxide nanoparticles (SPIONs). We examined whether the rotational nanoparticle movement could be used for remote induction of cell death by injuring lysosomal membrane structures. We further hypothesized that the shear forces created by the generation of oscillatory torques (incomplete rotation) of SPIONs bound to lysosomal membranes would cause membrane permeabilization, lead to extravasation of lysosomal contents into the cytoplasm, and induce apoptosis. To this end, we covalently conjugated SPIONs with antibodies targeting the lysosomal protein marker LAMP1 (LAMP1-SPION). Remote activation of slow rotation of LAMP1-SPIONs significantly improved the efficacy of cellular internalization of the nanoparticles. LAMP1-SPIONs then preferentially accumulated along the membrane in lysosomes in both rat insulinoma tumor cells and human pancreatic beta cells due to binding of LAMP1-SPIONs to endogenous LAMP1. Further activation of torques by the LAMP1-SPIONs bound to lysosomes resulted in rapid decrease in size and number of lysosomes, attributable to tearing of the lysosomal membrane by the shear force of the rotationally activated LAMP1-SPIONs. This remote activation resulted in an increased expression of early and late apoptotic markers and impaired cell growth. Our findings suggest that DMF treatment of lysosome-targeted nanoparticles offers a noninvasive tool to induce apoptosis remotely and could serve as an important platform technology for a wide range of biomedical applications. PMID:24597847

  12. Streptozotocin induces endoplasmic reticulum stress and apoptosis via disruption of calcium homeostasis in mouse pancreas.

    PubMed

    Ahn, Changhwan; An, Beum-Soo; Jeung, Eui-Bae

    2015-09-01

    Calcium homeostasis refers to the regulation of calcium ion concentration in the body. This concentration is tightly controlled by a stabilizing system consisting of calcium channels and calcium buffering proteins. Calcium homeostasis is crucial for cell survival. Various forms of cell death (e.g., necrosis and apoptosis) also share calcium signaling pathways and molecular effectors. Calcium acts not only as a ubiquitous second messenger involved in apoptosis along with various cell death inducers but also a regulator for the synthesis of enzymes/hormones such as insulin. We hypothesized that streptozotocin disrupts calcium homeostasis and the altered intracellular calcium levels may induce cell death. After streptozotocin administration, blood glucose level was increased while insulin levels decreased. The expression of insulin response markers also decreased relative to the vehicle group. L-type voltage-gated calcium channel expression and sarcoplasmic reticulum Ca(2+) ATPase were increased by streptozotocin. Calcium buffering protein calbindin-D9k and calmodulin family members were also increased. The expression of genes involved in transporting calcium ions to the endoplasmic reticulum (ER) was decrease while the expression of those affecting the removal of calcium from the ER was increased. Depletion of calcium from the ER leads to ER-stress and can induce apoptosis. In the streptozotocin-treatment group, apoptosis markers were increased. Taken together, these results imply that the disruption of calcium homeostasis by streptozotocin induces ER-stress and leads to the apoptosis of pancreatic cells. Additionally, findings from this study suggest that imbalances in calcium homeostasis could promote pancreatic beta cell death and result in type I diabetes. PMID:26003140

  13. Rapamycin protects against dominant negative-HNF1A-induced apoptosis in INS-1 cells.

    PubMed

    Farrelly, Angela M; Kilbride, Seán M; Bonner, Caroline; Prehn, Jochen H M; Byrne, Maria M

    2011-11-01

    HNF1A-maturity onset diabetes of the young (HNF1A-MODY) is caused by mutations in Hnf1a gene encoding the transcription factor hepatocyte nuclear factor 1alpha (HNF1A). An increased rate of apoptosis has been associated with the decrease in beta-cell mass that is a hallmark of HNF1A-MODY and other forms of diabetes. In a cellular model of HNF1A-MODY, we have recently shown that signalling through mammalian target of rapamycin (mTOR) is decreased by the overexpression of a dominant-negative mutant of HNF1A (DN-HNF1A). mTOR is a protein kinase which has important roles in cell metabolism and growth, but also in cell survival, where it has been shown to be both protective and detrimental. Here, we show that pharmacological inhibition of mTOR activity with rapamycin protected INS-1 cells against DN-HNF1A-induced apoptosis. Rapamycin also prevented DN-HNF1A-induced activation of AMP-activated protein kinase (AMPK), an intracellular energy sensor which we have previously shown to mediate DN-HNF1A-induced apoptosis. Conversely, activation of mTOR with leucine potentiated DN-HNF1A-induced apoptosis. Gene silencing of raptor (regulatory associated protein of mTOR), a subunit of mTOR complex 1 (mTORC1), also conferred protection on INS-1 cells against DN-HNF1A-induced apoptosis, confirming that mTORC1 mediates the protective effect. The potential relevance of this effect with regards to the clinical use of rapamycin as an immunosuppressant in diabetics post-transplantation is discussed. PMID:21874357

  14. Pattern of rise in subplasma membrane Ca{sup 2+} concentration determines type of fusing insulin granules in pancreatic {beta} cells

    SciTech Connect

    Ohara-Imaizumi, Mica; Aoyagi, Kyota; Nakamichi, Yoko; Nishiwaki, Chiyono; Sakurai, Takashi; Nagamatsu, Shinya

    2009-07-31

    We simultaneously analyzed insulin granule fusion with insulin fused to green fluorescent protein and the subplasma membrane Ca{sup 2+} concentration ([Ca{sup 2+}]{sub PM}) with the Ca{sup 2+} indicator Fura Red in rat {beta} cells by dual-color total internal reflection fluorescence microscopy. We found that rapid and marked elevation in [Ca{sup 2+}]{sub PM} caused insulin granule fusion mostly from previously docked granules during the high KCl-evoked release and high glucose-evoked first phase release. In contrast, the slow and sustained elevation in [Ca{sup 2+}]{sub PM} induced fusion from newcomers translocated from the internal pool during the low KCl-evoked release and glucose-evoked second phase release. These data suggest that the pattern of the [Ca{sup 2+}]{sub PM} rise directly determines the types of fusing granules.

  15. Dilation of the endoplasmic reticulum in beta cells due to molecular overcrowding? Kinetic simulations of extension limits and consequences on proinsulin synthesis.

    PubMed

    Despa, F

    2009-03-01

    Insulin regulates the energy homeostasis of the human body. This is synthesized in the endoplasmic reticulum (ER) of pancreatic beta cells from proinsulin. Chronic hyperglycemia increases considerably the proinsulin secretion, overcrowding the ER. Recent experimental evidence demonstrates that such states favor the proinsulin denaturation. The biophysical mechanism of this cellular dysfunction remains largely unknown. We use basic molecular principles and numerical simulations of time-dependent crowding conditions in the ER to show that crowding effects enhance the propensity of proinsulin molecules to (mis)fold in compressed, nonnative structures. Present results suggest: i) misfolding events and toxic accumulations increase dramatically if the proinsulin load exceeds 50% of the available space and ii) insufficient lag time for the relaxation of the ER between consecutive proinsulin uploads can cause irreversible alterations of folding capabilities. Present study may prove useful in generating new testable statements on circumstances leading to the development of diabetes. PMID:19121888

  16. Arsenite reduces insulin secretion in rat pancreatic {beta}-cells by decreasing the calcium-dependent calpain-10 proteolysis of SNAP-25

    SciTech Connect

    Diaz-Villasenor, Andrea; Burns, Anna L.; Salazar, Ana Maria; Sordo, Monserrat; Hiriart, Marcia; Cebrian, Mariano E.; Ostrosky-Wegman, Patricia

    2008-09-15

    An increase in the prevalence of type 2 diabetes has been consistently observed among residents of high arsenic exposure areas. We have previously shown that in rat pancreatic {beta}-cells, low arsenite doses impair the secretion of insulin without altering its synthesis. To further study the mechanism by which arsenite reduces insulin secretion, we evaluated the effects of arsenite on the calcium-calpain pathway that triggers insulin exocytosis in RINm5F cells. Cell cycle and proliferation analysis were also performed to complement the characterization. Free [Ca{sup 2+}]i oscillations needed for glucose-stimulated insulin secretion were abated in the presence of subchronic low arsenite doses (0.5-2 {mu}M). The global activity of calpains increased with 2 {mu}M arsenite. However, during the secretion of insulin stimulated with glucose (15.6 mM), 1 {mu}M arsenite decreased the activity of calpain-10, measured as SNAP-25 proteolysis. Both proteins are needed to fuse insulin granules with the membrane to produce insulin exocytosis. Arsenite also induced a slowdown in the {beta} cell line proliferation in a dose-dependent manner, reflected by a reduction of dividing cells and in their arrest in G2/M. Data obtained showed that one of the mechanisms by which arsenite impairs insulin secretion is by decreasing the oscillations of free [Ca{sup 2+}]i, thus reducing calcium-dependent calpain-10 partial proteolysis of SNAP-25. The effects in cell division and proliferation observed with arsenite exposure can be an indirect consequence of the decrease in insulin secretion.

  17. Ca2+-dependent dephosphorylation of kinesin heavy chain on beta-granules in pancreatic beta-cells. Implications for regulated beta-granule transport and insulin exocytosis

    NASA Technical Reports Server (NTRS)

    Donelan, Matthew J.; Morfini, Gerardo; Julyan, Richard; Sommers, Scott; Hays, Lori; Kajio, Hiroshi; Briaud, Isabelle; Easom, Richard A.; Molkentin, Jeffery D.; Brady, Scott T.; Rhodes, Christopher J.

    2002-01-01

    The specific biochemical steps required for glucose-regulated insulin exocytosis from beta-cells are not well defined. Elevation of glucose leads to increases in cytosolic [Ca2+]i and biphasic release of insulin from both a readily releasable and a storage pool of beta-granules. The effect of elevated [Ca2+]i on phosphorylation of isolated beta-granule membrane proteins was evaluated, and the phosphorylation of four proteins was found to be altered by [Ca2+]i. One (a 18/20-kDa doublet) was a Ca2+-dependent increase in phosphorylation, and, surprisingly, three others (138, 42, and 36 kDa) were Ca2+-dependent dephosphorylations. The 138-kDa beta-granule phosphoprotein was found to be kinesin heavy chain (KHC). At low levels of [Ca2+]i KHC was phosphorylated by casein kinase 2, but KHC was rapidly dephosphorylated by protein phosphatase 2B beta (PP2Bbeta) as [Ca2+]i increased. Inhibitors of PP2B specifically reduced the second, microtubule-dependent, phase of insulin secretion, suggesting that dephosphorylation of KHC was required for transport of beta-granules from the storage pool to replenish the readily releasable pool of beta-granules. This is distinct from synaptic vesicle exocytosis, because neurotransmitter release from synaptosomes did not require a Ca2+-dependent KHC dephosphorylation. These results suggest a novel mechanism for regulating KHC function and beta-granule transport in beta-cells that is mediated by casein kinase 2 and PP2B. They also implicate a novel regulatory role for PP2B/calcineurin in the control of insulin secretion downstream of a rise in [Ca2+]i.

  18. Correlations between beta-cells' calcium dynamics reveal differences in functional connectivity patterns in islets of Langerhans from pancreas tissue slices under low and high levels of glucose

    NASA Astrophysics Data System (ADS)

    Stožer, Andraž; Gosak, Marko; Korošak, Dean; Yakubo, Kousuke; Dolenšek, Jure; Marhl, Marko; Rupnik, Marjan Slak

    2012-08-01

    In the last decade, approach developed in the frame of complex network theory has presented very successful and popular tools for studying the structure and functioning of complex systems. A particularly attractive avenue in this context is the analysis of biological systems, since structural principles of complex networks have been identified at all scales of functioning of living organisms. In the present paper, we propose the construction of a complex network representation of a pancreatic islet. In this compact microorgan, under physiological conditions the release of the single most important anabolic hormone insulin is robustly controlled by an efficient cell-to-cell communication mediated by gap junctions. Here, we extract networks of insulin releasing beta-cells from experimentally measured time series data on calcium dynamics and from positional information obtained by confocal laser-scanning functional imaging of islets in acute pancreatic tissue slices. In particular, connectivity patterns are determined on the basis of correlations between calcium dynamics in the islet. The extracted networks are then scrutinized with conventional tools for network analysis, whereby particular importance is devoted to comparison of the network structure under low and high glucose levels, i.e. physiologically resting and stimulating conditions, respectively. We show that the cellular dynamics is more correlated under stimulation and that the networks obtained in both regimes display a different organization. The range of interactions among beta cells is significantly shorter in the case of a higher stimulation. Our results thus provide novel insights into the relationship between network topology and functional organization of pancreatic islets.

  19. Unprotected daily sun exposure is differently associated with central adiposity and beta-cell dysfunction by gender: The Korean national health and nutrition examination survey (KNHANES) V

    SciTech Connect

    Ohn, Jung Hun; Kwon, In Ho; Park, Juri; Ryu, Ohk Hyun; Lee, Seong Jin; Kim, Doo-Man; Ihm, Sung-Hee; Choi, Moon-Gi; Yoo, Hyung Joon; Hong, Eun-Gyoung

    2014-08-15

    Background: Ultraviolet irradiation by sun exposure has been associated with both harms and benefits to metabolic health. Objective: The objective of this study was to determine whether unprotected daily sun exposure is associated with the prevalence of diabetes and explore the underlying mechanism. Methods: We analyzed the Korean National Health and Nutrition Survey V from 2010 to 2011. Participants 19–60 years of age were asked about the average amount of time they had been exposed to direct sunlight per day since the age of 19. We categorized participants into three groups with different levels of lifetime daily sun exposure and explored the association of sun exposure with the prevalence of diabetes. Results: The risk of diabetes was higher in subjects with more than 5 h of unprotected sun exposure per day, with an odds ratio of 2.39 (95% CI 1.75–3.25), compared to those with less than 2 h of sun exposure, and the association remained significant after adjusting for diabetes risk factors. Long-term sun exposure was associated with increased central obesity and the possibility of an increase in visceral adiposity, especially among women, and with decrease in beta cell function and peripheral adiposity or percent body fat in men. Conclusions: Our study provides a cutoff for upper limit of sun exposure and suggests unprotected daily sun exposure for more than 5 h should be avoided to prevent diabetes. Increased central adiposity and decreased beta cell function were observed in women and men, respectively, who had long-term unprotected daily sun exposure. - Highlights: • Sun exposure for more than 5 h per day is associated with diabetes risk. • Insulin resistance associated with visceral adiposity may play a role in women. • Insulin secretory defect may explain diabetes risk in men.

  20. Doc2b Serves as a Scaffolding Platform for Concurrent Binding of Multiple Munc18 Isoforms in Pancreatic Islet Beta Cells

    PubMed Central

    Ramalingam, Latha; Lu, Jingping; Hudmon, Andy; Thurmond, Debbie C.

    2015-01-01

    Biphasic glucose-stimulated insulin secretion (GSIS) from pancreatic beta cells involves SNARE protein-regulated exocytosis. SNARE complex assembly further requires the regulatory proteins Munc18c, Munc18-1 and Doc2b. Munc18-1 and Munc18c are required for 1st- and 2nd-phase GSIS, respectively. These distinct Munc18-1 and Munc18c roles are related to their transient high-affinity binding with their cognate t-SNAREs; Syntaxin 1A and Syntaxin 4, respectively. Doc2b is essential for both phases of GSIS, yet the molecular basis for this remains unresolved. Because Doc2b binds to Munc18-1 and Munc18c via it’s distinct C2A and C2B domains, respectively, we hypothesized that Doc2b may provide a plasma membrane-localized scaffold/platform for transient docking of these Munc18 isoforms during GSIS. Toward this, macromolecular complexes composed of Munc18c, Doc2b, and Munc18-1 were detected in beta cells. In vitro interaction assays indicated that Doc2b is required to bridge the interaction between Munc18c and Munc18-1 in the macromolecular complex; Munc18c and Munc18-1 failed to associate in the absence of Doc2b. Competition-based GST-Doc2b interaction assays revealed that Doc2b could simultaneously bind both Munc18-1 and Munc18c. Hence, these data support a working model wherein Doc2b functions as a docking platform/scaffold for transient interactions with the multiple Munc18 isoforms operative in insulin release, promoting SNARE assembly. PMID:25190515

  1. Cooperation between HMGA1, PDX-1, and MafA is Essential for Glucose-Induced Insulin Transcription in Pancreatic Beta Cells

    PubMed Central

    Arcidiacono, Biagio; Iiritano, Stefania; Chiefari, Eusebio; Brunetti, Francesco S.; Gu, Guoqiang; Foti, Daniela Patrizia; Brunetti, Antonio

    2014-01-01

    The high-mobility group AT-hook 1 (HMGA1) protein is a nuclear architectural factor that can organize chromatin structures. It regulates gene expression by controlling the formation of stereospecific multiprotein complexes called “enhanceosomes” on the AT-rich regions of target gene promoters. Previously, we reported that defects in HMGA1 caused decreased insulin receptor expression and increased susceptibility to type 2 diabetes mellitus in humans and mice. Interestingly, mice with disrupted HMGA1 gene had significantly smaller islets and decreased insulin content in their pancreata, suggesting that HMGA1 may have a direct role in insulin transcription and secretion. Herein, we investigate the regulatory roles of HMGA1 in insulin transcription. We provide evidence that HMGA1 physically interacts with PDX-1 and MafA, two critical transcription factors for insulin gene expression and beta-cell function, both in vitro and in vivo. We then show that the overexpression of HMGA1 significantly improves the transactivating activity of PDX-1 and MafA on human and mouse insulin promoters, while HMGA1 knockdown considerably decreased this transactivating activity. Lastly, we demonstrate that high glucose stimulus significantly increases the binding of HMGA1 to the insulin (INS) gene promoter, suggesting that HMGA1 may act as a glucose-sensitive element controlling the transcription of the INS gene. Together, our findings provide evidence that HMGA1, by regulating PDX-1- and MafA-induced transactivation of the INS gene promoter, plays a critical role in pancreatic beta-cell function and insulin production. PMID:25628604

  2. A role for the intermediate affinity IL-2R in the protection against glucocorticoid-induced apoptosis.

    PubMed Central

    Rebollo, A; Pitton, C; García, A; Gómez, J; Silva, A

    1995-01-01

    Recent work has shown that T lymphocytes undergo apoptosis upon treatment with the glucocorticoid analogue dexamethasone. These cells can be protected from the effect of dexamethasone by interleukin-2 (IL-2) or IL-4. We were interested in analysing whether a transfected cell dependent on three different lymphokines could be protected by them from the effect of dexamethasone. In addition, we took advantage of our cellular system, in which we expressed intermediate- or high-affinity IL-2R independently, to analyse the role of these receptors in the protection from glucocorticoid-induced apoptosis. In this report we show that IL-2 rescues murine T cells expressing exogenous intermediate- (TS1 beta) or high-affinity (TS1 alpha beta) IL-2 receptor (IL-2R) from dexamethasone-induced apoptosis. This result suggests that intermediate-affinity IL-2R alone can replace high-affinity IL-2R for the protection from the effect of dexamethasone. In addition, IL-4 and IL-9 are rescue-factors, as well as IL-2, of glucocorticoid-treated TS1 beta and TS1 alpha beta cells. Our data suggest that the presence of the alpha-chain of the IL-2R is not required for rescue by IL-2 from the effect of dexamethasone. In addition, we show that proliferation is not required for preventing glucocorticoid-induced apoptosis. This result implies a new role for the intermediate-affinity IL-2R. Images Figure 4 Figure 7 PMID:7751021

  3. Geniposide promotes beta-cell regeneration and survival through regulating β-catenin/TCF7L2 pathway

    PubMed Central

    Yao, D D; Yang, L; Wang, Y; Liu, C; Wei, Y J; Jia, X B; Yin, W; Shu, L

    2015-01-01

    T-cell factor 7-like 2 (TCF7L2) is an important transcription factor of Wnt/β-catenin signaling, which has critical roles in β-cell survival and regeneration. In preliminary screening assay, we found geniposide, a naturally occurring compound, was able to increase TCF7L2 mRNA level in Min6 cells. Here we aimed to investigate the role of geniposide in β-cell and underlying mechanism involved. Geniposide was found to promote β-cell survival by increasing β-cell proliferation and decreasing β-cell apoptosis in cultured mouse islets after challenge with diabetic stimuli. Geniposide protected β-cell through activating Wnt signaling, enhanced expressions of TCF7L2 and GLP-1R, activated AKT, inhibited GSK3β activity, and promoted β-catenin nuclear translocation. The protective effect of geniposide was remarkably suppressed by siRNAs against β-catenin, or by ICG001 (β-catenin/TCF-mediated transcription inhibitor). Moreover, geniposide promoted β-cell regeneration in vivo to normalize blood glucose in high-fat diet and db/db mice. Increased β-cell proliferation was observed in pancreatic sections of geniposide-treated diabetic mice. Most importantly, geniposide triggered small islet-like cell clusters formation as a result of β-cell neogenesis from ductal epithelium, which was well correlated with the increase in TCF7L2 expression. In exocrine cells isolated from mouse pancreas, geniposide could induce duct cell differentiation through upregulating TCF7L2 expression and activating JAK2/STAT3 pathway. Taken together, we identified a novel role of geniposide in promoting β-cell survival and regeneration by mechanisms involving the activation of β-catenin/TCF7L2 signaling. Our finding highlights the potential value of geniposide as a possible treatment for type 2 diabetes. PMID:25950476

  4. Recombinant adenoviral expression of IL-10 protects beta cell from impairment induced by pro-inflammatory cytokine.

    PubMed

    Xu, Ai-Jing; Zhu, Wei; Tian, Fei; Yan, Li-Hua; Li, Tang

    2010-11-01

    Interleukin-10 (IL-10) is a pleiotropic immunosuppressive and immunostimulatory cytokine. In autoimmune diabetes of the nonobese diabetic (NOD) mouse, IL-10 has exhibited paradoxical effects. Systemic IL-10 expression prevented or delayed diabetes onset in NOD mice while local expression of IL-10 did not. As antigen-presenting cells (APCs) play a central role in the generation of primary T cell responses, the direct role of this gene in pancreatic beta (β) cell is not clear. The effects of IL-10 on the protection of β cells in vitro were examined. In the present study, we examined the effects of adenovirus vector-mediated murine IL-10 (mIL-10) gene transfer to islet cell line RINm5F cells in vitro and to explore if IL-10 overexpression may prevent cytokine-mediated cytotoxicity. We had established the recombinant adenovirus vector containing mIL-10 genes (Ad-mIL-10) successfully. After infection of Ad-mIL-10, both mRNA and protein were expressed in RINm5F cells. Moreover, RINm5F cells secreted IL-10 protein into culture medium. Ad-mIL-10 prevented IL-1β-mediated nitric oxide production from β cells in vitro as well as the suppression of β cells function as determined by glucose-stimulated insulin production. Furthermore, Ad-mIL-10 gene transfer led to a profound reduction of Fas-expressing β cells and caspase-3 activity which were induced by IL-1β and the apoptotic rates of Ad-mIL-10 group were decreased. These findings show that IL-10 gene transfer to β cells may be beneficial in maintaining cells function, protecting islet cells from apoptosis-mediated by factors, which showed the potential therapy for type 1 diabetes mellitus. PMID:20658311

  5. Mortalin, Apoptosis, and Neurodegeneration

    PubMed Central

    Londono, Carolina; Osorio, Cristina; Gama, Vivian; Alzate, Oscar

    2012-01-01

    Mortalin is a highly conserved heat-shock chaperone usually found in multiple subcellular locations. It has several binding partners and has been implicated in various functions ranging from stress response, control of cell proliferation, and inhibition/prevention of apoptosis. The activity of this protein involves different structural and functional mechanisms, and minor alterations in its expression level may lead to serious biological consequences, including neurodegeneration. In this article we review the most current data associated with mortalin’s binding partners and how these protein-protein interactions may be implicated in apoptosis and neurodegeneration. A complete understanding of the molecular pathways in which mortalin is involved is important for the development of therapeutic strategies for cancer and neurodegenerative diseases. PMID:24970131

  6. Cell death and impairment of glucose-stimulated insulin secretion induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the {beta}-cell line INS-1E

    SciTech Connect

    Piaggi, Simona; Novelli, Michela; Martino, Luisa; Masini, Matilde; Raggi, Chiara; Orciuolo, Enrico; Masiello, Pellegrino; Casini, Alessandro; De Tata, Vincenzo . E-mail: v.detata@ipg.med.unipi.it

    2007-05-01

    The aim of this research was to characterize 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity on the insulin-secreting {beta}-cell line INS-1E. A sharp decline of cell survival (below 20%) was observed after 1 h exposure to TCDD concentrations between 12.5 and 25 nM. Ultrastructurally, {beta}-cell death was characterized by extensive degranulation, appearance of autophagic vacuoles, and peripheral nuclear condensation. Cytotoxic concentrations of TCDD rapidly induced a dose-dependent increase in intracellular calcium concentration. Blocking calcium entry by EGTA significantly decreased TCDD cytotoxicity. TCDD was also able to rapidly induce mitochondrial depolarization. Interestingly, 1 h exposition of INS-1E cells to very low TCDD concentrations (0.05-1 nM) dramatically impaired glucose-stimulated but not KCl-stimulated insulin secretion. In conclusion, our results clearly show that TCDD exerts a direct {beta}-cell cytotoxic effect at concentrations of 15-25 nM, but also markedly impairs glucose-stimulated insulin secretion at concentrations 20 times lower than these. On the basis of this latter observation we suggest that pancreatic {beta}-cells could be considered a specific and sensitive target for dioxin toxicity.

  7. Presence or absence of a known DKA precipitant defines distinct syndromes of “A-β+” Ketosis-Prone Diabetes (KPD) based on long-term beta cell function, HLA class II alleles, and gender predilection

    PubMed Central

    Nalini, Ramaswami; Ozer, Kerem; Maldonado, Mario; Patel, Sanjeet G.; Hampe, Christiane S.; Guthikonda, Anu; Villanueva, Jesus; Smith, E. O'Brian; Gaur, Lakshmi K.; Balasubramanyam, Ashok

    2010-01-01

    Objective Ketosis prone diabetes (KPD) is heterogeneous. Longitudinal follow-up revealed that patients with “A-β+” KPD (absent autoantibodies and preserved beta cell function) segregated into two subgroups with distinct evolution of beta cell function and glycemic control. Generalized linear analysis demonstrated that the variable that most significantly differentiated them was presence of a clinically evident precipitating event for the index DKA. Hence we performed a comprehensive analysis of A-β+ KPD patients presenting with “provoked” compared to “unprovoked” DKA. Methods Clinical, biochemical and beta cell functional characteristics were compared between provoked and unprovoked A-β+ KPD patients followed prospectively for 1-8 years. HLA class II allele frequencies were compared between these two groups and population controls. Results Unprovoked A-β+ KPD patients (n=83) had greater BMI, male preponderance, higher frequency of women with oligo/anovulation, more frequent African-American ethnicity and less frequent family history of diabetes than provoked A-β+ KPD patients (n=64). The provoked group had higher frequencies of the HLA class II type 1 diabetes susceptibility alleles DQB1*0302 (than the unprovoked group or population controls), and DRB1*04 (than the unprovoked group), while the unprovoked group had a higher frequency of the protective allele DQB1*0602. Beta cell secretory reserve and glycemic control improved progressively in the unprovoked group, but declined in the provoked group. The differences persisted in comparisons restricted to patients with new-onset diabetes. Conclusions “Unprovoked” A-β+ KPD is a distinct syndrome characterized by reversible beta cell dysfunction with male predominance and increased frequency of DQB1*0602, while “provoked” A-β+ KPD is characterized by progressive loss of beta cell reserve and increased frequency of DQB1*0302 and DRB1*04. Unprovoked DKA predicts long-term beta cell functional

  8. Role of Calpain in Apoptosis

    PubMed Central

    Momeni, Hamid Reza

    2011-01-01

    Apoptosis, a form of programmed cell death that occurs under physiological as well as pathological conditions, is characterized by morphological and biochemical features. While the importance of caspases in apoptosis is established, several noncaspase proteases (Ca2+-dependent proteases) such as calpain may play a role in the execution of apoptosis. The calpain family consists of two major isoforms, calpain I and calpain II which require µM and mM Ca2+ concentrations to initiate their activity. An increase in intracellular Ca2+ level is thought to trigger a cascade of biochemical processes including calpain activation. Once activated, calpains degrade membrane, cytoplasmic and nuclear substrates, leading to the breakdown of cellular architecture and finally apoptosis. The activation of calpain has been implicated in neuronal apoptosis following spinal cord injuries and neurodegenerative diseases. This review focuses on calpain with an emphasis on its key role in the proteolysis of cellular protein substrates following apoptosis. PMID:23507938

  9. Pathophysiological Significance of Hepatic Apoptosis

    PubMed Central

    Wang, Kewei; Lin, Bingliang

    2013-01-01

    Apoptosis is a classical pathological feature in liver diseases caused by various etiological factors such as drugs, viruses, alcohol, and cholestasis. Hepatic apoptosis and its deleterious effects exacerbate liver function as well as involvement in fibrosis/cirrhosis and carcinogenesis. An imbalance between apoptotic and antiapoptotic capabilities is a prominent characteristic of liver injury. The regulation of apoptosis and antiapoptosis can be a pivotal step in the treatment of liver diseases. PMID:27335822

  10. JNK Signaling in Apoptosis

    PubMed Central

    Dhanasekaran, Danny N.; Reddy, E. Premkumar

    2011-01-01

    Jun N-terminal kinases or JNKs play a critical role in death receptor-initiated extrinsic as well as mitochondrial intrinsic apoptotic pathways. JNKs activate apoptotic signaling by the upregulation pro-apoptotic genes via the transactivation of specific transcription factors or by directly modulating the activities of mitochondrial pro- and anti-apoptotic proteins through distinct phosphorylation events. This review analyzes our present understanding of the role of JNK in apoptotic signaling and the various mechanisms by which JNK promotes apoptosis PMID:18931691

  11. A novel regulatory mechanism for trimeric GTP-binding proteins in the membrane and secretory granule fractions of human and rodent beta cells.

    PubMed Central

    Kowluru, A; Seavey, S E; Rhodes, C J; Metz, S A

    1996-01-01

    Recently we described roles for heterotrimeric and low-molecular-mass GTP-binding proteins in insulin release from normal rat islets. During these studies, we observed that a protein with an apparent molecular mass (37 kDa) similar to that of the beta subunit of trimeric GTP-binding proteins underwent phosphorylation in each of five classes of insulin-secreting cells. Incubation of the beta cell total membrane fraction or the isolated secretory granule fraction (but not the cytosolic fraction) with [gamma-32P]ATP or [gamma-32P]GTP resulted in the phosphorylation of this protein, which was selectively immunoprecipitated by an anti-serum directed against the common beta subunit of trimeric G-proteins. Disruption of the alpha beta gamma trimer (by pretreatment with either fluoroaluminate or guanosine 5'(-)[gamma-thio]triphosphate) prevented beta subunit phosphorylation. Based on differential sensitivities to pH, heat and the histidine-selective reagent diethyl pyrocarbonate (and reversal of the latter by hydroxylamine), the phosphorylated amino acid was presumptively identified as histidine. Incubation of pure beta subunit alone or in combination with the exogenous purified alpha subunit of transducin did not result in the phosphorylation of the beta subunit, but addition of the islet cell membrane fraction did support this event, suggesting that membrane localization (or a membrane-associated factor) is required for beta subunit phosphorylation. Incubation of phosphorylated beta subunit with G alpha.GDP accelerated the dephosphorylation of the beta subunit, accompanied by the formation of G alpha-GTP. Immunoblotting detected multiple alpha subunits (of Gi, G(o) and Gq) and at least one beta subunit in the secretory granule fraction of normal rat islets and insulinoma cells. These data describe a potential alternative mechanism for the activation of GTP-binding proteins in beta cells which contrasts with the classical receptor-agonist mechanism: G beta undergoes

  12. Biomarkers of apoptosis

    PubMed Central

    Ward, T H; Cummings, J; Dean, E; Greystoke, A; Hou, J M; Backen, A; Ranson, M; Dive, C

    2008-01-01

    Within the era of molecularly targeted anticancer agents, it has become increasingly important to provide proof of mechanism as early on as possible in the drug development cycle, especially in the clinic. Selective activation of apoptosis is often cited as one of the major goals of cancer chemotherapy. Thus, the present minireview focuses on a discussion of the pros and cons of a variety of methodological approaches to detect different components of the apoptotic cascade as potential biomarkers of programmed cell death. The bulk of the discussion centres on serological assays utilising the technique of ELISA, since here there is an obvious advantage of sampling multiple time points. Potential biomarkers of apoptosis including circulating tumour cells, cytokeratins and DNA nucleosomes are discussed at length. However, accepting that a single biomarker may not have the power to predict proof of concept and patient outcome, it is clear that in the future more emphasis will be placed on technologies that can analyse panels of biomarkers in small volumes of samples. To this end the increased throughput afforded by multiplex ELISA technologies is discussed. PMID:19238626

  13. Microcephaly with Simplified Gyration, Epilepsy, and Infantile Diabetes Linked to Inappropriate Apoptosis of Neural Progenitors

    PubMed Central

    Poulton, Cathryn J.; Schot, Rachel; Kia, Sima Kheradmand; Jones, Marta; Verheijen, Frans W.; Venselaar, Hanka; de Wit, Marie-Claire Y.; de Graaff, Esther; Bertoli-Avella, Aida M.; Mancini, Grazia M.S.

    2011-01-01

    We describe a syndrome of primary microcephaly with simplified gyral pattern in combination with severe infantile epileptic encephalopathy and early-onset permanent diabetes in two unrelated consanguineous families with at least three affected children. Linkage analysis revealed a region on chromosome 18 with a significant LOD score of 4.3. In this area, two homozygous nonconserved missense mutations in immediate early response 3 interacting protein 1 (IER3IP1) were found in patients from both families. IER3IP1 is highly expressed in the fetal brain cortex and fetal pancreas and is thought to be involved in endoplasmic reticulum stress response. We reported one of these families previously in a paper on Wolcott-Rallison syndrome (WRS). WRS is characterized by increased apoptotic cell death as part of an uncontrolled unfolded protein response. Increased apoptosis has been shown to be a cause of microcephaly in animal models. An autopsy specimen from one patient showed increased apoptosis in the cerebral cortex and pancreas beta cells, implicating premature cell death as the pathogenetic mechanism. Both patient fibroblasts and control fibroblasts treated with siRNA specific for IER3IP1 showed an increased susceptibility to apoptotic cell death under stress conditions in comparison to controls. This directly implicates IER3IP1 in the regulation of cell survival. Identification of IER3IP1 mutations sheds light on the mechanisms of brain development and on the pathogenesis of infantile epilepsy and early-onset permanent diabetes. PMID:21835305

  14. Apoptosis and the Airway Epithelium

    PubMed Central

    White, Steven R.

    2011-01-01

    The airway epithelium functions as a barrier and front line of host defense in the lung. Apoptosis or programmed cell death can be elicited in the epithelium as a response to viral infection, exposure to allergen or to environmental toxins, or to drugs. While apoptosis can be induced via activation of death receptors on the cell surface or by disruption of mitochondrial polarity, epithelial cells compared to inflammatory cells are more resistant to apoptotic stimuli. This paper focuses on the response of airway epithelium to apoptosis in the normal state, apoptosis as a potential regulator of the number and types of epithelial cells in the airway, and the contribution of epithelial cell apoptosis in important airways diseases. PMID:22203854

  15. Regeneration of beta cells in islets of Langerhans of pancreas of alloxan diabetic rats by acetone extract of Momordica charantia (Linn.) (bitter gourd) fruits.

    PubMed

    Singh, Neera; Gupta, Manushma

    2007-12-01

    Acetone extract of whole fruit powder of M. charantia (bitter gourd) in doses 25, 50 and 75 mg/100 g body weight lowered the blood glucose from 13.30 to 50% after 8 to 30 days treatment in alloxan diabetic albino rats, confirming antihyperglycemic effect of this plant in diabetic animals and humans. Histological observations with acetone extract showed different phases of recovery of beta cells of the islets of Langerhans of pancreas, which in the untreated diabetic rats were less in number and showed varied degree of atrophy. The most important finding of the present study was observation of the presence of small scattered islets among the acinar tissue in some experimental animals, which may reflect neoformation of islets from pre-existing islet cells. The liver of alloxan diabetic rats showed hydropic degeneration, fatty change and necrosis at some places but liver of extract treated animals was normal. Glycogen localization in liver of diabetic rats was faint but after 30 days treatment with different doses of extract, normal to heavy glycogen localization was observed. PMID:18254212

  16. Low dose monoethyl phthalate (MEP) exposure triggers proliferation by activating PDX-1 at 1.1B4 human pancreatic beta cells.

    PubMed

    Güven, Celal; Dal, Fulya; Aydoğan Ahbab, Müfide; Taskin, Eylem; Ahbab, Süleyman; Adin Çinar, Suzan; Sırma Ekmekçi, Sema; Güleç, Çağrı; Abacı, Neslihan; Akçakaya, Handan

    2016-07-01

    Phthalate plasticizers used in a wide range of common plastic products are released into the environment and may pose a risk of increased incidence of type 2 diabetes. In this work, we studied the effects of monoethyl phthalate (MEP), the metabolite of diethyl phthalate, exposure on 1.1B4 human pancreatic beta cells at low doses (1-1000 nM). We showed that MEP treatment induced proliferation in 1.1B4 cells. Also PCNA protein expression levels were increased related to proliferation induction. It has been noted that phthalates can exert estrogen mediated response by interacting with ER. In our study 24 h MEP treatment decreased ERα protein expression level conversely it increased the same protein expression level after 72 h treatment. Also MEP treatment decreased ERβ expression after 72 h at 1.1B4 cells. Our results further show that insulin content of 1.1B4 cells were increased with low dose MEP treatment. Along with our insulin content results, PDX- 1 expression levels were also increased at 1.1B4 cells with MEP treatment. These findings suggest that MEP acts as an estrogenic compound and PPARγ agonist at lower concentrations. Also it should be noted that PDX-1 may be a critical regulator of 1.1B4 cells treated with MEP. PMID:27133914

  17. Aqueous leaf extract of Passiflora alata Curtis promotes antioxidant and anti-inflammatory effects and consequently preservation of NOD mice beta cells (non-obese diabetic).

    PubMed

    Figueiredo, D; Colomeu, Talita Cristina; Schumacher, Nayara Simon Gonzalez; Stivanin-Silva, L G; Cazarin, Cinthia Baú Betim; Meletti, Laura Maria Molina; Fernandes, Luís Gustavo Romani; Prado, Marcelo Alexandre; Zollner, R L

    2016-06-01

    Passiflora alata Curtis (P. alata) leaves have anti-inflammatory properties; the present study aimed to investigate the anti-diabetogenic properties of P. alata aqueous leaf extract. HPLC analysis identified the phenolic compounds catechin, epicatechin and rutin. The aqueous extract was administered for 30weeks to non-obese diabetic (NOD) mice presenting a decrease of 28.6% in diabetes incidence and the number of inflammatory cells in pancreatic islets, when compared with the control group (water). The P. alata group presented an antioxidant effect and decreased lipid peroxidation in the serum of NOD mice. Increased numbers of insulin-positive cells were also observed in the pancreatic islets of the treated group. The diabetic group exhibited higher levels in the glucose tolerance test and glycemic index, in comparison to the P. alata-treated group and non-diabetic control BALB/c mice. In addition, the P. alata extract reduced the percentage and the proliferation index of NOD mice lymphocytes submitted to in vitro dose/response mitogenic stimulation assays. These results suggest that the aqueous extract of P. alata has anti-inflammatory properties, contributing to the protection of beta cells in pancreatic islets in NOD mice, and presents potential for use a supporting approach to treat type 1 diabetes. PMID:27039211

  18. Long-term glycemic control using polymer-encapsulated human stem cell-derived beta cells in immune-competent mice.

    PubMed

    Vegas, Arturo J; Veiseh, Omid; Gürtler, Mads; Millman, Jeffrey R; Pagliuca, Felicia W; Bader, Andrew R; Doloff, Joshua C; Li, Jie; Chen, Michael; Olejnik, Karsten; Tam, Hok Hei; Jhunjhunwala, Siddharth; Langan, Erin; Aresta-Dasilva, Stephanie; Gandham, Srujan; McGarrigle, James J; Bochenek, Matthew A; Hollister-Lock, Jennifer; Oberholzer, Jose; Greiner, Dale L; Weir, Gordon C; Melton, Douglas A; Langer, Robert; Anderson, Daniel G

    2016-03-01

    The transplantation of glucose-responsive, insulin-producing cells offers the potential for restoring glycemic control in individuals with diabetes. Pancreas transplantation and the infusion of cadaveric islets are currently implemented clinically, but these approaches are limited by the adverse effects of immunosuppressive therapy over the lifetime of the recipient and the limited supply of donor tissue. The latter concern may be addressed by recently described glucose-responsive mature beta cells that are derived from human embryonic stem cells (referred to as SC-β cells), which may represent an unlimited source of human cells for pancreas replacement therapy. Strategies to address the immunosuppression concerns include immunoisolation of insulin-producing cells with porous biomaterials that function as an immune barrier. However, clinical implementation has been challenging because of host immune responses to the implant materials. Here we report the first long-term glycemic correction of a diabetic, immunocompetent animal model using human SC-β cells. SC-β cells were encapsulated with alginate derivatives capable of mitigating foreign-body responses in vivo and implanted into the intraperitoneal space of C57BL/6J mice treated with streptozotocin, which is an animal model for chemically induced type 1 diabetes. These implants induced glycemic correction without any immunosuppression until their removal at 174 d after implantation. Human C-peptide concentrations and in vivo glucose responsiveness demonstrated therapeutically relevant glycemic control. Implants retrieved after 174 d contained viable insulin-producing cells. PMID:26808346

  19. Adipose Tissue-Derived Mesenchymal Stem Cells Exert In Vitro Immunomodulatory and Beta Cell Protective Functions in Streptozotocin-Induced Diabetic Mice Model

    PubMed Central

    Rahavi, Hossein; Hashemi, Seyed Mahmoud; Soleimani, Masoud; Mohammadi, Jamal; Tajik, Nader

    2015-01-01

    Regenerative and immunomodulatory properties of mesenchymal stem cells (MSCs) might be applied for type 1 diabetes mellitus (T1DM) treatment. Thus, we proposed in vitro assessment of adipose tissue-derived MSCs (AT-MSCs) immunomodulation on autoimmune response along with beta cell protection in streptozotocin- (STZ-) induced diabetic C57BL/6 mice model. MSCs were extracted from abdominal adipose tissue of normal mice and cultured to proliferate. Diabetic mice were prepared by administration of multiple low-doses of streptozotocin. Pancreatic islets were isolated from normal mice and splenocytes prepared from normal and diabetic mice. Proliferation, cytokine production, and insulin secretion assays were performed in coculture experiments. AT-MSCs inhibited splenocytes proliferative response to specific (islet lysate) and nonspecific (PHA) triggers in a dose-dependent manner (P < 0.05). Decreased production of proinflammatory cytokines, such as IFN-γ, IL-2, and IL-17, and increased secretion of regulatory cytokines such as TGF-β, IL-4, IL-10, and IL-13 by stimulated splenocytes were also shown in response to islet lysate or PHA stimulants (P < 0.05). Finally, we demonstrated that AT-MSCs could effectively sustain viability as well as insulin secretion potential of pancreatic islets in the presence of reactive splenocytes (P < 0.05). In conclusion, it seems that MSCs may provide a new horizon for T1DM cell therapy and islet transplantation in the future. PMID:25893202

  20. Positron emission tomography study on pancreatic somatostatin receptors in normal and diabetic rats with {sup 68}Ga-DOTA-octreotide: A potential PET tracer for beta cell mass measurement

    SciTech Connect

    Sako, Takeo; Hasegawa, Koki; Nishimura, Mie; Kanayama, Yousuke; Wada, Yasuhiro; Hayashinaka, Emi; Cui, Yilong; Kataoka, Yosky; Senda, Michio; Watanabe, Yasuyoshi

    2013-12-06

    Highlights: •PET images showed high uptake of {sup 68}Ga-DOTA-octreotide in the normal pancreas. •{sup 68}Ga-DOTA-octreotide specifically binds to somatostatin receptors in the pancreas. •The pancreatic uptake of {sup 68}Ga-DOTA-octreotide was decreased in the diabetic rats. •{sup 68}Ga-DOTA-octreotide could be a candidate PET probe to measure the beta cell mass. -- Abstract: Diabetes mellitus (DM) is a metabolic disorder characterized by hyperglycemia, and the loss or dysfunction of pancreatic beta cells has been reported before the appearance of clinical symptoms and hyperglycemia. To evaluate beta cell mass (BCM) for improving the detection and treatment of DM at earlier stages, we focused on somatostatin receptors that are highly expressed in the pancreatic beta cells, and developed a positron emission tomography (PET) probe derived from octreotide, a metabolically stable somatostatin analog. Octreotide was conjugated with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), a chelating agent, and labeled with {sup 68}Gallium ({sup 68}Ga). After intravenous injection of {sup 68}Ga-DOTA-octreotide, a 90-min emission scan of the abdomen was performed in normal and DM model rats. The PET studies showed that {sup 68}Ga-DOTA-octreotide radioactivity was highly accumulated in the pancreas of normal rats and that the pancreatic accumulation was significantly reduced in the rats administered with an excess amount of unlabeled octreotide or after treatment with streptozotocin, which was used for the chemical induction of DM in rats. These results were in good agreement with the ex vivo biodistribution data. These results indicated that the pancreatic accumulation of {sup 68}Ga-DOTA-octreotide represented specific binding to the somatostatin receptors and reflected BCM. Therefore, PET imaging with {sup 68}Ga-DOTA-octreotide could be a potential tool for evaluating BCM.

  1. Antibody Response to Serpin B13 Induces Adaptive Changes in Mouse Pancreatic Islets and Slows Down the Decline in the Residual Beta Cell Function in Children with Recent Onset of Type 1 Diabetes Mellitus.

    PubMed

    Kryvalap, Yury; Lo, Chi-Wen; Manuylova, Ekaterina; Baldzizhar, Raman; Jospe, Nicholas; Czyzyk, Jan

    2016-01-01

    Type 1 diabetes mellitus (T1D) is characterized by a heightened antibody (Ab) response to pancreatic islet self-antigens, which is a biomarker of progressive islet pathology. We recently identified a novel antibody to clade B serpin that reduces islet-associated T cell accumulation and is linked to the delayed onset of T1D. As natural immunity to clade B arises early in life, we hypothesized that it may influence islet development during that time. To test this possibility healthy young Balb/c male mice were injected with serpin B13 mAb or IgG control and examined for the number and cellularity of pancreatic islets by immunofluorescence and FACS. Beta cell proliferation was assessed by measuring nucleotide analog 5-ethynyl-2'-deoxyuridine (5-EdU) incorporation into the DNA and islet Reg gene expression was measured by real time PCR. Human studies involved measuring anti-serpin B13 autoantibodies by Luminex. We found that injecting anti-serpin B13 monoclonal Ab enhanced beta cell proliferation and Reg gene expression, induced the generation of ∼80 pancreatic islets per animal, and ultimately led to increase in the beta cell mass. These findings are relevant to human T1D because our analysis of subjects just diagnosed with T1D revealed an association between baseline anti-serpin activity and slower residual beta cell function decline in the first year after the onset of diabetes. Our findings reveal a new role for the anti-serpin immunological response in promoting adaptive changes in the endocrine pancreas and suggests that enhancement of this response could potentially help impede the progression of T1D in humans. PMID:26578518

  2. Effects of Liraglutide Combined with Short-Term Continuous Subcutaneous Insulin Infusion on Glycemic Control and Beta Cell Function in Patients with Newly Diagnosed Type 2 Diabetes Mellitus: A Pilot Study

    PubMed Central

    Ke, Weijian; Liu, Liehua; Liu, Juan; Chen, Ailing; Deng, Wanping; Zhang, Pengyuan; Cao, Xiaopei; Liao, Zhihong; Xiao, Haipeng; Liu, Jianbin; Li, Yanbing

    2016-01-01

    The objective of this paper is to investigate the effects of liraglutide in combination with short-term continuous subcutaneous insulin infusion (CSII) therapy on glycemic control and beta cell function in patients with newly diagnosed type 2 diabetes mellitus (T2DM). Thirty-nine eligible newly diagnosed T2DM patients were recruited and randomized to receive either of two therapies: short-term CSII alone (CSII alone group) or CSII in combination with liraglutide (CSII + Lira group) for 12 weeks. Blood glucose control, homeostasis model assessment (HOMA) indices, and acute insulin response (AIR) were compared between the two groups. The patients in CSII + Lira group achieved euglycemia with equivalent insulin dosage in shorter time (1 (0) versus 2 (3) days, P = 0.039). HbA1c at the end of study was comparable between two groups (6.3 ± 0.7% versus 6.0 ± 0.5%, for CSII alone group and CSII + Lira group, resp., P = 0.325). The increment of AIR was higher in CSII + Lira group (177.58 (351.57) μU·min/mL versus 58.15 (51.30) μU·min/mL, P < 0.001). However, after stopping liraglutide, its effect on beta cell function disappeared completely. Liraglutide combined with short-term CSII was effective in further improving beta cell function, but the beneficial effects did not sustain after suspension of the therapy. PMID:26640805

  3. [Apoptosis and its biomedical significance].

    PubMed

    Ortega-Camarillo, C; Díaz-Flores, M; Avalos-Rodríguez, A; Vergara-Onofre, M; Rosales-Torres, A M

    2001-01-01

    Cell death can occur through apoptotic or necrotic death pathways. Membrane disruption leads to inflammation, a typical feature of necrosis. Apoptosis constitutes a genetically controlled physiologic process of cell removal. It is characterized by cell shrinkage, chromatin condensation, and DNA cleavage. Apoptotic cells are rapidly recognized and engulfed by phagocytes thus inhibiting an inflammatory response following necrosis. Apoptosis has been proposed as a basic event to protect tissue homeostasis. This paper analyzes the genetic, biochemical, and morphologic characteristics related to apoptosis, as well as its relationship to certain illnesses. PMID:11766462

  4. Validation of the BETA-2 Score: An Improved Tool to Estimate Beta Cell Function After Clinical Islet Transplantation Using a Single Fasting Blood Sample.

    PubMed

    Forbes, S; Oram, R A; Smith, A; Lam, A; Olateju, T; Imes, S; Malcolm, A J; Shapiro, A M J; Senior, P A

    2016-09-01

    The beta score, a composite measure of beta cell function after islet transplantation, has limited sensitivity because of its categorical nature and requires a mixed-meal tolerance test (MMTT). We developed a novel score based on a single fasting blood sample. The BETA-2 score used stepwise forward linear regression incorporating glucose (in millimoles per liter), C-peptide (in nanomoles per liter), hemoglobin A1c (as a percentage) and insulin dose (U/kg per day) as continuous variables from the original beta score data set (n = 183 MMTTs). Primary and secondary analyses assessed the score's ability to detect glucose intolerance (90-min MMTT glucose ≥8 mmol/L) and insulin independence, respectively. A validation cohort of islet transplant recipients (n = 114 MMTTs) examined 12 mo after transplantation was used to compare the score's ability to detect these outcomes. The BETA-2 score was expressed as follows (range 0-42): [Formula: see text] A score <20 and ≥15 detected glucose intolerance and insulin independence, respectively, with >82% sensitivity and specificity. The BETA-2 score demonstrated greater discrimination than the beta score for these outcomes (p < 0.05). Using a fasting blood sample, the BETA-2 score estimates graft function as a continuous variable and shows greater discrimination of glucose intolerance and insulin independence after transplantation versus the beta score, allowing frequent assessments of graft function. Studies examining its utility to track long-term graft function are required. PMID:27017888

  5. Mitochondria and apoptosis: emerging concepts

    PubMed Central

    Li, Mark Xiang

    2015-01-01

    As mitochondria are the powerhouses of the cell, their damage during the cell suicide process of apoptosis is essentially responsible for cellular demise in most cells. A key family of proteins, the B-cell lymphoma-2 (BCL-2) family, determines the integrity of mitochondria in the face of apoptotic insult. A comprehensive understanding of the molecular details of how apoptosis is initiated and how it culminates is essential if apoptosis is to fulfil its undoubted potential as a therapeutic target to treat diseases ranging from cancer to neurodegenerative conditions. Recent advances have provided significant insight into the control of this fundamental process while prompting a re-evaluation of what was considered dogma in the field. Emerging evidence also points to a potential overarching control network that governs not only apoptosis but other fundamental mitochondrial processes, including mitochondrial fission/fusion and quality control. PMID:26097715

  6. Beyond Apoptosis in Lupus

    PubMed Central

    Colonna, Lucrezia; Lood, Christian; Elkon, Keith B.

    2014-01-01

    Purpose of review Systemic lupus erythematosus (SLE) is characterized by autoantibodies directed against nuclear autoantigens normally concealed from immune recognition in healthy individuals. Here we summarize recently identified mechanisms of abnormal cell death leading to exposure and aberrant processing of nucleoprotein self antigens, and discuss their role in the SLE pathogenesis. Recent findings During the past few years, the unveiling of several new forms of cell death has expanded our understanding beyond the simple view of “apoptotic” versus “necrotic” cell death. SLE patients show abnormalities in cell death at several levels, including increased rates of apoptosis, necrosis, and autophagy, as well as reduced clearance of dying cells. These abnormalities lead to an increased autoantigen burden and also antigen modifications, such as nucleic acid oxidation that increase the inflammatory properties of self antigens. Recent investigations have highlighted the role of opsonins in determining the immunogenic versus tolerogenic characteristics of self antigens. Summary Dysregulation of different forms of programmed cell death contributes to increased exposure, availability, and immunogenic characteristic of intracellular self antigens, which all participate in development of lupus autoimmunity. As our understanding of abnormalities of cell death in SLE advances, potential therapeutic opportunities await human implementation. PMID:25036095

  7. Cytoskeleton and apoptosis.

    PubMed

    Ndozangue-Touriguine, Olivia; Hamelin, Jocelyne; Bréard, Jacqueline

    2008-07-01

    Apoptosis is a genetically programmed and physiological mode of cell death that leads to the removal of unwanted or abnormal cells. Cysteine-proteases called caspases are responsible for the apoptotic execution phase which is characterized by specific biochemical events as well as morphological changes. These changes, which lead to the orderly dismantling of the apoptotic cell, include cell contraction, dynamic membrane blebbing, chromatin condensation, nuclear disintegration, cell fragmentation followed by phagocytosis of the dying cell. They involve major modifications of the cytoskeleton which are largely mediated by cleavage of several of its components by caspases. For example, dynamic membrane blebbing is due to the increased contractility of the acto-myosin system following myosin light chain (MLC) phosphorylation. MLC phosphorylation is a consequence of the cleavage of a Rho GTPase effector, the kinase ROCK I, by caspase-3. This cleavage induces a constitutive kinase activity by removal of an inhibitory domain. Chromatin condensation is facilitated by the processing of lamins by caspases. Collapse of the cytokeratin network is mediated by cleavage of keratin 18. On another hand, the actin cytoskeleton rearrangement needed in the phagocyte for engulfment of the dying cell is due to the activation of the small GTPase Rac, a GTPase of the Rho family that induces actin polymerisation and formation of lamellipodia. In addition to mediating the morphological modifications of the apoptotic cell, several proteins of the cytoskeleton such as actin and keratins are also involved in the regulation of apoptotic signaling. PMID:18462707

  8. Spontaneous apoptosis in human thymocytes.

    PubMed Central

    Tiso, M.; Gangemi, R.; Bargellesi Severi, A.; Pizzolitto, S.; Fabbi, M.; Risso, A.

    1995-01-01

    Apoptosis seems to be involved in different stages of immune cell development. In particular, experimental evidence suggests that it is a major form of cell death in the thymus. The present analysis of human thymocytes reveals that a fraction of these cells, cultured in vitro, undergoes spontaneous apoptosis. This observation is based both on molecular (DNA fragmentation) and morphological (electron microscopic) investigations of the cells. The apoptotic thymocytes are CD3- or CD3lo, CD4lo, and CD8lo and do not express Bcl-2 protein. Furthermore, thymocytes die by apoptosis when exposed to pharmacological stimuli, such as tumor necrosis factor-alpha, dexamethasone, ATP, or Ca++ ionophore. Thus the apoptotic machinery in thymocytes can be triggered by an imbalance in growth factors in the in vitro culture media and can be modulated by various biochemical signals. The process of spontaneous apoptosis is independent of mRNA or protein synthesis, as actinomycin D and cycloheximide fail to inhibit this phenomenon. Furthermore, apoptosis seems to require active oxidative phosphorylation, as it is prevented by incubation of the cells with inhibitors of the respiratory chain. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 6 Figure 7 Figure 8 PMID:7639336

  9. Natural history of beta-cell autoimmunity in young children with increased genetic susceptibility to type 1 diabetes recruited from the general population.

    PubMed

    Kimpimäki, T; Kulmala, P; Savola, K; Kupila, A; Korhonen, S; Simell, T; Ilonen, J; Simell, O; Knip, M

    2002-10-01

    thoroughly standardized assay appear to be more specific for the screening of beta-cell autoimmunity in young children with increased genetic susceptibility to type 1 diabetes in the Finnish population, which has the highest incidence of type 1 diabetes in the world. PMID:12364437

  10. Apoptosis deregulation in myeloproliferative neoplasms

    PubMed Central

    Tognon, Raquel; Nunes, Natália de Souza; de Castro, Fabíola Attié

    2013-01-01

    ABSTRACT Philadelphia-chromosome negative chronic myeloproliferative neoplasms are clonal hematologic diseases characterized by hematopoietic progenitor independence from or hypersensitivity to cytokines. The cellular and molecular mechanisms involved in the pathophysiology of myeloproliferative neoplasms have not yet been fully clarified. Pathophysiologic findings relevant for myeloproliferative neoplasms are associated with genetic alterations, such as, somatic mutation in the gene that codifies JAK-2 (JAK V617F). Deregulation of the process of programmed cellular death, called apoptosis, seems to participate in the pathogenesis of these disorders. It is known that expression deregulation of pro- and anti-apoptotic genes promotes cell resistance to apoptosis, culminating with the accumulation of myeloid cells and establishing neoplasms. This review will focus on the alterations in apoptosis regulation in myeloproliferative neoplasms, and the importance of a better understanding of this mechanism for the development of new therapies for these diseases. PMID:24488400

  11. Molecular mechanisms of hepatic apoptosis

    PubMed Central

    Wang, K

    2014-01-01

    Apoptosis is a prominent feature of liver diseases. Causative factors such as alcohol, viruses, toxic bile acids, fatty acids, drugs, and immune response, can induce apoptotic cell death via membrane receptors and intracellular stress. Apoptotic signaling network, including membrane death receptor-mediated cascade, reactive oxygen species (ROS) generation, endoplasmic reticulum (ER) stress, lysosomal permeabilization, and mitochondrial dysfunction, is intermixed each other, but one mechanism may dominate at a particular stage. Mechanisms of hepatic apoptosis are complicated by multiple signaling pathways. The progression of liver disease is affected by the balance between apoptotic and antiapoptotic capabilities. Therapeutic options of liver injury are impacted by the clear understanding toward mechanisms of hepatic apoptosis. PMID:24434519

  12. CREB is a regulatory target for the protein kinase Akt/PKB in the differentiation of pancreatic ductal cells into islet {beta}-cells mediated by hepatocyte growth factor

    SciTech Connect

    Li, Xin-Yu; Zhan, Xiao-Rong; Liu, Xiao-Min; Wang, Xiao-Chen

    2011-01-14

    Research highlights: {yields} CREB is a regulatory target for the protein kinase Akt/PKB in pancreatic duct cells. {yields} Activation of the PI3K/AKT/CREB pathway plays a critical role in the HGF-mediated differentiation of pancreatic duct cells in vivo. {yields} CREB was causally linked to the expression of transcription factors during PDEC differentiation induced by HGF. -- Abstract: We have previously reported that the PI3K/Akt signaling pathway is involved in hepatocyte growth factor (HGF)-induced differentiation of adult rat pancreatic ductal epithelial cells (PDECs) into islet {beta}-cells in vitro. The transcription factor CREB is one of the downstream key effectors of the PI3K/Akt signaling pathway. Recent studies showing that CREB is required for the survival of certain cell types prompted us to examine whether CREB is a nuclear target for activation via the HGF-dependent Ser/Thr kinase Akt/PKB in the differentiation of pancreatic duct cell into islet {beta}-cells. In this study, we first attempted to examine whether HGF modulates the Akt-dependent activation of target gene CREB and then investigated whether CREB activity affects the differentiation of HGF-induced PDECs. Finally, we studied the role of CREB in modulating the expression of transcription factors in PDECs during the differentiation of HGF-induced PDECs. Our results demonstrated that CREB is a regulatory target for the protein kinase Akt/PKB in the differentiation of pancreatic ductal cells into islet {beta}-cells mediated by HGF.

  13. Monitoring apoptosis in real time.

    PubMed

    Green, Allan M; Steinmetz, Neil D

    2002-01-01

    Many therapeutically active anticancer treatments exert their effect by the induction of apoptosis and necrosis. Serial biopsies in breast cancer patients have suggested that response to therapy correlates with early posttreatment increases in tumor apoptotic index. Radiolabeled technetium Tc 99m-recombinant human (rh) annexin V provides a noninvasive technique for imaging treatment-induced cell death. Annexin V is a naturally occurring human protein that binds avidly to membrane-associated phosphatidylserine (PS). PS is normally found only on the inner leaflet of the cell membrane double layer, but it is actively transported to the outer layer as an early event in apoptosis and becomes available for annexin binding. Annexin also gains access to PS as a result of the membrane fragmentation associated with necrosis. In vitro studies of apoptosis using fluorescein annexin have shown good correlation with assessments of apoptosis documented by nuclear DNA degradation and caspase activation. In vivo localization of intravenously administered Tc 99m-annexin V has been demonstrated in numerous preclinical models of apoptosis, including anti-Fas-mediated hepatic apoptosis, rejection of allogeneic heterotopic cardiac allografts, cyclophosphamide treatment of murine lymphoma, cyclophosphamide-induced apoptosis in bone marrow, and leukocyte apoptosis associated with abscess formation. Scintigraphic studies in humans using Tc 99m-rh annexin V have demonstrated the feasibility of imaging cell death in acute myocardial infarction, in tumors with a high apoptotic index, and in response to anti-tumor chemotherapy of non-small cell lung cancer, small-cell lung cancer, breast cancer, lymphoma, and sarcoma. Increased localization of Tc 99m-rh annexin V within 1 to 3 days of chemotherapy has been noted in some, but not all, subjects with these tumors. To date, most subjects showing increased Tc 99m-rh annexin V uptake after the first course of chemotherapy have shown objective

  14. Pancreatic carcinogenesis: apoptosis and angiogenesis.

    PubMed

    Onizuka, Shinya; Kawakami, Shunsuke; Taniguchi, Ken; Fujioka, Hikaru; Miyashita, Kosei

    2004-04-01

    Apoptosis and angiogenesis are critical biologic processes that are altered during carcinogenesis. Both apoptosis and angiogenesis may play an important role in pancreatic carcinogenesis. Despite numerous advances in the diagnosis and treatment of pancreatic cancer, its prognosis remains dismal and a new therapeutic approach is much needed. Recent research has revealed that apoptosis and angiogenesis are closely interrelated. Several reports show that a tumor suppresser gene that is expressed in pancreatic carcinoma and related to malignant potential can induce apoptosis and also inhibit angiogenesis. At present, it is generally accepted that tumor growth in cancers, including pancreatic cancer, depends on angiogenesis. We have identified 2 new angiogenesis inhibitors from a conditioned medium of human pancreatic carcinoma cell line (BxPC-3): antiangiogenic antithrombin III (aaAT-III) and vitamin D binding protein-macrophage activating factor (DBP-maf). These molecules were able to regress tumors in severe combined immunodeficiency disease (SCID) mice, demonstrating potent inhibition of endothelial cell proliferation. Moreover, the angiogenesis inhibitors induced tumor dormancy in the animal model. These results suggest that antiangiogenic therapy using angiogenesis inhibitors may become a new strategy for treatment of pancreatic cancer in the near future. PMID:15084979

  15. Fluorescence Lifetime Imaging of Apoptosis

    PubMed Central

    Xiao, Annie; Gibbons, Anne E.; Luker, Kathryn E.; Luker, Gary D.

    2015-01-01

    Genetically-encoded fluorescence resonance energy transfer (FRET) reporters are powerful tools to analyze cell signaling and function at single cell resolution in standard two-dimensional cell cultures, but these reporters rarely have been applied to three-dimensional environments. FRET interactions between donor and acceptor molecules typically are determined by changes in relative fluorescence intensities, but wavelength-dependent differences in absorption of light complicate this analysis method in three-dimensional settings. Here we report fluorescence lifetime imaging microscopy (FLIM) with phasor analysis, a method that displays fluorescence lifetimes on a pixel-wise basis in real time, to quantify apoptosis in breast cancer cells stably expressing a genetically encoded FRET reporter. This microscopic imaging technology allowed us to identify treatment-induced apoptosis in single breast cancer cells in environments ranging from two-dimensional cell culture, spheroids with cancer and bone marrow stromal cells, and living mice with orthotopic human breast cancer xenografts. Using this imaging strategy, we showed that combined metabolic therapy targeting glycolysis and glutamine pathways significantly reduced overall breast cancer metabolism and induced apoptosis. We also determined that distinct subpopulations of bone marrow stromal cells control resistance of breast cancer cells to chemotherapy, suggesting heterogeneity of treatment responses of malignant cells in different bone marrow niches. Overall, this study establishes FLIM with phasor analysis as an imaging tool for apoptosis in cell-based assays and living mice, enabling real-time, cellular-level assessment of treatment efficacy and heterogeneity. PMID:26771007

  16. APOPTOSIS IN WHOLE MOUSE OVARIES

    EPA Science Inventory

    Apoptosis in Whole Mouse Ovaries
    Robert M. Zucker Susan C. Jeffay and Sally D. Perreault
    Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina, 27711.

  17. Calmodulin antagonists induce platelet apoptosis.

    PubMed

    Wang, Zhicheng; Li, Suping; Shi, Quanwei; Yan, Rong; Liu, Guanglei; Dai, Kesheng

    2010-04-01

    Calmodulin (CaM) antagonists induce apoptosis in various tumor models and inhibit tumor cell invasion and metastasis, thus some of which have been extensively used as anti-cancer agents. In platelets, CaM has been found to bind directly to the cytoplasmic domains of several platelet receptors. Incubation of platelets with CaM antagonists impairs the receptors-related platelet functions. However, it is still unknown whether CaM antagonists induce platelet apoptosis. Here we show that CaM antagonists N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W7), tamoxifen (TMX), and trifluoperazine (TFP) induce apoptotic events in human platelets, including depolarization of mitochondrial inner transmembrane potential, caspase-3 activation, and phosphatidylserine exposure. CaM antagonists did not incur platelet activation as detected by P-selectin surface expression and PAC-1 binding. However, ADP-, botrocetin-, and alpha-thrombin-induced platelet aggregation, platelet adhesion and spreading on von Willebrand factor surface were significantly reduced in platelets pre-treated with CaM antagonists. Furthermore, cytosolic Ca(2+) levels were obviously elevated by both W7 and TMX, and membrane-permeable Ca(2+) chelator BAPTA-AM significantly reduced apoptotic events in platelets induced by W7. Therefore, these findings indicate that CaM antagonists induce platelet apoptosis. The elevation of the cytosolic Ca(2+) levels may be involved in the regulation of CaM antagonists-induced platelet apoptosis. PMID:20172594

  18. Pin1 in Neuronal Apoptosis

    PubMed Central

    Becker, Esther B.E.; Bonni, Azad

    2009-01-01

    While the role of the prolyl isomerase Pin1 in dividing cells has long been recognized, Pin1’s function in postmitotic neurons is poorly understood. We have identified a novel mechanism by which Pin1 mediates activation of the mitochondrial cell death machinery specifically in neurons. This perspective presents a sophisticated signaling pathway that triggers neuronal apoptosis upon JNK-mediated phosphorylation of the BH3-only protein BIMEL at serine 65. Pin1 is enriched at the mitochondria in neurons together with BIMEL and components of a neuron-specific JNK signaling complex and functions as a molecular switch that couples the phosphorylation of BIMEL by JNK to apoptosis specifically in neurons. We discuss how these findings relate to our understanding of the development of the nervous system and the pathogenesis of neurologic disorders. PMID:17568190

  19. Inhibitors of apoptosis catch ubiquitin.

    PubMed

    Rajalingam, Krishnaraj; Dikic, Ivan

    2009-01-01

    IAP (inhibitor of apoptosis) proteins are a class of anti-apoptotic regulators characterized by the presence of BIR (baculoviral IAP repeat) domains. Some of the IAPs also possess a RING (really interesting new gene) domain with E3 ubiquitin ligase activity. In this issue of the Biochemical Journal, Blankenship et al. unveil the presence of an UBA (ubiquitin-associated domain) in several IAPs. UBAs in c-IAPs (cellular IAPs) bind to monoubiquitin and ubiquitin chains and are implicated in degradation of c-IAPs by promoting their interaction with proteasomes as well as in regulation of TNF-alpha (tumour necrosis factor-alpha)-induced apoptosis. These novel observations establish IAPs as ubiquitin-interacting proteins and opens up new lines of investigation. PMID:19061481

  20. Apoptosis in irradiated murine tumors.

    PubMed

    Stephens, L C; Ang, K K; Schultheiss, T E; Milas, L; Meyn, R E

    1991-09-01

    Early radiation responses of transplantable murine ovarian (OCaI) and hepatocellular (HCaI) carcinomas were examined at 6, 24, 48, 96, and 144 h after single photon doses of 25, 35, or 45 Gy. Previous studies using tumor growth delay and tumor radiocurability assays had shown OCaI tumors to be relatively radiosensitive and HCaI tumors to be radioresistant. At 6 h, approximately 20% of nuclei in OCaI tumors showed aberrations characteristic of cell death by apoptosis. This contrasted to an incidence of 3% in HCaI tumors. Mitotic activity was eliminated in OCaI tumors but was only transiently suppressed in HCaI tumors. At 24-96 h, OCaI tumors continued to display apoptosis and progressive necrosis, whereas HCaI tumors responded by exhibiting marked pleomorphism. Factors other than mitotic activity may influence tumor radiosensitivity, and one of these may be susceptibility to induction of apoptosis (programmed cell death), because this was a prominent early radiation response by the radiosensitive OCaI tumors. PMID:1886987

  1. Apoptosis of V beta 8.2+ T lymphocytes in the spinal cord during recovery from experimental autoimmune encephalomyelitis induced in Lewis rats by inoculation with myelin basic protein.

    PubMed

    McCombe, P A; Nickson, I; Tabi, Z; Pender, M P

    1996-07-01

    To study T cell apoptosis during spontaneous recovery from experimental autoimmune encephalomyelitis (EAE), we extracted lymphocytes from the spinal cords of Lewis rats with EAE induced by inoculation with myelin basic protein (MBP) and adjuvants. Using flow cytometry we assessed the numbers of CD5+ and TCR alpha beta + lymphocytes, as well as V beta 8.2+ lymphocytes, which constitute the predominant encephalitogenic MBP-reactive cells in Lewis rats. Rats developed neurological signs of disease 10-12 days after inoculation. The peak of disease was on day 14 after inoculation and was followed by clinical recovery. The numbers of CD5+, TCR alpha beta + and V beta 8.2+ cells obtained from the spinal cord were greatest on day 13. During spontaneous clinical recovery, there was a decline in the numbers of all the cells studied, with a selective loss of V beta 8.2+ cells from the CD5+ and TCR alpha beta + populations. To determine whether the decline in lymphocyte numbers was due to apoptosis, we used simultaneous surface labelling and propidium iodide staining of the DNA of the cells extracted from the spinal cord. From day 14 onwards, there was selective enrichment of V beta 8.2+ cells in the apoptotic population, and the percentage of V beta 8.2+ cells undergoing apoptosis was greater than the percentages of CD5+ and TCR alpha beta + cells undergoing apoptosis. These findings indicate that recovery from acute EAE is associated with the selective apoptosis, in the central nervous system, of these disease-relevant cells. The findings in this study of actively induced EAE are similar to those of our previous study of EAE induced by transfer of encephalitogenic MBP-specific T cells (Z. Tabi et al., Eur. J. Immunol. 24: 2609-2617, 1994) and further support the hypothesis that selective apoptosis of autoreactive T cells in the central nervous system is of primary importance in spontaneous recovery from EAE. PMID:8836965

  2. Optogenetic apoptosis: light-triggered cell death.

    PubMed

    Hughes, Robert M; Freeman, David J; Lamb, Kelsey N; Pollet, Rebecca M; Smith, Weston J; Lawrence, David S

    2015-10-01

    An optogenetic Bax has been designed that facilitates light-induced apoptosis. We demonstrate that mitochondrial recruitment of a genetically encoded light-responsive Bax results in the release of mitochondrial proteins, downstream caspase-3 cleavage, changes in cellular morphology, and ultimately cell death. Mutagenesis of a key phosphorylatable residue or modification of the C-terminus mitigates background (dark) levels of apoptosis that result from Bax overexpression. The mechanism of optogenetic Bax-mediated apoptosis was explored using a series of small molecules known to interfere with various steps in programmed cell death. Optogenetic Bax appears to form a mitochondrial apoptosis-induced channel analogous to that of endogenous Bax. PMID:26418181

  3. Apoptosis and Molecular Targeting Therapy in Cancer

    PubMed Central

    Hassan, Mohamed; Watari, Hidemichi; AbuAlmaaty, Ali; Ohba, Yusuke; Sakuragi, Noriaki

    2014-01-01

    Apoptosis is the programmed cell death which maintains the healthy survival/death balance in metazoan cells. Defect in apoptosis can cause cancer or autoimmunity, while enhanced apoptosis may cause degenerative diseases. The apoptotic signals contribute into safeguarding the genomic integrity while defective apoptosis may promote carcinogenesis. The apoptotic signals are complicated and they are regulated at several levels. The signals of carcinogenesis modulate the central control points of the apoptotic pathways, including inhibitor of apoptosis (IAP) proteins and FLICE-inhibitory protein (c-FLIP). The tumor cells may use some of several molecular mechanisms to suppress apoptosis and acquire resistance to apoptotic agents, for example, by the expression of antiapoptotic proteins such as Bcl-2 or by the downregulation or mutation of proapoptotic proteins such as BAX. In this review, we provide the main regulatory molecules that govern the main basic mechanisms, extrinsic and intrinsic, of apoptosis in normal cells. We discuss how carcinogenesis could be developed via defective apoptotic pathways or their convergence. We listed some molecules which could be targeted to stimulate apoptosis in different cancers. Together, we briefly discuss the development of some promising cancer treatment strategies which target apoptotic inhibitors including Bcl-2 family proteins, IAPs, and c-FLIP for apoptosis induction. PMID:25013758

  4. Death-Defining Immune Responses After Apoptosis

    PubMed Central

    Campisi, L.; Cummings, R. J.; Blander, J. Magarian

    2014-01-01

    Apoptosis is a programmed form of cell death whereby characteristic internal cellular dismantling is accompanied by the preservation of plasma membrane integrity. Maintaining this order during apoptosis prevents the release of cellular contents and ensures a noninflammatory death. Here, we consider examples of apoptosis in different contexts and discuss how the same form of cell death could have different immunological consequences. Multiple parameters such as cell death as a result of microbial infection, the nature of the inflammatory microenvironment, the type of responding phagocytic cells and the genetic background of the host organism all differentially influence the immunological consequences of apoptosis. PMID:24903539

  5. Curcumin enhances recovery of pancreatic islets from cellular stress induced inflammation and apoptosis in diabetic rats

    SciTech Connect

    Rashid, Kahkashan; Sil, Parames C.

    2015-02-01

    The phytochemical, curcumin, has been reported to play many beneficial roles. However, under diabetic conditions, the detail mechanism of its beneficial action in the glucose homeostasis regulatory organ, pancreas, is poorly understood. The present study has been designed and carried out to explore the role of curcumin in the pancreatic tissue of STZ induced and cellular stress mediated diabetes in eight weeks old male Wistar rats. Diabetes was induced with a single intraperitoneal dose of STZ (65 mg/kg body weight). Post to diabetes induction, animals were treated with curcumin at a dose of 100 mg/kg body weight for eight weeks. Underlying molecular and cellular mechanism was determined using various biochemical assays, DNA fragmentation, FACS, histology, immunoblotting and ELISA. Treatment with curcumin reduced blood glucose level, increased plasma insulin and mitigated oxidative stress related markers. In vivo and in vitro experimental results revealed increased levels of proinflammatory cytokines (TNF-α, IL1-β and IFN-γ), reduced level of cellular defense proteins (Nrf-2 and HO-1) and glucose transporter (GLUT-2) along with enhanced levels of signaling molecules of ER stress dependent and independent apoptosis (cleaved Caspase-12/9/8/3) in STZ administered group. Treatment with curcumin ameliorated all the adverse changes and helps the organ back to its normal physiology. Results suggest that curcumin protects pancreatic beta-cells by attenuating inflammatory responses, and inhibiting ER/mitochondrial dependent and independent pathways of apoptosis and crosstalk between them. This uniqueness and absence of any detectable adverse effect proposes the possibility of using this molecule as an effective protector in the cellular stress mediated diabetes mellitus. - Highlights: • STZ induced cellular stress plays a vital role in pancreatic dysfunction. • Cellular stress causes inflammation, pancreatic islet cell death and diabetes. • Deregulation of Nrf-2

  6. Insulin exerts metabolic and growth-promoting effects by a direct action on the liver in vivo: clarification of the functional significance of the portal vascular link between the beta cells of the pancreatic islets and the liver.

    PubMed Central

    Griffen, S C; Russell, S M; Katz, L S; Nicoll, C S

    1987-01-01

    The functional significance of the portal vascular link between the beta cells of the pancreatic islets and the liver has not been established. Previous studies indicated that insulin does not acutely regulate glucose metabolism by a direct hepatic effect. More recent observations suggest that the role of insulin in regulating body growth may be mediated, at least in part, by the liver. Our experiments were designed to test whether insulin can promote body growth and regulate glucose metabolism by a direct hepatic action in vivo. Rats were made diabetic by injections of streptozotocin, and insulin or solvent was infused into the jugular vein (JV) or the hepatic portal vein (HPV) for 14 days using catheters that were attached to osmotic minipumps. Infusion of a low dose of insulin (2 units per kg per day) into the JV had no effects on the hyperglycemia, body weight gain, tail growth, tibial epiphysial cartilage plate thickness, or serum levels of somatomedin C in the diabetic rats. However, the same dose given into the HPV caused a 30% reduction of blood glucose and stimulated a significant degree of growth, as determined by all indices. Infusion of a higher dose of insulin (5 units per kg per day) into either vein caused full restoration of body weight gain and tail growth and it restored the glycemic status almost to normal. However, it did not increase the tibial epiphysial plate width or serum somatomedin C levels above those of the rats given the low dose of the hormone into the HPV. These results indicate that insulin can act directly on the liver to promote body growth and to regulate glucose metabolism. The significance of direct delivery of insulin from the pancreatic beta cells to the liver may be as much for growth control as for glucose homeostasis. Images PMID:3313390

  7. Therapeutic approaches to the modulation of apoptosis.

    PubMed

    Murphy, Finbarr J; Seery, Liam T; Hayes, Ian

    2003-01-01

    The appreciation of the role of apoptosis in the vast majority of diseases affecting humans has revolutionized the discovery and development of drugs targeting inflammation and oncology. Novel therapeutic approaches to modulate disease by regulating apoptosis are currently being tested in preclinical and clinical settings. Enthusiasm for some of these therapies is reflected in the fact that they have received U.S. Food and Drug Administration approval in record time. Approaches include the traditional use of small molecules to target specific players in the apoptosis cascade. They also include radical new approaches such as using antisense molecules to inhibit production of the Bcl-2 protein or antibodies that ligate either death receptors, such as TRAIL (tumour necrosis factor-related apoptosis-inducing ligand), or the MHC (HLA-DR), resulting in the initiation of apoptosis of target cells. Antibodies targeting cell-specific antigens are being used in conjunction with radioactive isotopes to deliver a more specific chemotherapy, particularly in the case of B-cell lymphomas. Other therapies target mitochondria, a key organelle in the apoptosis cascade. This diverse range of therapies includes photodynamic therapy, retinoic acid and arsenic trioxide, all of which induce apoptosis by generating reactive oxygen species. As our understanding of apoptosis increases, further opportunities will arise for tailor-made therapies that will result in improved clinical outcome without the devastating side effects of current interventions. PMID:14585079

  8. Apoptosis in mammalian oocytes: a review.

    PubMed

    Tiwari, Meenakshi; Prasad, Shilpa; Tripathi, Anima; Pandey, Ashutosh N; Ali, Irfan; Singh, Arvind K; Shrivastav, Tulsidas G; Chaube, Shail K

    2015-08-01

    Apoptosis causes elimination of more than 99% of germ cells from cohort of ovary through follicular atresia. Less than 1% of germ cells, which are culminated in oocytes further undergo apoptosis during last phases of oogenesis and depletes ovarian reserve in most of the mammalian species including human. There are several players that induce apoptosis directly or indirectly in oocytes at various stages of meiotic cell cycle. Premature removal of encircling granulosa cells from immature oocytes, reduced levels of adenosine 3',5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate, increased levels of calcium (Ca(2+)) and oxidants, sustained reduced level of maturation promoting factor, depletion of survival factors, nutrients and cell cycle proteins, reduced meiotic competency, increased levels of proapoptotic as well as apoptotic factors lead to oocyte apoptosis. The BH3-only proteins also act as key regulators of apoptosis in oocyte within the ovary. Both intrinsic (mitochondria-mediated) as well as extrinsic (cell surface death receptor-mediated) pathways are involved in oocyte apoptosis. BID, a BH3-only protein act as a bridge between both apoptotic pathways and its cleavage activates cell death machinery of both the pathways inside the follicular microenvironment. Oocyte apoptosis leads to the depletion of ovarian reserve that directly affects reproductive outcome of various mammals including human. In this review article, we highlight some of the important players and describe the pathways involved during oocyte apoptosis in mammals. PMID:25958165

  9. CHCHD2 connects mitochondrial metabolism to apoptosis

    PubMed Central

    Liu, Yong; Zhang, Yanping

    2015-01-01

    As the powerhouse of cells and gatekeeper for apoptosis, mitochondria control life and death. CHCHD2, a mitochondrial protein previously known to regulate metabolism, has recently been identified as an apoptosis inhibitor. New data suggest a model in which CHCHD2 performs a prosurvival function by acting as both a reactive oxygen species scavenger and BCL-XL activator. PMID:27308501

  10. THE ROLE OF APOPTOSIS IN NEUROTOXICOLOGY.

    EPA Science Inventory

    The role of apoptosis in neurodegeneration in developing animals and in adults has become increasingly apparent in the past ten years. Normal apoptosis occurs in the CNS from the embryonic stage through senescence, with different cells in each region of the nervous system having ...

  11. Analysis of apoptosis in Caenorhabditis elegans.

    PubMed

    Lant, Benjamin; Derry, W Brent

    2014-05-01

    The nematode worm Caenorhabditis elegans has provided researchers with a wealth of information on the molecular mechanisms controlling programmed cell death (apoptosis). Its genetic tractability, optical clarity, and relatively short lifespan are key advantages for rapid assessment of apoptosis in vivo. The use of forward and reverse genetics methodology, coupled with in vivo imaging, has provided deep insights into how a multicellular organism orchestrates the self-destruction of specific cells during development and in response to exogenous stresses. Strains of C. elegans carrying mutations in the core elements of the apoptotic pathway, or in tissue-specific regulators of apoptosis, can be used for genetic analyses to reveal conserved mechanisms by which apoptosis is regulated in the somatic and reproductive (germline) tissue. Here we present an introduction to the study of apoptosis in C. elegans, including current techniques for visualization, analysis, and screening. PMID:24786497

  12. Induction of apoptosis by Shiga toxins

    PubMed Central

    Tesh, Vernon L

    2010-01-01

    Shiga toxins comprise a family of structurally and functionally related protein toxins expressed by Shigella dysenteriae serotype 1 and multiple serotypes of Escherichia coli. While the capacity of Shiga toxins to inhibit protein synthesis by catalytic inactivation of eukaryotic ribosomes has been well described, it is also apparent that Shiga toxins trigger apoptosis in many cell types. This review presents evidence that Shiga toxins induce apoptosis of epithelial, endothelial, leukocytic, lymphoid and neuronal cells. Apoptotic signaling pathways activated by the toxins are reviewed with an emphasis on signaling mechanisms that are shared among different cell types. Data suggesting that Shiga toxins induce apoptosis through the endoplasmic reticulum stress response and clinical evidence demonstrating apoptosis in humans infected with Shiga toxin-producing bacteria are briefly discussed. The potential for use of Shiga toxins to induce apoptosis in cancer cells is briefly reviewed. PMID:20210553

  13. Apoptosis inducers in chronic lymphocytic leukemia

    PubMed Central

    Billard, Christian

    2014-01-01

    Chronic lymphocytic leukemia (CLL) is characterized by a typical defect in apoptosis and is still an incurable disease. Numerous apoptosis inducers have been described. These synthetic compounds and natural products (mainly derived from plants) display antileukemic properties in vitro and in vivo and some have even been tested in the clinic in CLL. They act through several different mechanisms. Most of them involve proteins of the Bcl-2 family, which are the key regulators in triggering the mitochondrial pathway of caspase-dependent apoptosis. Thus, the Mcl-1/Noxa axis appeared as a target. Here I overview natural and synthetic apoptosis inducers and their mechanisms of action in CLL cells. Opportunities for developing novel, apoptosis-based therapeutics are presented. PMID:24525395

  14. Lymphocyte apoptosis in murine Pneumocystis pneumonia

    PubMed Central

    Shi, Xin; LeCapitaine, Nicole J; Rudner, Xiaowen L; Ruan, Sanbao; Shellito, Judd E

    2009-01-01

    Background Apoptosis of lymphocytes is important in the termination of an immune response to infection but has also been shown to have detrimental effects in animal models of systemic infection and sepsis. We sought to characterize lymphocyte apoptosis in an animal model of pneumonia due to Pneumocystis murina, an infection localized to the lungs. Methods Control mice and mice depleted of CD4+ lymphocytes were inoculated with Pneumocystis. Apoptosis of lung and spleen lymphocytes was assayed by flow cytometry and PCR assay of apoptotic proteins. Results In control mice, apoptosis of lung lymphocytes was maximal just after the infection was cleared from lung tissue and then declined. However, in CD4-depleted mice, apoptosis was also upregulated in recruited lymphocytes in spite of progressive infection. In splenic lymphocytes, apoptosis was observed early at 1 week after inoculation and then declined. Apoptosis of lung lymphocytes in control mice was associated with a decrease in mRNA for Bcl-2 and an increase in mRNA for Bim. In CD4-depleted mice, lavaged CD8+ cells did change intracellular Bcl-2 but showed increased mRNA for Bim. Conclusion Apoptosis of both pulmonary and extrapulmonary lymphocytes is part of the normal host response to Pneumocystis but is also triggered in CD4-deficient animals with progressive infection. In normal mice apoptosis of pulmonary lymphocytes may serve to terminate the immune response in lung tissue. Apoptosis of lung lymphocytes takes place via both the intrinsic and extrinsic apoptotic pathways and is associated with changes in both pro- and anti-apoptotic proteins. PMID:19558669

  15. Corticosteroid treatment of experimental autoimmune encephalomyelitis in the Lewis rat results in loss of V beta 8.2+ and myelin basic protein-reactive cells from the spinal cord, with increased total T-cell apoptosis but reduced apoptosis of V beta 8.2+ cells.

    PubMed

    McCombe, P A; Nickson, I; Tabi, Z; Pender, M P

    1996-11-01

    We have studied the effects of corticosteroid treatment on the numbers of lymphocytes obtained from the spinal cords of Lewis rats with acute experimental autoimmune encephalomyelitis (EAE) induced by inoculation with myelin basic protein (MBP) and adjuvants. Flow cytometric studies showed that treatment with dexamethasone (4 mg/kg) 8-12 h prior to study on day 14 after inoculation resulted in a reduction in the numbers of CD5+, TCR alpha beta + and V beta 8.2+ cells in the spinal cord. Limiting dilution analysis indicated that dexamethasone treatment 12 h prior to study on day 12 after inoculation reduced the frequencies of MBP-reactive and interleukin-2-responsive lymphocytes in the spinal cord to low levels, but reduced the frequency of concanavalin-A-responsive lymphocytes to a lesser extent. Using propidium iodide staining of nuclear chromatin we also studied lymphocyte apoptosis. Greater numbers of apoptotic cells were found in the cells extracted from the spinal cords of rats, examined on day 14, that had been treated 1-12 h previously with dexamethasone, than in saline-treated controls. This increased level of apoptosis was observed in the CD5+ and TCR alpha beta + cell populations. At 1-4 h after dexamethasone treatment there was a reduction in the selective apoptosis of V beta 8.2+ cells that normally occurs during spontaneous recovery from EAE. Therefore apoptosis of V beta 8.2+ cells cannot explain the reduction in the numbers of V beta 8.2+ cells and MBP-reactive cells in the CNS after dexamethasone treatment. By 8-12 h after dexamethasone treatment the selectivity of the apoptotic process was restored. These studies suggest that a reduction in the number of T-lymphocytes in the central nervous system contributes to the beneficial effects of corticosteroids in EAE. PMID:8898717

  16. Scoparia dulcis, a traditional antidiabetic plant, protects against streptozotocin induced oxidative stress and apoptosis in vitro and in vivo.

    PubMed

    Latha, Muniappan; Pari, Leelavinothan; Sitasawad, Sandhya; Bhonde, Ramesh

    2004-01-01

    Oxidative stress is implicated in the pathogenesis of diabetic complications. The experiments were performed on normal and experimental male Wistar rats treated with Scoparia dulcis plant extract (SPEt). The effect of SPEt was tested on streptozotocin (STZ) treated Rat insulinoma cell lines (RINm5F cells) and isolated islets in vitro. Administration of an aqueous extract of Scoparia dulcis by intragastric intubation (po) at a dose of 200 mg/kg body weight significantly decreased the blood glucose and lipid peroxidative marker thiobarbituric acid reactive substances (TBARS) with significant increase in the activities of plasma insulin, pancreatic superoxide dismutase (SOD), catalase (CAT), and reduced glutathione (GSH) in streptozotocin diabetic rats at the end of 15 days treatment. Streptozotocin at a dose of 10 mug/mL evoked 6-fold stimulation of insulin secretion from isolated islets indicating its insulin secretagogue activity. The extract markedly reduced the STZ-induced lipidperoxidation in RINm5F cells. Further, SPEt protected STZ-mediated cytotoxicity and nitric oxide (NO) production in RINm5F cells. Treatment of RINm5F cells with 5 mM STZ and 10 mug of SPEt completely abrogated apoptosis induced by STZ, suggesting the involvement of oxidative stress. Flow cytometric assessment on the level of intracellular peroxides using fluorescent probe 2'7'-dichlorofluorescein diacetate (DCF-DA) confirmed that STZ (46%) induced an intracellular oxidative stress in RINm5F cells, which was suppressed by SPEt (21%). In addition, SPEt also reduced (33%) the STZ-induced apoptosis (72%) in RINm5F cells indicating the mode of protection of SPEt on RIN m5Fcells, islets, and pancreatic beta-cell mass (histopathological observations). Present study thus confirms antihyperglycemic effect of SPEt and also demonstrated the consistently strong antioxidant properties of Scoparia dulcis used in the traditional medicine. PMID:15549711

  17. Honey and Apoptosis in Human Gastric Mucosa

    PubMed Central

    Ghaffari, Aida; Somi, Mohammad H; Safaiyan, Abdolrasoul; Modaresi, Jabiz; Ostadrahimi, Alireza

    2012-01-01

    Background: Gastric cancer is the fourth most common malignancy in the world. Honey is a complex mixture of special biological active constituents. Honey possesses antioxidant and antitumor properties. Nutritional studies have indicated that consumption of honey modulates the risk of developing gastric cancer. On the other hand, apoptosis has been reported to play a decisive role in precancerous changes. Our chief study was conducted to assess the relationship between consumption of honey and apoptosis in human gastric mucosa. Method: This cross-sectional study was conducted on 98 subjects over 18 years old, referred to two hospitals in Tabriz, Iran. Subjects were undergone an upper gastrointestinal endoscopy, 62 subjects were finally enrolled. Honey consumption was assessed by a Food Frequency Questionnaire (FFQ) and apoptosis was detected by TUNEL technique. We tested polynomial curve to find the best fit between honey consumption and apoptosis. Results: A positive relation between honey consumption and apoptosis was found (P=0.024). Our results indicated that the final and the best fit curve was: apoptosis = 1.714+1.648(honey amount) - 0.533(honey amount)2 +1.833×10-5(honey amount)7. Conclusion: Honey consumption had positive effects on gastric cancer by inducing apoptosis in gastric mucosa. PMID:24688918

  18. The Role of Mitochondria in Apoptosis*

    PubMed Central

    Wang, Chunxin; Youle, Richard J.

    2016-01-01

    Mitochondria play key roles in activating apoptosis in mammalian cells. Bcl-2 family members regulate the release of proteins from the space between the mitochondrial inner and outer membrane that, once in the cytosol, activate caspase proteases that dismantle cells and signal efficient phagocytosis of cell corpses. Here we review the extensive literature on proteins released from the intermembrane space and consider genetic evidence for and against their roles in apoptosis activation. We also compare and contrast apoptosis pathways in Caenorhabditis elegans, Drosophila melanogaster, and mammals that indicate major mysteries remaining to be solved. PMID:19659442

  19. [The comeback of mitochondria in Drosophila apoptosis].

    PubMed

    Clavier, Amandine; Rincheval-Arnold, Aurore; Mignotte, Bernard; Guénal, Isabelle

    2016-05-01

    The role of the mitochondrion in mammalian cell apoptosis has been established since the mid-1990s. However, the importance of this organelle in non-mammalian apoptosis has long been regarded as minor, notably because of the absence of a crucial role for cytochrome c in caspase activation. Recent results indicate that the control of caspase activation and apoptosis in Drosophila cell death occurs at the mitochondrial level. Numerous proteins that appear key for Drosophila apoptosis regulation constitutively or transiently bind to mitochondria. They participate in the cell death process at different levels such as degradation of an IAP caspase inhibitor, production of mitochondrial reactive oxygen species or stimulation of the mitochondrial fission machinery. The aim of this review is to take stock of these events that might have their counterpart in humans. PMID:27225920

  20. Autophagy and apoptosis in liver injury

    PubMed Central

    Wang, Kewei

    2015-01-01

    Apoptosis is a primary characteristic in the pathogenesis of liver disease. Hepatic apoptosis is regulated by autophagic activity. However, mechanisms mediating their interaction remain to be determined. Basal level of autophagy ensures the physiological turnover of old and damaged organelles. Autophagy also is an adaptive response under stressful conditions. Autophagy can control cell fate through different cross-talk signals. A complex interplay between hepatic autophagy and apoptosis determines the degree of hepatic apoptosis and the progression of liver disease as demonstrated by pre-clinical models and clinical trials. This review summarizes recent advances on roles of autophagy that plays in pathophysiology of liver. The autophagic pathway can be a novel therapeutic target for liver disease. PMID:25927598

  1. Noninvasive real-time imaging of apoptosis.

    PubMed

    Laxman, Bharathi; Hall, Daniel E; Bhojani, Mahaveer Swaroop; Hamstra, Daniel A; Chenevert, Thomas L; Ross, Brian D; Rehemtulla, Alnawaz

    2002-12-24

    Strict coordination of proliferation and programmed cell death (apoptosis) is essential for normal physiology. An imbalance in these two opposing processes results in various diseases including AIDS, neurodegenerative disorders, myelodysplastic syndromes, ischemiareperfusion injury, cancer, autoimmune disease, among others. Objective and quantitative noninvasive imaging of apoptosis would be a significant advance for rapid and dynamic screening as well as validation of experimental therapeutic agents. Here, we report the development of a recombinant luciferase reporter molecule that when expressed in mammalian cells has attenuated levels of reporter activity. In cells undergoing apoptosis, a caspase-3-specific cleavage of the recombinant product occurs, resulting in the restoration of luciferase activity that can be detected in living animals with bioluminescence imaging. The ability to image apoptosis noninvasively and dynamically over time provides an opportunity for high-throughput screening of proapoptotic and antiapoptotic compounds and for target validation in vivo in both cell lines and transgenic animals. PMID:12475931

  2. Autophagy and apoptosis in liver injury.

    PubMed

    Wang, Kewei

    2015-01-01

    Apoptosis is a primary characteristic in the pathogenesis of liver disease. Hepatic apoptosis is regulated by autophagic activity. However, mechanisms mediating their interaction remain to be determined. Basal level of autophagy ensures the physiological turnover of old and damaged organelles. Autophagy also is an adaptive response under stressful conditions. Autophagy can control cell fate through different cross-talk signals. A complex interplay between hepatic autophagy and apoptosis determines the degree of hepatic apoptosis and the progression of liver disease as demonstrated by pre-clinical models and clinical trials. This review summarizes recent advances on roles of autophagy that plays in pathophysiology of liver. The autophagic pathway can be a novel therapeutic target for liver disease. PMID:25927598

  3. An AIF orthologue regulates apoptosis in yeast

    PubMed Central

    Wissing, Silke; Ludovico, Paula; Herker, Eva; Büttner, Sabrina; Engelhardt, Silvia M.; Decker, Thorsten; Link, Alexander; Proksch, Astrid; Rodrigues, Fernando; Corte-Real, Manuela; Fröhlich, Kai-Uwe; Manns, Joachim; Candé, Céline; Sigrist, Stephan J.; Kroemer, Guido; Madeo, Frank

    2004-01-01

    Apoptosis-inducing factor (AIF), a key regulator of cell death, is essential for normal mammalian development and participates in pathological apoptosis. The proapoptotic nature of AIF and its mode of action are controversial. Here, we show that the yeast AIF homologue Ynr074cp controls yeast apoptosis. Similar to mammalian AIF, Ynr074cp is located in mitochondria and translocates to the nucleus of yeast cells in response to apoptotic stimuli. Purified Ynr074cp degrades yeast nuclei and plasmid DNA. YNR074C disruption rescues yeast cells from oxygen stress and delays age-induced apoptosis. Conversely, overexpression of Ynr074cp strongly stimulates apoptotic cell death induced by hydrogen peroxide and this effect is attenuated by disruption of cyclophilin A or the yeast caspase YCA1. We conclude that Ynr074cp is a cell death effector in yeast and rename it AIF-1 (Aif1p, gene AIF1). PMID:15381687

  4. Regulation of apoptosis by peroxisome proliferators.

    PubMed

    Roberts, Ruth A; Michel, Cecile; Coyle, Beth; Freathy, Caroline; Cain, Kelvin; Boitier, Eric

    2004-04-01

    Peroxisome proliferators (PPs) constitute a large and chemically diverse family of non-genotoxic rodent hepatocarcinogens that activate the PP-activated receptor alpha (PPARalpha). In order to investigate the hypothesis that PPs elicit their carcinogenic effects through the suppression of apoptosis, we established an in vitro assay for apoptosis using both primary rat hepatocytes and the FaO rat hepatoma cell line. Apoptosis was induced by transforming growth factor beta1 (TGFbeta1), the physiological negative regulator of liver growth. In this system, PPs could suppress both spontaneous and TGFbeta1-induced apoptosis. In order to understand the mechanisms of this regulation of apoptosis, we conducted microarray analysis followed by pathway-specific gene clustering in TGFbeta1-treated cells. After treatment, 76 genes were up-regulated and 185 were down-regulated more than 1.5-fold. Cluster analysis of up-regulated genes revealed three clusters, A-C. Cluster A (4h) was associated with 12% apoptosis and consisted of genes mainly of the cytoskeleton and extracellular matrix such as troponin and the proteoglycan SDC4. In cluster B (8h; 25% apoptosis), there were many pro- and anti-apoptotic genes such as XIAP, BAK1 and BAD, whereas at 16h (40% apoptosis) the regulated genes were mainly those of the cellular stress pathways such as the genes implicated in the activation of the transcription factor NFkappab. Genes found down-regulated in response to TGFbeta1 were mainly those associated with oxidative stress and several genes implicated in glutathione production and maintenance. Thus, TGFbeta1 may induce apoptosis via a down regulation of oxidant defence leading to the generation of reactive oxygen species. The ability of PPs to impact on these apoptosis pathways remains to be determined. To approach this question, we have developed a technique using laser capture microdissection of livers treated with the PP, clofibric acid coupled with gene expression array analysis

  5. The Relationship of Metabolic Syndrome Traits with Beta-Cell Function and Insulin Sensitivity by Oral Minimal Model Assessment in South Asian and European Families Residing in the Netherlands

    PubMed Central

    Jainandunsing, Sjaam; de Rooij, Felix W. M.

    2016-01-01

    Background. There are different metabolic syndrome traits among patients with different ethnicities. Methods. We investigated this by studying 44 South Asians and 54 Europeans and classified them in three groups according to the occurrence of metabolic syndrome (MetS) and Type 2 Diabetes (T2D). Insulin sensitivity index (ISI), static, dynamic, and total beta-cell responsivity indices (Φ), and disposition indices (DIs) were calculated with the use of oral minimal model (OMM). Results. In both ethnicities, ISI was lower in the subgroup with MetS and T2D as compared to the subgroup without MetS nor T2D (P < 0.004). South Asians without MetS were more insulin resistant than Europeans without MetS (P = 0.033). In the South Asians, ISI, dynamic DI, and static DI were associated significantly (P < 0.006) with high-density lipoprotein cholesterol and triglycerides. In the Europeans, ISI was associated with waist-to-hip ratio (P = 0.005) and systolic and diastolic blood pressure (P < 0.005), while static DI was related to the systolic blood pressure (P = 0.005). Conclusions. MetS was linked with insulin resistance and reduced capacity to handle glucose regardless of ethnicity. ISI and DIs were associated with lipid traits in South Asians and with blood pressure in Europeans suggesting that insulin resistance enhances different metabolic syndrome traits among different ethnicities. PMID:27597980

  6. The Relationship of Metabolic Syndrome Traits with Beta-Cell Function and Insulin Sensitivity by Oral Minimal Model Assessment in South Asian and European Families Residing in the Netherlands.

    PubMed

    Geragotou, Thekla; Jainandunsing, Sjaam; Özcan, Behiye; de Rooij, Felix W M; Kokkinos, Alexander; Tentolouris, Nicholas; Sijbrands, Eric J G

    2016-01-01

    Background. There are different metabolic syndrome traits among patients with different ethnicities. Methods. We investigated this by studying 44 South Asians and 54 Europeans and classified them in three groups according to the occurrence of metabolic syndrome (MetS) and Type 2 Diabetes (T2D). Insulin sensitivity index (ISI), static, dynamic, and total beta-cell responsivity indices (Φ), and disposition indices (DIs) were calculated with the use of oral minimal model (OMM). Results. In both ethnicities, ISI was lower in the subgroup with MetS and T2D as compared to the subgroup without MetS nor T2D (P < 0.004). South Asians without MetS were more insulin resistant than Europeans without MetS (P = 0.033). In the South Asians, ISI, dynamic DI, and static DI were associated significantly (P < 0.006) with high-density lipoprotein cholesterol and triglycerides. In the Europeans, ISI was associated with waist-to-hip ratio (P = 0.005) and systolic and diastolic blood pressure (P < 0.005), while static DI was related to the systolic blood pressure (P = 0.005). Conclusions. MetS was linked with insulin resistance and reduced capacity to handle glucose regardless of ethnicity. ISI and DIs were associated with lipid traits in South Asians and with blood pressure in Europeans suggesting that insulin resistance enhances different metabolic syndrome traits among different ethnicities. PMID:27597980

  7. Human mature erythroblasts are resistant to apoptosis.

    PubMed

    Hristoskova, Sashka; Holzgreve, Wolfgang; Hahn, Sinuhe; Rusterholz, Corinne

    2007-03-10

    Apoptosis plays an important role in red blood cell development, notably by regulating the fate of early erythroid progenitors. We show here that, by contrast, mature erythroblasts are resistant to apoptosis. Treatment of these cells with several apoptosis-inducing agents failed to trigger caspase activation and oligonucleosomal DNA fragmentation. Interestingly, we find that cytochrome c levels are dramatically reduced even though the cells contain mitochondria. Supplementation of cytosolic extracts from mature erythroblasts with cytochrome c, however, did not rescue caspase activation. This was not due to the presence of inhibitors of apoptosis, as these proteins were also missing in these cells. We also show that cytochrome c depletion is a normal event during erythroblast differentiation, which follows transient, developmentally induced caspase activation and correlates with the loss of response to cytokine withdrawal or drug-induced apoptosis. Our data therefore suggest that erythroblasts acquire resistance to apoptosis during maturation through the developmentally induced depletion of cytochrome c and other crucial regulators of the apoptotic machinery. PMID:17289021

  8. Umbelliprenin Induces Apoptosis in CLL Cell Lines

    PubMed Central

    Ziai, Seyed Ali; Gholami, Omid; Iranshahi, Mehrdad; Zamani, Amir Hassan; Jeddi-Tehrani, Mahmood

    2012-01-01

    Chronic lymphocytic leukemia (CLL) remains an incurable disease that requires innovative new approaches to improve therapeutic outcome. Many Ferula species, including F. asa-foetida, synthesize terpenyloxy coumarins. One of these coumarins is umbelliprenin, which has been implicated with induction of apoptosis in some cancer cell lines. In this study induction of apoptosis by umbelliprenin on Jurkat T-CLL and Raji B-CLL cell lines was studied. In this regard, cells were incubated with various concentrations of umbelliprenin in-vitro for different times and assayed for apoptosis with annexin V–FITC/PI double staining flowcytometry method. Results showed that umbelliprenin induced apoptosis in leukemic cells in a dose- and time-dependent manner and that CLL cells were more susceptible to umbelliprenin induced cell death than normal peripheral blood mononuclear cell (PBMCs). Moreover, we study the induction of apoptosis in Jurkat cells by umbelliprenin in the presence of interleukin 4 (IL-4) as an agent that causes resistance to apoptosis in CLL cells, was also student. We showed that IL-4 can not reduce apoptotic effect of umbelliprenin. The preferential toxicity of umbelliprenin for CLL cells, supports the hypothesis that oral administration of umbelliprenin in the form of foods or folk medicines containing this coumarin, might enhance protection against the development of CLL in man with little side effects. In conclusion, umbelliprenin may be an effective therapeutic agent in the treatment of CLL, and thus clinical studies with umbelliprenin may be appropriate. PMID:24250490

  9. Ultrastructural definition of apoptosis in heart failure.

    PubMed

    Arbustini, Eloisa; Brega, Agnese; Narula, Jagat

    2008-06-01

    Cardiac myocytes die through apoptosis, oncosis, and autophagy. Apoptosis affects single cells and is morphologically characterized by nuclear fragmentation with generation of apoptotic bodies that can be seen either within dying cells or free in the interstitial spaces. Dead myocytes are removed by macrophages through phagocytosis without triggering inflammation. The circulating markers of myocyte necrosis are not increased by apoptosis. The morphologic changes of the induction and early execution phases are seen at electron microscopy while late fragmentation is visible on both light and electron microscopy. Immunoelectron microscopy provides combined functional and structural information showing cytochrome c immuno-labelling release from mitochondria, TUNEL labelling of apoptotic nuclei, annexin V translocation in the outer plasma cell layer. Oncosis is characterized by specific morphologic features that may coexist with apoptosis, especially in ischemic myocardium. Autophagy is a defense process that is associated with significant myocardial damage and necrosis when removal of the lysosomal content is impaired. Morphological features of apoptosis, oncosis, and autophagocytosis may coexist at the same time. Although dead myocytes showing characteristics of autophagy and apoptosis are rarely observed in human decompensated hearts, autophagic vacuoles, and early apoptotic changes may be seen more often in morphologically viable myocytes. Such features may occur in failing hearts of both ischemic and non-ischemic etiology. The shared mode of cardiac myocyte death in failing human hearts of different etiologies suggests that preservation of myocyte integrity may be possible by similar therapeutic strategies. PMID:18172761

  10. Morphologic criteria and detection of apoptosis.

    PubMed

    Saraste, A

    1999-05-01

    Apoptosis is an organized, energy dependent process, which leads to cell death. Its definition is based on distinct morphological features [10] and demonstration of internucleosomal DNA degradation [27], executed by selectively activated DNAses [4, 22]. The morphologic hallmarks of apoptosis include chromatic margination, nuclear condensation and fragmentation, and condensation of the cell with preservation of organelles. The process is followed by fragmentation of the cell into membrane-bound apoptotic bodies, which undergo phagocytosis by nearby cells without associated inflammation [10, 11]. Apoptosis characteristically occurs in insolated single cells. The duration of apoptosis is estimated to be from 12 to 24 hours, but in cell culture visible morphologic changes are accomplished in less than two hours [10, 16]. Non-apoptotic cell death, a prototype of which is cell death due to ischemia (oncosis), is characterized by depletion of intracellular ATP stores, swelling of the cell with disruption of organelles and rupture of the plasma membrane [15]. Groups of necrotic cells and inflammation are found in tissues [10, 15]. The significance of apoptosis has mostly been studied using the TUNEL assay that detects DNA strand breaks in tissue sections and allows quantification of apoptotic cells by light microscopy [6]. Common experience seems to be that the TUNEL assay is prone to false positive or negative findings. This has been explained by the dependence of the staining kinetics on the reagent concentration [17], fixation of the tissue [2] and the extent of proteolysis [17]. Active RNA synthesis [12] and DNA damage in necrotic cells [17, 19] may cause non-specific staining. To obtain reliable and reproducible results, TUNEL assay should be carefully standardized by using tissue sections treated with DNAse (positive control of apoptosis). Quantification of apoptosis should include enough microscopic fields and identification of the cell type undergoing apoptosis

  11. Trauma patients’ elevated Tumor Necrosis Related Apoptosis Inducing Ligand (TRAIL) contributes to increased T cell apoptosis

    PubMed Central

    Bandyopadhyay, Gautam; Bankey, Paul E.; Miller-Graziano, Carol L.

    2012-01-01

    Immunosuppression resulting from excessive post-trauma apoptosis of hyperactivated Tcells is controversial. TRAIL mediated Tcell apoptosis decreases highly activated Tcells’ responses. Caspase-10, a particular TRAIL target, was increased in trauma patients’ Tcells with concomitantly elevated plasma TRAIL levels. These patients’ Tcells developed anergy, implicating increased TRAIL-mediated Tcell apoptosis in post-trauma Tcell anergy. Control Tcells cultured with patients’ sera containing high TRAIL levels increased their Caspase-10 activity and apoptosis. Stimulated primary Tcells are TRAIL apoptosis resistant. Increased plasma Thrombospondin-1 and Tcell expression of CD47, a Thrombospondin-1 receptor, preceded patients’ Tcell anergy. CD47 triggering of Tcells increased their sensitivity to TRAIL-induced apoptosis. Augmentation of Tcell TRAIL-induced apoptosis was secondary to CD47 triggered activation of the Src homology-containing phosphatase-1(SHP-1) and was partially blocked by a SHP-1 inhibitor. We suggest that combined post-trauma CD47 triggering, SHP-1 mediated NFκB suppression, and elevated TRAIL levels increase patients’ CD47 expressing Tcell apoptosis, thus contributing to subsequent Tcell anergy. PMID:22926077

  12. Chondrocyte Apoptosis in the Pathogenesis of Osteoarthritis

    PubMed Central

    Hwang, Hyun Sook; Kim, Hyun Ah

    2015-01-01

    Apoptosis is a highly-regulated, active process of cell death involved in development, homeostasis and aging. Dysregulation of apoptosis leads to pathological states, such as cancer, developmental anomalies and degenerative diseases. Osteoarthritis (OA), the most common chronic joint disease in the elderly population, is characterized by progressive destruction of articular cartilage, resulting in significant disability. Because articular cartilage depends solely on its resident cells, the chondrocytes, for the maintenance of extracellular matrix, the compromising of chondrocyte function and survival would lead to the failure of the articular cartilage. The role of subchondral bone in the maintenance of proper cartilage matrix has been suggested as well, and it has been proposed that both articular cartilage and subchondral bone interact with each other in the maintenance of articular integrity and physiology. Some investigators include both articular cartilage and subchondral bone as targets for repairing joint degeneration. In late-stage OA, the cartilage becomes hypocellular, often accompanied by lacunar emptying, which has been considered as evidence that chondrocyte death is a central feature in OA progression. Apoptosis clearly occurs in osteoarthritic cartilage; however, the relative contribution of chondrocyte apoptosis in the pathogenesis of OA is difficult to evaluate, and contradictory reports exist on the rate of apoptotic chondrocytes in osteoarthritic cartilage. It is not clear whether chondrocyte apoptosis is the inducer of cartilage degeneration or a byproduct of cartilage destruction. Chondrocyte death and matrix loss may form a vicious cycle, with the progression of one aggravating the other, and the literature reveals that there is a definite correlation between the degree of cartilage damage and chondrocyte apoptosis. Because current treatments for OA act only on symptoms and do not prevent or cure OA, chondrocyte apoptosis would be a valid

  13. Chondrocyte Apoptosis in the Pathogenesis of Osteoarthritis.

    PubMed

    Hwang, Hyun Sook; Kim, Hyun Ah

    2015-01-01

    Apoptosis is a highly-regulated, active process of cell death involved in development, homeostasis and aging. Dysregulation of apoptosis leads to pathological states, such as cancer, developmental anomalies and degenerative diseases. Osteoarthritis (OA), the most common chronic joint disease in the elderly population, is characterized by progressive destruction of articular cartilage, resulting in significant disability. Because articular cartilage depends solely on its resident cells, the chondrocytes, for the maintenance of extracellular matrix, the compromising of chondrocyte function and survival would lead to the failure of the articular cartilage. The role of subchondral bone in the maintenance of proper cartilage matrix has been suggested as well, and it has been proposed that both articular cartilage and subchondral bone interact with each other in the maintenance of articular integrity and physiology. Some investigators include both articular cartilage and subchondral bone as targets for repairing joint degeneration. In late-stage OA, the cartilage becomes hypocellular, often accompanied by lacunar emptying, which has been considered as evidence that chondrocyte death is a central feature in OA progression. Apoptosis clearly occurs in osteoarthritic cartilage; however, the relative contribution of chondrocyte apoptosis in the pathogenesis of OA is difficult to evaluate, and contradictory reports exist on the rate of apoptotic chondrocytes in osteoarthritic cartilage. It is not clear whether chondrocyte apoptosis is the inducer of cartilage degeneration or a byproduct of cartilage destruction. Chondrocyte death and matrix loss may form a vicious cycle, with the progression of one aggravating the other, and the literature reveals that there is a definite correlation between the degree of cartilage damage and chondrocyte apoptosis. Because current treatments for OA act only on symptoms and do not prevent or cure OA, chondrocyte apoptosis would be a valid

  14. Solamargine triggers hepatoma cell death through apoptosis

    PubMed Central

    XIE, XIAODONG; ZHU, HAITAO; YANG, HUIJIAN; HUANG, WENSI; WU, YINGYING; WANG, YING; LUO, YANLING; WANG, DONGQING; SHAO, GENBAO

    2015-01-01

    Solamargine (SM), a steroidal alkaloid glycoside extracted from the traditional Chinese herb Solanum incanum, has been evidenced to inhibit the growth and induce apoptosis in a number of human cancer cell lines. In the present study, the anticancer effect of SM and underlying molecular mechanism of SM-induced apoptosis were investigated on the human hepatocellular carcinoma cells, SMMC7721 and HepG2. The proliferation effects of SM on the SMMC7721 and HepG2 cell lines were evaluated using MTT and colony formation assays. In addition, the percentage of apoptosis was measured using an Annexin V/propidium iodide staining method and the cell cycle distribution mediated by SM was analyzed using flow cytometry. The expression levels of B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), caspase-3, caspase-9, proliferating cell nuclear antigen (pcna) and Ki67 proteins were examined to further demonstrate the proliferate and apoptosis effects of SM on the hepatoma cells. The results indicated that SM effectively inhibited hepatoma cell proliferation and promoted apoptosis. SM resulted in cell cycle arrest at the G2/M phase in the two cell lines. In addition, SM downregulated the levels of proliferation-associated (Ki67 and pcna) and anti-apoptotic (Bcl-2) proteins, and promoted the activity of apoptosis-associated proteins (Bax, caspase-3 and caspase-9). Therefore, the activation of the Bcl-2/Bax and caspase signaling pathways may be involved in the SM-induced apoptosis of hepatoma cells. PMID:26170994

  15. Training-induced apoptosis in skeletal muscle.

    PubMed

    Boffi, F M; Cittar, J; Balskus, G; Muriel, M; Desmaras, E

    2002-09-01

    Apoptosis or programmed cell death is a genetically controlled response of cells to commit suicide and is associated with DNA fragmentation or laddering. The common inducers of apoptosis include Ca2+i and oxygen free radicals/oxidative stress, which are also implicated in the pathogenesis of exercise-induced myopathies. To examine training-induced apoptosis, Thoroughbred horses were subjected to 3 months training programme on a treadmill. At the end of the training programme venous blood samples were taken for a creatine kinase (CK) assay. In addition, muscle biopsy samples were obtained for a membrane lipid peroxidation measurement by malondialdehyde (MDA) assay and for apoptosis detection. Apoptosis was studied by visualising the apoptotic myocytes on the paraffin sections by the modified TUNEL method. DNA laddering was evaluated by subjecting the DNA obtained from the biopsies to 1.5% agarose gel electrophoresis. There was a significant increase (P<0.05) of protein-bound MDA, and a nonsignificant trend (P = 0.14) for the control group to have higher levels of CK compared to the trained group. Under light microscopy, percentage of the TUNEL positive cells was higher (P<0.001) in the training group. This result was corroborated with the findings of DNA fragmentation by gel electrophoresis, which showed higher ladders of DNA band at the same group. In conclusion, these results clearly demonstrate that there is training-induced apoptosis in skeletal muscle. It is probable that apoptosis allows the work/recovery/rebound/supercompensation cycle, when unaccustomed muscle cells activate programmed cell death and are replaced by new and stronger cells, which is the mechanism for training-induced increases in fitness. PMID:12405700

  16. Evaluation of Apoptosis in Immunotoxicity Testing

    PubMed Central

    Nagarkatti, Mitzi; Rieder, Sadiye Amcaoglu; Vakharia, Dilip; Nagarkatti, Prakash S.

    2014-01-01

    Immunotoxicity testing is important in determining the toxic effects of chemical substances, medicinal products, airborne pollutants, cosmetics, medical devices, and food additives. The immune system of the host is a direct target of these toxicants, and the adverse effects include serious health complications such as susceptibility to infections, cancer, allergic reactions, and autoimmune diseases. One way to investigate the harmful effects of different chemicals is to study apoptosis in immune cell populations. Apoptosis is defined as the programmed cell death, and in general, this process helps in development and maintains homeostasis. However, in the case of an insult by a toxicant, apoptosis of the immune cells can lead to immunosuppression resulting in the development of cancer and the inability to fight infections. Apoptosis is characterized by cell shrinkage, nuclear condensation, changes in cell membrane and mitochondria, DNA fragmentation into 200 base oligomers, and protein degradation by caspases. Various methods are employed in order to investigate apoptosis. These methods include direct measurement of apoptotic cells with flow cytometry and in situ labeling, as well as RNA, DNA, and protein assays that are indicative of apoptotic molecules. PMID:19967519

  17. Sodium nitroprusside induces apoptosis of rabbit chondrocytes

    NASA Astrophysics Data System (ADS)

    Liang, Qian; Wang, Xiao-Ping; Chen, Tong-Sheng

    2013-02-01

    Osteoarthritis (OA) is characterized by a slowly progressing degradation of the matrix and destruction of articular cartilage. Apoptosis of chondrocyte is accounted for the mechanism of OA. Nitric oxide (NO), as a stimulus, has been shown to induce chondrocyte apoptosis by activating the matrix metalloproteinases (MMPs), increasing the expression of cyclooxygenase 2 (COX-2) and the level of prostaglandin E2 (PGE2), inhibiting the proteoglycan synthesis and type II collagen expression. In this study, sodium nitroprusside (SNP) was administered to be the NO donor to explore the mechanism of NO-induced apoptosis of rabbit chondrocytes obtained from six weeks old New Zealand rabbits. CCK-8 assay revealed the inhibitory effect of SNP on cell viability. We used flow cytometry (FCM) to assess the form of cell death by Annexin-V/propidium iodide (PI) double staining, and evaluate the change of mitochondrial membrane potential (ΔΨm). We found that the SNP induced chondrocyte apoptosis in a dose- and time-dependent manner and an observable reduction of ΔΨm. In conclusion, our findings indicate that SNP induces apoptosis of rabbit chondrocytes via a mitochondria-mediated pathway.

  18. Targeting the Apoptosis Pathway in Hematologic Malignancies

    PubMed Central

    Zaman, Shadia; Wang, Rui; Gandhi, Varsha

    2014-01-01

    Apoptosis is a cell death program that is well-orchestrated for normal tissue homeostasis and for removal of damaged, old, or infected cells. It is regulated by intrinsic and extrinsic pathways. The intrinsic pathway responds to signals such as ultraviolet radiation or DNA damage and activates “executioner” caspases through a mitochondria-dependent pathway. The extrinsic pathway is activated by death signals induced, for example, by an infection that activates the immune system or receptor-mediated pathways. The extrinsic pathway signals also cascade down to executioner caspases that cleave target proteins and lead to cell death. Strict control of cellular apoptosis is important for the hematopoietic system as it has a high turnover rate. However, the apoptosis program is often deregulated in hematologic malignancies leading to the accumulation of malignant cells. Therefore, apoptosis pathways have been identified for development of anticancer therapeutics. We review here the proteins that have been targeted for anticancer drug development in hematologic malignancies. These include BCL-2 family proteins, death ligands and receptors, inhibitor of apoptosis family proteins, and caspases. Except for caspase activators, drugs that target each of these classes of proteins have advanced into clinical trials. PMID:24295132

  19. NMR exposure sensitizes tumor cells to apoptosis.

    PubMed

    Ghibelli, L; Cerella, C; Cordisco, S; Clavarino, G; Marazzi, S; De Nicola, M; Nuccitelli, S; D'Alessio, M; Magrini, A; Bergamaschi, A; Guerrisi, V; Porfiri, L M

    2006-03-01

    NMR technology has dramatically contributed to the revolution of image diagnostic. NMR apparatuses use combinations of microwaves over a homogeneous strong (1 Tesla) static magnetic field. We had previously shown that low intensity (0.3-66 mT) static magnetic fields deeply affect apoptosis in a Ca2+ dependent fashion (Fanelli et al., 1999 FASEBJ., 13;95-102). The rationale of the present study is to examine whether exposure to the static magnetic fields of NMR can affect apoptosis induced on reporter tumor cells of haematopoietic origin. The impressive result was the strong increase (1.8-2.5 fold) of damage-induced apoptosis by NMR. This potentiation is due to cytosolic Ca2+ overload consequent to NMR-promoted Ca2+ influx, since it is prevented by intracellular (BAPTA-AM) and extracellular (EGTA) Ca2+ chelation or by inhibition of plasma membrane L-type Ca2+ channels. Three-days follow up of treated cultures shows that NMR decrease long term cell survival, thus increasing the efficiency of cytocidal treatments. Importantly, mononuclear white blood cells are not sensitised to apoptosis by NMR, showing that NMR may increase the differential cytotoxicity of antitumor drugs on tumor vs normal cells. This strong, differential potentiating effect of NMR on tumor cell apoptosis may have important implications, being in fact a possible adjuvant for antitumor therapies. PMID:16528477

  20. Lysosomal destabilization in p53-induced apoptosis

    PubMed Central

    Yuan, Xi-Ming; Li, Wei; Dalen, Helge; Lotem, Joseph; Kama, Rachel; Sachs, Leo; Brunk, Ulf T.

    2002-01-01

    The tumor suppressor wild-type p53 can induce apoptosis. M1-t-p53 myeloid leukemic cells have a temperature-sensitive p53 protein that changes its conformation to wild-type p53 after transfer from 37°C to 32°C. We have now found that these cells showed an early lysosomal rupture after transfer to 32°C. Mitochondrial damage, including decreased membrane potential and release of cytochrome c, and the appearance of apoptotic cells occurred later. Lysosomal rupture, mitochondrial damage, and apoptosis were all inhibited by the cytokine IL-6. Some other compounds can also inhibit apoptosis induced by p53. The protease inhibitor N-tosyl-l-phenylalanine chloromethyl ketone inhibited the decrease in mitochondrial membrane potential and cytochrome c release, the Ca2+-ATPase inhibitor thapsigargin inhibited only cytochrome c release, and the antioxidant butylated hydroxyanisole inhibited only the decrease in mitochondrial membrane potential. In contrast to IL-6, these other compounds that inhibited some of the later occurring mitochondrial damage did not inhibit the earlier p53-induced lysosomal damage. The results indicate that apoptosis is induced by p53 through a lysosomal-mitochondrial pathway that is initiated by lysosomal destabilization, and that this pathway can be dissected by using different apoptosis inhibitors. These findings on the induction of p53-induced lysosomal destabilization can also help to formulate new therapies for diseases with apoptotic disorders. PMID:11959917

  1. Wharton's jelly mesenchymal stem cells as candidates for beta cells regeneration: extending the differentiative and immunomodulatory benefits of adult mesenchymal stem cells for the treatment of type 1 diabetes.

    PubMed

    Anzalone, Rita; Lo Iacono, Melania; Loria, Tiziana; Di Stefano, Antonino; Giannuzzi, Pantaleo; Farina, Felicia; La Rocca, Giampiero

    2011-06-01

    Mesenchymal stem cells (MSC) are uniquely capable of crossing germinative layers borders (i.e. are able to differentiate towards ectoderm-, mesoderm- and endoderm-derived cytotypes) and are viewed as promising cells for regenerative medicine approaches in several diseases. Type I diabetes therapy should potentially benefit from such differentiated cells: the search for alternatives to organ/islet transplantation strategies via stem cells differentiation is an ongoing task, significant goals having been achieved in most experimental settings (e.g. insulin production and euglycaemia restoration), though caution is still needed to ensure safe and durable effects in vivo. MSC are obtainable in high numbers via ex vivo culture and can be differentiated towards insulin-producing cells (IPC). Moreover, recent reports evidenced that MSC possess immunomodulatory activities (acting on both innate and acquired immunity effectors) which should result in a reduction of the immunogenicity of transplanted cells, thus limiting rejection. Moreover it has been proposed that MSC administration should be used to attenuate the autoimmune processes which lead to the destruction of beta cells. This review illustrates the recent advances made in differentiating human MSC to IPC. In particular, we compare the effectiveness of the differentiation protocols applied, the markers and functional assays used to characterize differentiated progeny, and the in vivo controls. We further speculate on how MSC derived from Wharton's jelly of human umbilical cord may represent a more promising regenerative medicine tool, as recently demonstrated for endoderm-derived organs (as liver) in human subjects, also considering their peculiar immunomodulatory features compared to other MSC populations. PMID:20972649

  2. Apoptosis in Drosophila: which role for mitochondria?

    PubMed

    Clavier, Amandine; Rincheval-Arnold, Aurore; Colin, Jessie; Mignotte, Bernard; Guénal, Isabelle

    2016-03-01

    It is now well established that the mitochondrion is a central regulator of mammalian cell apoptosis. However, the importance of this organelle in non-mammalian apoptosis has long been regarded as minor, mainly because of the absence of a crucial role for cytochrome c in caspase activation. Recent results indicate that the control of caspase activation and cell death in Drosophila occurs at the mitochondrial level. Numerous proteins, including RHG proteins and proteins of the Bcl-2 family that are key regulators of Drosophila apoptosis, constitutively or transiently localize in mitochondria. These proteins participate in the cell death process at different levels such as degradation of Diap1, a Drosophila IAP, production of mitochondrial reactive oxygen species or stimulation of the mitochondrial fission machinery. Here, we review these mitochondrial events that might have their counterpart in human. PMID:26679112

  3. Lipid metabolism, apoptosis and cancer therapy.

    PubMed

    Huang, Chunfa; Freter, Carl

    2015-01-01

    Lipid metabolism is regulated by multiple signaling pathways, and generates a variety of bioactive lipid molecules. These bioactive lipid molecules known as signaling molecules, such as fatty acid, eicosanoids, diacylglycerol, phosphatidic acid, lysophophatidic acid, ceramide, sphingosine, sphingosine-1-phosphate, phosphatidylinositol-3 phosphate, and cholesterol, are involved in the activation or regulation of different signaling pathways. Lipid metabolism participates in the regulation of many cellular processes such as cell growth, proliferation, differentiation, survival, apoptosis, inflammation, motility, membrane homeostasis, chemotherapy response, and drug resistance. Bioactive lipid molecules promote apoptosis via the intrinsic pathway by modulating mitochondrial membrane permeability and activating different enzymes including caspases. In this review, we discuss recent data in the fields of lipid metabolism, lipid-mediated apoptosis, and cancer therapy. In conclusion, understanding the underlying molecular mechanism of lipid metabolism and the function of different lipid molecules could provide the basis for cancer cell death rationale, discover novel and potential targets, and develop new anticancer drugs for cancer therapy. PMID:25561239

  4. Control of apoptosis by Drosophila DCAF12.

    PubMed

    Hwangbo, Dae-Sung; Biteau, Benoit; Rath, Sneha; Kim, Jihyun; Jasper, Heinrich

    2016-05-01

    Regulated Apoptosis (Programmed Cell Death, PCD) maintains tissue homeostasis in adults, and ensures proper growth and morphogenesis of tissues during development of metazoans. Accordingly, defects in cellular processes triggering or executing apoptotic programs have been implicated in a variety of degenerative and neoplastic diseases. Here, we report the identification of DCAF12, an evolutionary conserved member of the WD40-motif repeat family of proteins, as a new regulator of apoptosis in Drosophila. We find that DCAF12 is required for Diap1 cleavage in response to pro-apoptotic signals, and is thus necessary and sufficient for RHG (Reaper, Hid, and Grim)-mediated apoptosis. Loss of DCAF12 perturbs the elimination of supernumerary or proliferation-impaired cells during development, and enhances tumor growth induced by loss of neoplastic tumor suppressors, highlighting the wide requirement for DCAF12 in PCD. PMID:26972874

  5. Downregulation of Reactive Oxygen Species in Apoptosis

    PubMed Central

    Jeong, Chul-Ho; Joo, Sang Hoon

    2016-01-01

    Generation of reactive oxygen species (ROS) by diverse anti-cancer drugs or phytochemicals has been closely related with the induction of apoptosis in cancers. Also, the downregulation of ROS by these chemicals has been found to block initiation of carcinogenesis. Therefore, modulation of ROS by phytochemicals emerges as a crucial mechanism to regulate apoptosis in cancer prevention or therapy. This review summarizes the current understanding of the selected chemical compounds and related cellular components that modulate ROS during apoptotic process. Metformin, quercetin, curcumin, vitamin C, and other compounds have been shown to downregulate ROS in the cellular apoptotic process, and some of them even induce apoptosis in cancer cells. The cellular components mediating the downregulation of ROS include nuclear factor erythroid 2-related factor 2 antioxidant signaling pathway, thioredoxin, catalase, glutathione, heme oxygenase-1, and uncoupling proteins. The present review provides information on the relationship between these compounds and the cellular components in modulating ROS in apoptotic cancer cells. PMID:27051644

  6. Apoptosis induced by dioscin in Hela cells.

    PubMed

    Cai, Jing; Liu, Mingjie; Wang, Zhao; Ju, Yong

    2002-02-01

    Dioscin, a saponin extracted from the root of Polygonatum Zanlanscianense Pamp, markedly inhibited proliferation of Hela cells. The results indicated that Hela cells underwent apoptosis in dose- and time-dependent manners when treated with Dioscin. Caspase-3, -8 and -9 activities were also detected. The low enzymatic activity of caspase-8 and high activity of caspase-9 showed that the mitochondrial pathway was activated in apoptosis. The reduced expression of the survival protein Bcl-2 also confirmed this result. These studies may be significant in finding a new drug to treat human cervical cancer. PMID:11853164

  7. Apoptosis after reperfused myocardial infarction: Role of angiotensin II

    PubMed Central

    Jugdutt, Bodh I

    2004-01-01

    Angiotensin II (Ang II) plays a significant role in apoptosis after myocardial infarction (MI) and reperfused MI. Cumulative evidence suggests that Ang II is a major contributor to cardiomyocyte (CM) apoptosis and left ventricular (LV) dysfunction after acute reperfused MI and that apoptosis mediates a major portion of early LV dysfunction. Importantly, blockade of the Ang II type 1 receptor (AT1R) limits CM apoptosis and LV dysfunction after acute reperfused MI. Ang II type 2 receptor activation during AT1R blockade contributes to these beneficial effects. The role of Ang II and apoptosis in chronic LV remodelling, healing and post-MI heart failure is more complex and involves effects on the CMs, fibroblasts and vascular cells. The long-term effects of agents targeting apoptosis after reperfused MI, including AT1R blockade, on apoptosis in different cell types, windows of enhanced apoptosis and the appropriate timing of therapy need to be considered. PMID:19641712

  8. Noncoding RNAs in Beta Cell Biology

    PubMed Central

    Singer, Ruth A.; Arnes, Luis; Sussel, Lori

    2015-01-01

    Purpose of Review The identification and characterization of essential islet transcription factors have improved our understanding of β cell development, provided insights into many of the cellular dysfunctions related to diabetes, and facilitated the successful generation of β cells from alternative cell sources. Recently, noncoding RNAs have emerged as a novel set of molecules that may represent missing components of the known islet regulatory pathways. The purpose of this review is to highlight studies that have implicated noncoding RNAs as important regulators of pancreas cell development and β cell function. Recent Findings Disruption of essential components of the microRNA processing machinery, in addition to misregulation of individual miRNAs, has revealed the importance of microRNAs in pancreas development and β cell function. Furthermore, over 1000 islet-specific long noncoding RNAs have been identified in mouse and human islets, suggesting that this class of noncoding molecules will also play important functional roles in the β cell. Summary The analysis of noncoding RNAs in the pancreas will provide important new insights into pancreatic regulatory processes that will improve our ability to understand and treat diabetes and may facilitate the generation of replacement β cells from alternative cell sources. PMID:25692923

  9. A novel method for detection of apoptosis

    SciTech Connect

    Zagariya, Alexander M.

    2012-04-15

    There are two different Angiotensin II (ANG II) peptides in nature: Human type (ANG II) and Bovine type (ANG II*). These eight amino acid peptides differ only at position 5 where Valine is replaced by Isoleucine in the Bovine type. They are present in all species studied so far. These amino acids are different by only one atom of carbon. This difference is so small, that it will allow any of ANG II, Bovine or Human antibodies to interact with all species and create a universal method for apoptosis detection. ANG II concentrations are found at substantially higher levels in apoptotic, compared to non-apoptotic, tissues. ANG II accumulation can lead to DNA damage, mutations, carcinogenesis and cell death. We demonstrate that Bovine antiserum can be used for universal detection of apoptosis. In 2010, the worldwide market for apoptosis detection reached the $20 billion mark and significantly increases each year. Most commercially available methods are related to Annexin V and TUNNEL. Our new method based on ANG II is more widely known to physicians and scientists compared to previously used methods. Our approach offers a novel alternative for assessing apoptosis activity with enhanced sensitivity, at a lower cost and ease of use.

  10. Farnesol-induced apoptosis in Candida albicans.

    PubMed

    Shirtliff, Mark E; Krom, Bastiaan P; Meijering, Roelien A M; Peters, Brian M; Zhu, Jingsong; Scheper, Mark A; Harris, Megan L; Jabra-Rizk, Mary Ann

    2009-06-01

    Farnesol, a precursor in the isoprenoid/sterol pathway, was recently identified as a quorum-sensing molecule produced by the fungal pathogen Candida albicans. Farnesol is involved in the inhibition of germination and biofilm formation by C. albicans and can be cytotoxic at certain concentrations. In addition, we have shown that farnesol can trigger apoptosis in mammalian cells via the classical apoptotic pathways. In order to elucidate the mechanism behind farnesol cytotoxicity in C. albicans, the response to farnesol was investigated, using proteomic analysis. Global protein expression profiles demonstrated significant changes in protein expression resulting from farnesol exposure. Among the downregulated proteins were those involved in metabolism, glycolysis, protein synthesis, and mitochondrial electron transport and the respiratory chain, whereas proteins involved in folding, protection against environmental and oxidative stress, actin cytoskeleton reorganization, and apoptosis were upregulated. Cellular changes that accompany apoptosis (regulated cell death) were further analyzed using fluorescent microscopy and gene expression analysis. The results indicated reactive oxygen species accumulation, mitochondrial degradation, and positive terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) in the farnesol-exposed cells concurrent with increased expression of antioxidant-encoding and drug response genes. More importantly, the results demonstrated farnesol-induced upregulation of the caspase gene MCA1 and the intracellular presence of activated caspases. In conclusion, this study demonstrated that farnesol promotes apoptosis in C. albicans through caspase activation, implying an important physiological role for farnesol in the fungal cell life cycle with important implications for adaptation and survival. PMID:19364863

  11. THE ROLE OF APOPTOSIS IN NEUROTOXICOLOGY

    EPA Science Inventory

    Apoptosis, a form of programmed cell death, occurs in the nervous system throughout development, but with a preponderance of cell death occurring during the prenatal and perinatal periods. Aberrant periods of increased or decreased cell death, induced by toxicants in air, water,...

  12. Fluorescence spectroscopy to assess apoptosis in myocardium

    NASA Astrophysics Data System (ADS)

    Ranji, Mahsa; Matsubara, Muneaki; Grosso, Michael A.; Jaggard, Dwight L.; Chance, Britton; Gorman, Robert C.; Gorman, Joseph H., III

    2007-02-01

    Apoptosis induced mitochondrial destruction and dysfunction has been shown to play an important role in the pathogenesis of both acute cardiac ischemia-reperfusion injury and chronic myocardial infarction-induced ventricular remodeling. Unfortunately this understanding has not translated into effective therapeutic strategies for either condition-mostly due to an inability to assess mitochondrial dysfunction/apoptosis effectively in humans. All current measures of apoptosis are pseudo-quantitative and require invasive tissue biopsy. Our group has developed an optical, non-tissue destructive catheter based device that allows the quantitative regional assessment of this pathological process in vivo. This instrument has been designed to acquire fluorescence signals of intrinsic mitochondrial fluorophores, Nicotinamide Adenine Dinucleotide (NAD) and Flavoprotein (FP). The normalized ratio of these fluorophores (FP/FP+NADH) called the redox ratio, is an indicator of the in vivo mitochondrial dysfunction. 1-3 We have demonstrated in a rabbit reperfusion model of apoptotic myocyte injury that this redox ratio is drastically increased which is consistent with profound apoptosis-induced "unhinging" of the mitochondrial respiratory function.

  13. Techniques to Distinguish Apoptosis from Necroptosis.

    PubMed

    Feoktistova, Maria; Wallberg, Fredrik; Tenev, Tencho; Geserick, Peter; Leverkus, Martin; Meier, Pascal

    2016-04-01

    The processes by which cells die are as tightly regulated as those that govern cell growth and proliferation. Recent studies of the molecular pathways that regulate and execute cell death have uncovered a plethora of signaling cascades that lead to distinct modes of cell death, including "apoptosis," "necrosis," "autophagic cell death," and "mitotic catastrophe." Cells can readily switch from one form of death to another; therefore, it is vital to have the ability to monitor the form of death that cells are undergoing. A number of techniques are available that allow the detection of cell death and when combined with either knockdown approaches or inhibitors of specific signaling pathways, such as caspase or RIP kinase pathways, they allow the rapid dissection of divergent cell death pathways. However, techniques that reveal the end point of cell death cannot reconstruct the sequence of events that have led to death; therefore, they need to be complemented with methods that can distinguish all forms of cell death. Apoptotic cells frequently undergo secondary necrosis under in vitro culture conditions; therefore, novel methods relying on high-throughput time-lapse fluorescence video microscopy are necessary to provide temporal resolution to cell death events. Further, visualizing the assembly of multiprotein signaling hubs that can execute apoptosis or necroptosis helps to explore the underlying processes. Here we introduce a suite of techniques that reliably distinguish necrosis from apoptosis and secondary necrosis, and that enable investigation of signaling platforms capable of instructing apoptosis or necroptosis. PMID:27037077

  14. Ceramide in apoptosis: a revisited role.

    PubMed

    Levade, Thierry; Malagarie-Cazenave, Sophie; Gouazé, Valérie; Ségui, Bruno; Tardy, Claudine; Betito, Susan; Andrieu-Abadie, Nathalie; Cuvillier, Olivier

    2002-08-01

    The sphingolipid ceramide has recently emerged as a new transducer or modulator of apoptotic cell death. This function, however, has recently been challenged. Here, in the light of recent observations, the role of ceramide in apoptosis signaling is discussed. PMID:12374195

  15. Effect of hyaluronic acid on chondrocyte apoptosis

    PubMed Central

    Barreto, Ronald Bispo; Sadigursky, David; de Rezende, Marcia Uchoa; Hernandez, Arnaldo José

    2015-01-01

    OBJECTIVE: To determine the percentage of apoptotic cells in a contusion model of osteoarthritis (OA) and to assess whether intra-articular injection of high doses of hyaluronic acid (HA) immediately after trauma reduces chondrocyte apoptosis. METHODS: Forty knees from adult rabbits were impacted thrice with a 1 kg block released through a 1 meter tall cylinder (29.4 Joules). Subsequently, 2 mL of HA was injected in one knee and 2 mL saline in the contra-lateral knee. Medication were administered twice a week for 30 days, when animals were sacrificed. Specimens were prepared for optical microscopy exam and terminal deoxynucleotidyl transferase end labeling assay (TUNEL). RESULTS: The apoptosis rate in the contusion model was 68.01% (± 19.73%), a higher rate than previously described. HA significantly reduced the rate of apoptosis to 53.52% (± 18.09) (p <0.001). CONCLUSION: Intra-articular HA administration started immediately after trauma reduces impact-induced chondrocyte apoptosis rates in rabbits. Level of Evidence I, Experimental Study. PMID:27069407

  16. Sodium orthovanadate inhibits p53-mediated apoptosis.

    PubMed

    Morita, Akinori; Yamamoto, Shinichi; Wang, Bing; Tanaka, Kaoru; Suzuki, Norio; Aoki, Shin; Ito, Azusa; Nanao, Tomohisa; Ohya, Soichiro; Yoshino, Minako; Zhu, Jin; Enomoto, Atsushi; Matsumoto, Yoshihisa; Funatsu, Osamu; Hosoi, Yoshio; Ikekita, Masahiko

    2010-01-01

    Sodium orthovanadate (vanadate) inhibits the DNA-binding activity of p53, but its precise effects on p53 function have not been examined. Here, we show that vanadate exerts a potent antiapoptotic activity through both transcription-dependent and transcription-independent mechanisms relative to other p53 inhibitors, including pifithrin (PFT) alpha. We compared the effects of vanadate to PFTalpha and PFTmicro, an inhibitor of transcription-independent apoptosis by p53. Vanadate suppressed p53-associated apoptotic events at the mitochondria, including the loss of mitochondrial membrane potential, the conformational change of Bax and Bak, the mitochondrial translocation of p53, and the interaction of p53 with Bcl-2. Similarly, vanadate suppressed the apoptosis-inducing activity of a mitochondrially targeted temperature-sensitive p53 in stable transfectants of SaOS-2 cells. In radioprotection assays, which rely on p53, vanadate completely protected mice from a sublethal dose of 8 Gy and partially from a lethal dose of 12 Gy. Together, our findings indicated that vanadate effectively suppresses p53-mediated apoptosis by both transcription-dependent and transcription-independent pathways, and suggested that both pathways must be inhibited to completely block p53-mediated apoptosis. PMID:20048077

  17. PECAM-1, apoptosis and CD34+ precursors.

    PubMed

    Zocchi, Maria R; Poggi, A

    2004-11-01

    Apoptosis is a physiological process that controls tissue homeostasis, in combination with survival signals delivered by distinct receptors that bind hormones, growth factors or extracellular matrix components. The extrinsic pathway of apoptosis is due to the triggering of death receptors and the activation of the caspase cascade; the intrinsic pathway is due to withdrawal of growth factors and mainly related to mitochondrial metabolism. The choice between survival or apoptosis, which is the result of such different integrated environmental signals, is crucial for the maintainance of bone marrow reservoir of hematopoietic precursors (HPC). CD34+ HPC can receive multiple survival signals during homing and maturation, due to different interactions with adhesion molecules expressed on endothelial and bone marrow stromal cells, proteins of the extracellular matrix and chemokines or growth factors. Among them, the signal delivered via platelet endothelial cell adhesion molecule-1 (PECAM-1) seems to contribute to the resistance of this cell population to starvation, and it is related to the maintainance of mitochondrial metabolism. Indeed, this molecule, originally described as an adhesion receptor belonging to the immunoglobulin superfamily, capable of homophilic and heterophilic interactions, turned out to be a signalling molecule, containing an immunoreceptor tyrosine-based inhibitory motifs (ITIM) within its cytoplasmic domain. In particular, it has been shown that PECAM-1 binds to different kinases and phosphatases, including the phosphatidylinositide-3-kinase that phosphorylates Akt, which, in turn can upregulate transcription and function of antiapoptotic proteins, such as Bcl-2 and Bcl-x or A1, responsible for the rescue from mitochondrial apoptosis. The possible role of PECAM-1 engagement in the prevention of starvation-induced apoptosis of HPC precursors and in the maintainance of their survival is discussed. PMID:15512808

  18. Erythrocyte ion channels in regulation of apoptosis.

    PubMed

    Lang, Florian; Birka, Christina; Myssina, Svetlana; Lang, Karl S; Lang, Philipp A; Tanneur, Valerie; Duranton, Christophe; Wieder, Thomas; Huber, Stephan M

    2004-01-01

    Erythrocytes lack mitochondria and nuclei, key organelles in the regulation of apoptosis. Until recently, erythrocytes were thus not considered subject to this type of cell death. However, exposure of erythrocytes to the Ca2+ ionophore ionomycin was shown to induce cell shrinkage, cell membrane blebbing and breakdown of phosphatidylserine asymmetry with subsequent phosphatidylserine exposure at the cell surface, all typical features of apoptosis. Further studies revealed the participation of ion channels in the regulation of erythrocyte "apoptosis." Osmotic shock, oxidative stress and energy depletion all activate a Ca2(+)-permeable non-selective cation channel in the erythrocyte cell membrane. The subsequent increase of Ca2+ concentration stimulates a scramblase leading to breakdown of cell membrane phosphatidylserine asymmetry and activates Ca2+ sensitive K+ (Gardos) channels leading to KCl loss and (further) cell shrinkage. Phosphatidylserine exposure and cell shrinkage are blunted in the nominal absence of extracellular Ca2+, in the presence of the cation channel inhibitors amiloride or ethylisopropylamiloride, at increased extracellular K+ or in the presence of the Gardos channel inhibitors clotrimazole or charybdotoxin. Thus, increase of cytosolic Ca2+ and cellular loss of K+ participate in the triggering of erythrocyte scramblase. Nevertheless, phosphatidylserine exposure is not completely abrogated in the nominal absence of Ca2+, pointing to additional Ca2(+)-independent pathways. One of those is activation of sphingomyelinase with subsequent formation of ceramide which in turn leads to stimulation of erythrocyte scramblase. The exposure of phosphatidylserine at the extracellular face of the cell membrane stimulates phagocytes to engulf the apoptotic erythrocytes. Thus, sustained activation of the cation channels eventually leads to clearance of affected erythrocytes from peripheral blood. Erythropoietin inhibits the non-selective cation channel and thus

  19. Key regulators of apoptosis execution as biomarker candidates in melanoma

    PubMed Central

    Charles, Emilie M; Rehm, Markus

    2014-01-01

    Resistance to apoptosis is frequently detected in malignant melanoma, a skin cancer with rapidly growing incidence rates. Apoptosis resistance may develop with disease progression and may be associated with the poor responsiveness of metastatic melanoma to apoptosis-inducing treatments, such as genotoxic chemotherapy and radiotherapy. Likewise, the efficacy of novel treatment options (targeted kinase inhibitors and immunotherapeutics) that indirectly lead to cell death may depend on the susceptibility of melanoma to apoptosis. At its core, apoptosis execution is regulated by the interplay between a comparatively small number of pro- and anti-apoptotic proteins, and consequently numerous studies have investigated the potential of these players as biomarker candidates. Here, we provide a comprehensive overview of biomarker discovery studies focusing on key regulators of apoptosis execution, critically review the findings of these studies, and outline strategies that address current limitations and challenges in exploiting regulators of apoptosis execution as prognostic or predictive biomarkers in melanoma. PMID:27308353

  20. Biomarkers of Chondrocyte Apoptosis and Autophagy in Osteoarthritis

    PubMed Central

    Musumeci, Giuseppe; Castrogiovanni, Paola; Trovato, Francesca Maria; Weinberg, Annelie Martina; Al-Wasiyah, Mohammad K.; Alqahtani, Mohammed H.; Mobasheri, Ali

    2015-01-01

    Cell death with morphological and molecular features of apoptosis has been detected in osteoarthritic (OA) cartilage, which suggests a key role for chondrocyte death/survival in the pathogenesis of OA. Identification of biomarkers of chondrocyte apoptosis may facilitate the development of novel therapies that may eliminate the cause or, at least, slow down the degenerative processes in OA. The aim of this review was to explore the molecular markers and signals that induce chondrocyte apoptosis in OA. A literature search was conducted in PubMed, Scopus, Web of Science and Google Scholar using the keywords chondrocyte death, apoptosis, osteoarthritis, autophagy and biomarker. Several molecules considered to be markers of chondrocyte apoptosis will be discussed in this brief review. Molecular markers and signalling pathways associated with chondroycte apoptosis may turn out to be therapeutic targets in OA and approaches aimed at neutralizing apoptosis-inducing molecules may at least delay the progression of cartilage degeneration in OA. PMID:26334269

  1. Rabies virus matrix protein induces apoptosis by targeting mitochondria.

    PubMed

    Zan, Jie; Liu, Juan; Zhou, Jian-Wei; Wang, Hai-Long; Mo, Kai-Kun; Yan, Yan; Xu, Yun-Bin; Liao, Min; Su, Shuo; Hu, Rong-Liang; Zhou, Ji-Yong

    2016-09-10

    Apoptosis, as an innate antiviral defense, not only functions to limit viral replication by eliminating infected cells, but also contribute to viral dissemination, particularly at the late stages of infection. A highly neurotropic CVS strain of rabies virus induces apoptosis both in vitro and in vivo. However, the detailed mechanism of CVS-mediated neuronal apoptosis is not entirely clear. Here, we show that CVS induces apoptosis through mitochondrial pathway by dissipating mitochondrial membrane potential, release of cytochrome c and AIF. CVS blocks Bax activation at the early stages of infection; while M protein partially targets mitochondria and induces mitochondrial apoptosis at the late stages of infection. The α-helix structure spanning 67-79 amino acids of M protein is essential for mitochondrial targeting and induction of apoptosis. These results suggest that CVS functions on mitochondria to regulate apoptosis at different stages of infection, so as to for viral replication and dissemination. PMID:27426727

  2. Sustained adenosine exposure causes lung endothelial apoptosis: a possible contributor to cigarette smoke-induced endothelial apoptosis and lung injury

    PubMed Central

    Sakhatskyy, Pavlo; Newton, Julie; Shamirian, Paul; Hsiao, Vivian; Curren, Sean; Gabino Miranda, Gustavo Andres; Pedroza, Mesias; Blackburn, Michael R.; Rounds, Sharon

    2013-01-01

    Pulmonary endothelial cell (EC) apoptosis has been implicated in the pathogenesis of emphysema. Cigarette smoke (CS) causes lung EC apoptosis and emphysema. In this study, we show that CS exposure increased lung tissue adenosine levels in mice, an effect associated with increased lung EC apoptosis and the development of emphysema. Adenosine has a protective effect against apoptosis via adenosine receptor-mediated signaling. However, sustained elevated adenosine increases alveolar cell apoptosis in adenosine deaminase-deficient mice. We established an in vitro model of sustained adenosine exposure by incubating lung EC with adenosine in the presence of an adenosine deaminase inhibitor, deoxycoformicin. We demonstrated that sustained adenosine exposure caused lung EC apoptosis via nucleoside transporter-facilitated intracellular adenosine uptake, subsequent activation of p38 and JNK in mitochondria, and ultimately mitochondrial defects and activation of the mitochondria-mediated intrinsic pathway of apoptosis. Our results suggest that sustained elevated adenosine may contribute to CS-induced lung EC apoptosis and emphysema. Our data also reconcile the paradoxical effects of adenosine on apoptosis, demonstrating that prolonged exposure causes apoptosis via nucleoside transporter-mediated intracellular adenosine signaling, whereas acute exposure protects against apoptosis via activation of adenosine receptors. Inhibition of adenosine uptake may become a new therapeutic target in treatment of CS-induced lung diseases. PMID:23316066

  3. Quantitative trait loci on chromosome 8q24 for pancreatic beta-cell function and 7q11 for insulin sensitivity in obese nondiabetic white and black families: evidence from genome-wide linkage scans in the NHLBI Hypertension Genetic Epidemiology Network (HyperGEN) study.

    PubMed

    An, Ping; Freedman, Barry I; Rich, Stephen S; Mandel, Stephen A; Arnett, Donna K; Myers, Richard H; Chen, Yii-Der I; Hunt, Steven C; Rao, D C

    2006-02-01

    Genome-wide linkage scans were carried out using a multipoint variance components method in white and black families of the NHLBI Hypertension Genetic Epidemiology Network (HyperGEN) study to identify quantitative trait loci (QTLs) for pancreatic beta-cell function and insulin sensitivity estimated through the newly released nonlinear computer version of homeostasis model assessment 2. Participants fasting <8 h, with diagnosed type 2 diabetes, or taking blood glucose or blood lipid-lowering medications were excluded. Both phenotypes were adjusted separately by race and sex for the effects of age, BMI, and field center before linkage scans using 370 microsatellite markers were performed. A total of 685 white families (1,180 sibpairs) and 773 black families (775 sibpairs) were evaluated as well as subsets including 267 obese white families (757 sibpairs) and 427 obese black families (599 sibpairs) identified through tree-linkage analyses using interacting covariates of age, sex, and BMI. For beta-cell function in the obese white families, significant (logarithm of odds [LOD] score >3.6) evidence supporting linkages was detected on chromosome 8q24 at D8S1179 (135 cM, LOD score 4.2, empirical P = 0.002) and at D8S1128 (140 cM, LOD score 3.7, empirical P = 0.003). In addition, two regions supported linkage for insulin sensitivity index in the obese black families on chromosome 7q11 at D7S3046 (79 cM, LOD score 3.0, empirical P = 0.018) and on chromosome 6q26 at D6S1277 (173 cM, LOD score 3.0, empirical P = 0.018). Reducing clinical heterogeneity using obesity data and improved estimates of beta-cell function and insulin sensitivity may have permitted identification of a QTL on chromosome 8q24 for beta-cell function in the presence of estimated insulin resistance and a QTL on chromosome 7q11 for insulin sensitivity. These regions replicate previous reports for type 2 diabetes-associated traits. PMID:16443794

  4. Autophagy and apoptosis dysfunction in neurodegenerative disorders.

    PubMed

    Ghavami, Saeid; Shojaei, Shahla; Yeganeh, Behzad; Ande, Sudharsana R; Jangamreddy, Jaganmohan R; Mehrpour, Maryam; Christoffersson, Jonas; Chaabane, Wiem; Moghadam, Adel Rezaei; Kashani, Hessam H; Hashemi, Mohammad; Owji, Ali A; Łos, Marek J

    2014-01-01

    Autophagy and apoptosis are basic physiologic processes contributing to the maintenance of cellular homeostasis. Autophagy encompasses pathways that target long-lived cytosolic proteins and damaged organelles. It involves a sequential set of events including double membrane formation, elongation, vesicle maturation and finally delivery of the targeted materials to the lysosome. Apoptotic cell death is best described through its morphology. It is characterized by cell rounding, membrane blebbing, cytoskeletal collapse, cytoplasmic condensation, and fragmentation, nuclear pyknosis, chromatin condensation/fragmentation, and formation of membrane-enveloped apoptotic bodies, that are rapidly phagocytosed by macrophages or neighboring cells. Neurodegenerative disorders are becoming increasingly prevalent, especially in the Western societies, with larger percentage of members living to an older age. They have to be seen not only as a health problem, but since they are care-intensive, they also carry a significant economic burden. Deregulation of autophagy plays a pivotal role in the etiology and/or progress of many of these diseases. Herein, we briefly review the latest findings that indicate the involvement of autophagy in neurodegenerative diseases. We provide a brief introduction to autophagy and apoptosis pathways focusing on the role of mitochondria and lysosomes. We then briefly highlight pathophysiology of common neurodegenerative disorders like Alzheimer's diseases, Parkinson's disease, Huntington's disease and Amyotrophic lateral sclerosis. Then, we describe functions of autophagy and apoptosis in brain homeostasis, especially in the context of the aforementioned disorders. Finally, we discuss different ways that autophagy and apoptosis modulation may be employed for therapeutic intervention during the maintenance of neurodegenerative disorders. PMID:24211851

  5. HIV-1 protease-induced apoptosis

    PubMed Central

    2014-01-01

    Background Apoptosis is one of the presumptive causes of CD4+ T cell depletion during HIV infection and progression to AIDS. However, the precise role of HIV-1 in this process remains unexplained. HIV-1 protease (PR) has been suggested as a possible factor, but a direct link between HIV-1 PR enzymatic activity and apoptosis has not been established. Results Here, we show that expression of active HIV-1 PR induces death in HeLa and HEK-293 cells via the mitochondrial apoptotic pathway. This conclusion is based on in vivo observations of the direct localization of HIV-1 PR in mitochondria, a key player in triggering apoptosis. Moreover, we observed an HIV-1 PR concentration-dependent decrease in mitochondrial membrane potential and the role of HIV-1 PR in activation of caspase 9, PARP cleavage and DNA fragmentation. In addition, in vitro data demonstrated that HIV-1 PR mediates cleavage of mitochondrial proteins Tom22, VDAC and ANT, leading to release of AIF and Hsp60 proteins. By using yeast two-hybrid screening, we also identified a new HIV-1 PR interaction partner, breast carcinoma-associated protein 3 (BCA3). We found that BCA3 accelerates p53 transcriptional activity on the bax promoter, thus elevating the cellular level of pro-apoptotic Bax protein. Conclusion In summary, our results describe the involvement of HIV-1 PR in apoptosis, which is caused either by a direct effect of HIV-1 PR on mitochondrial membrane integrity or by its interaction with cellular protein BCA3. PMID:24886575

  6. Control of apoptosis by asymmetric cell division.

    PubMed

    Hatzold, Julia; Conradt, Barbara

    2008-04-01

    Asymmetric cell division and apoptosis (programmed cell death) are two fundamental processes that are important for the development and function of multicellular organisms. We have found that the processes of asymmetric cell division and apoptosis can be functionally linked. Specifically, we show that asymmetric cell division in the nematode Caenorhabditis elegans is mediated by a pathway involving three genes, dnj-11 MIDA1, ces-2 HLF, and ces-1 Snail, that directly control the enzymatic machinery responsible for apoptosis. Interestingly, the MIDA1-like protein GlsA of the alga Volvox carteri, as well as the Snail-related proteins Snail, Escargot, and Worniu of Drosophila melanogaster, have previously been implicated in asymmetric cell division. Therefore, C. elegans dnj-11 MIDA1, ces-2 HLF, and ces-1 Snail may be components of a pathway involved in asymmetric cell division that is conserved throughout the plant and animal kingdoms. Furthermore, based on our results, we propose that this pathway directly controls the apoptotic fate in C. elegans, and possibly other animals as well. PMID:18399720

  7. Lithium protects ethanol-induced neuronal apoptosis

    SciTech Connect

    Zhong Jin . E-mail: jizhong@iupui.edu; Yang Xianlin; Yao Weiguo; Lee Weihua

    2006-12-01

    Lithium is widely used for the treatment of bipolar disorder. Recent studies have demonstrated its neuroprotective effect. Ethanol is a potent neurotoxin that is particularly harmful to the developing nervous system. In this study, we evaluated lithium's neuroprotection against ethanol-induced apoptosis. Transient exposure of infant mice to ethanol caused apoptotic cell death in brain, which was prevented significantly by administering a low dose of lithium 15 min later. In cultured cerebellar granule neurons, ethanol-induced apoptosis and activation of caspase-3/9, both of which were prevented by lithium. However, lithium's protection is not mediated by its commonly known inhibition of glycogen synthase3{beta}, because neither ethanol nor lithium has significant effects on the phosphorylation of Akt (ser473) or GSK3{beta} (ser9). In addition, the selective GSK-3{beta} inhibitor SB-415286 was unable to prevent ethanol-induced apoptosis. These data suggest lithium may be used as a potential preventive measure for ethanol-induced neurological deficits.

  8. Alcohol and Apoptosis: Friends or Foes?

    PubMed Central

    Rodriguez, Ana; Chawla, Karan; Umoh, Nsini A.; Cousins, Valerie M.; Ketegou, Assama; Reddy, Madhumati G.; AlRubaiee, Mustafa; Haddad, Georges E.; Burke, Mark W.

    2015-01-01

    Alcohol abuse causes 79,000 deaths stemming from severe organ damage in the United States every year. Clinical manifestations of long-term alcohol abuse on the cardiac muscle include defective contractility with the development of dilated cardiomyopathy and low-output heart failure; which has poor prognosis with less than 25% survival for more than three years. In contrast, low alcohol consumption has been associated with reduced risk of cardiovascular disease, however the mechanism of this phenomenon remains elusive. The aim of this study was to determine the significance of apoptosis as a mediating factor in cardiac function following chronic high alcohol versus low alcohol exposure. Adult rats were provided 5 mM (low alcohol), 100 mM (high alcohol) or pair-fed non-alcohol controls for 4–5 months. The hearts were dissected, sectioned and stained with cresyl violet or immunohistochemically for caspase-3, a putative marker for apoptosis. Cardiomyocytes were isolated to determine the effects of alcohol exposure on cell contraction and relaxation. High alcohol animals displayed a marked thinning of the left ventricular wall combined with elevated caspase-3 activity and decreased contractility. In contrast, low alcohol was associated with increased contractility and decreased apoptosis suggesting an overall protective mechanism induced by low levels of alcohol exposure. PMID:26610584

  9. Increased small intestinal apoptosis in coeliac disease.

    PubMed Central

    Moss, S F; Attia, L; Scholes, J V; Walters, J R; Holt, P R

    1996-01-01

    BACKGROUND: Coeliac disease (CD) mucosa is flattened despite epithelial hyperproliferation. AIMS: To establish mechanisms of cell loss in CD. PATIENTS: 14 controls, 17 active CD patients, and 16 maintained with gluten free diet. METHODS: Programmed cell death was examined in small intestinal biopsy specimens by staining fragmented DNA using terminal uridine deoxynucleotidyl nick end labelling (TUNEL), in comparison with haematoxylin and eosin stained adjacent sections. Double staining with anti-CD45 antibodies determined the origin of apoptotic cells. Apoptosis was graded from 1-3 (< 5, 5-20, > 20% respectively). Proliferating cells, immunostained by Ki-67 (MIB-1) antibody, were counted. RESULTS: Apoptotic cells were seen rarely by haematoxylin and eosin but more readily by TUNEL. In controls, 1.4 +/- 0.2% of epithelial cells were apoptotic (mean grade 1.1), mainly located in the upper villus. In active CD, frequent apoptotic cells were distributed throughout the crypt-villus unit (mean grade 2.4), decreasing after treatment to 1.1 (p < 0.001) even when still histologically abnormal. CD45 antibodies rarely stained apoptotic cells in active CD. The number of TUNEL positive cells correlated with proliferating cell number (p < 0.001). CONCLUSION: Enterocyte apoptosis is greatly increased in untreated CD, correlates with proliferation, and falls to normal with a gluten free diet, before histological improvement. Increased apoptosis may be responsible for villous atrophy in CD. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:9038662

  10. Ion channels and apoptosis in cancer

    PubMed Central

    Bortner, Carl D.; Cidlowski, John A.

    2014-01-01

    Humans maintain a constant cell number throughout their lifespan. This equilibrium of cell number is accomplished when cell proliferation and cell death are kept balanced, achieving a steady-state cell number. Abnormalities in cell growth or cell death can lead to an overabundance of cells known as neoplasm or tumours. While the perception of cancer is often that of an uncontrollable rate of cell growth or increased proliferation, a decrease in cell death can also lead to tumour formation. Most cells when detached from their normal tissue die. However, cancer cells evade cell death, tipping the balance to an overabundance of cell number. Therefore, overcoming this resistance to cell death is a decisive factor in the treatment of cancer. Ion channels play a critical role in cancer in regards to cell proliferation, malignant angiogenesis, migration and metastasis. Additionally, ion channels are also known to be critical components of apoptosis. In this review, we discuss the modes of cell death focusing on the ability of cancer cells to evade apoptosis. Specifically, we focus on the role ion channels play in controlling and regulating life/death decisions and how they can be used to overcome resistance to apoptosis in the treatment of cancer. PMID:24493752

  11. Apoptosis signal-regulating kinase 1 mediates denbinobin-induced apoptosis in human lung adenocarcinoma cells

    PubMed Central

    Kuo, Chen-Tzu; Chen, Bing-Chang; Yu, Chung-Chi; Weng, Chih-Ming; Hsu, Ming-Jen; Chen, Chien-Chih; Chen, Mei-Chieh; Teng, Che-Ming; Pan, Shiow-Lin; Bien, Mauo-Ying; Shih, Chung-Hung; Lin, Chien-Huang

    2009-01-01

    In the present study, we explore the role of apoptosis signal-regulating kinase 1 (ASK1) in denbinobin-induced apoptosis in human lung adenocarcinoma (A549) cells. Denbinobin-induced cell apoptosis was attenuated by an ASK1 dominant-negative mutant (ASK1DN), two antioxidants (N-acetyl-L-cysteine (NAC) and glutathione (GSH)), a c-Jun N-terminal kinase (JNK) inhibitor (SP600125), and an activator protein-1 (AP-1) inhibitor (curcumin). Treatment of A549 cells with denbinobin caused increases in ASK1 activity and reactive oxygen species (ROS) production, and these effects were inhibited by NAC and GSH. Stimulation of A549 cells with denbinobin caused JNK activation; this effect was markedly inhibited by NAC, GSH, and ASK1DN. Denbinobin induced c-Jun phosphorylation, the formation of an AP-1-specific DNA-protein complex, and Bim expression. Bim knockdown using a bim short interfering RNA strategy also reduced denbinobin-induced A549 cell apoptosis. The denbinobin-mediated increases in c-Jun phosphorylation and Bim expression were inhibited by NAC, GSH, SP600125, ASK1DN, JNK1DN, and JNK2DN. These results suggest that denbinobin might activate ASK1 through ROS production to cause JNK/AP-1 activation, which in turn induces Bim expression, and ultimately results in A549 cell apoptosis. PMID:19405983

  12. Artesunate induces AIF-dependent apoptosis in A549 cells

    NASA Astrophysics Data System (ADS)

    Zhou, Chen-juan; Chen, Tong-Sheng

    2012-03-01

    Artesunate (ART), a semi-synthetic derivative of the sesquiterpene artemisinin extracted from the Chinese herb Artemisia annua, exerts a broad spectrum of clinical activity against human cancers. It has been shown that ART induces cancer cells death through apoptosis pathway. This study investigated whether ART treatment induced reactive oxygen species (ROS)-dependent cell death in the apoptosis fashion in human lung adenocarconoma A549 cell line and the proapoptotic protein apoptosis inducing factor (AIF) is involved in ART-induced apoptosis. Cells treated with ART exhibited typical apoptotic morphology as chromatin condensation, margination and shrunken nucleus. ART treatment also induced a loss of mitochondrial membrane potential and AIF release from mitochondria. Silencing AIF can remarkable attenuated ART-induced apoptosis. Collectively, ART induces apoptosis by caspase-independent intrinsic pathway in A549 cells.

  13. Apoptosis as a Mechanism for Liver Disease Progression

    PubMed Central

    Guicciardi, Maria Eugenia; Gores, Gregory J.

    2011-01-01

    Hepatocyte injury is ubiquitous in clinical practice, and the mode of cell death associated with this injury is often apoptosis, especially by death receptors. Information from experimental systems demonstrates that hepatocyte apoptosis is sufficient to cause liver hepatic fibrogenesis. The mechanisms linking hepatocyte apoptosis to hepatic fibrosis remain incompletely understood, but likely relate to engulfment of apoptotic bodies by professional phagocytic cells and stellate cells, and release of mediators by cells undergoing apoptosis. Inhibition of apoptosis with caspase inhibitors has demonstrated beneficial effects in murine models of hepatic fibrosis. Recent studies implicating Toll-like receptor 9 (TLR9) in liver injury and fibrosis are also of particular interest. Engulfment of apoptotic bodies is one mechanism by which the TLR9 ligand (CpG DNA motifs) could be delivered to this intracellular receptor. These concepts suggest therapy focused on interrupting the cellular mechanisms linking apoptosis to fibrosis would be useful in human liver diseases. PMID:20960379

  14. CD45 regulates apoptosis in peripheral T lymphocytes.

    PubMed

    Liu, Zhe; Dawes, Ritu; Petrova, Svetla; Beverley, Peter C L; Tchilian, Elma Z

    2006-06-01

    Programmed cell death (apoptosis) is a key mechanism for regulating lymphocyte numbers. Murine lymph node lymphocytes cultured in vitro without added stimuli show significant levels of apoptosis over 24 h, detectable by staining with Annexin V. CD4 and CD8 T lymphocytes from transgenic (Tg) mice expressing single CD45RABC or CD45RO isoforms show increased apoptosis and the extent of apoptosis is inversely correlated with the level of CD45 expression. CD45 Tg cells exhibit phosphatidyl serine translocation and DNA oligonucleosome formation, and can be partially rescued from apoptosis by culture in caspase inhibitors or common gamma-chain-binding cytokines. We conclude that CD45 is an important regulator of spontaneous apoptosis in T lymphocytes and this mechanism may contribute to the disease associations reported for individuals expressing CD45 variant alleles. PMID:16621865