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Sample records for islet gip receptor

  1. Reversal of islet GIP receptor down-regulation and resistance to GIP by reducing hyperglycemia in the Zucker rat

    SciTech Connect

    Piteau, Shalea; Olver, Amy; Kim, Su-Jin; Winter, Kyle; Pospisilik, John Andrew; Lynn, Francis; Manhart, Susanne; Demuth, Hans-Ulrich; Speck, Madeleine; Pederson, Raymond A.; McIntosh, Christopher H.S.

    2007-11-03

    In type 2 diabetes (T2DM) {beta}-cell responsiveness to glucose-dependent insulinotropic polypeptide (GIP) is reduced. In a model of T2DM, the VDF Zucker rat, GIP receptor mRNA and protein levels were shown to be down-regulated. Possible restoration of responsiveness to GIP in Zucker rats by reducing hyperglycemia has been examined. ZDF rats with extreme hyperglycemia demonstrated greater islet GIP receptor mRNA down-regulation (94.3 {+-} 3.8%) than ZF rats (48.8 {+-} 22.8%). GIP receptor mRNA levels in ZDF rats returned to 83.0 {+-} 17.9% of lean following normalization of hyperglycemia by phlorizin treatment and pancreas perfusions demonstrated markedly improved GIP responsiveness. Treatment of VDF rats with a DP IV inhibitor (P32/98) resulted in improved glucose tolerance and restored sensitivity to GIP in isolated pancreata. These findings support the proposal that GIP receptor down-regulation in rodent T2DM is secondary to chronic hyperglycemia and that normalization of glycemia can restore GIP sensitivity.

  2. A naturally occurring GIP receptor variant undergoes enhanced agonist-induced desensitization, which impairs GIP control of adipose insulin sensitivity.

    PubMed

    Mohammad, Sameer; Patel, Rajesh T; Bruno, Joanne; Panhwar, Muhammad Siyab; Wen, Jennifer; McGraw, Timothy E

    2014-10-01

    Glucose-dependent insulinotropic polypeptide (GIP), an incretin hormone secreted from gastrointestinal K cells in response to food intake, has an important role in the control of whole-body metabolism. GIP signals through activation of the GIP receptor (GIPR), a G-protein-coupled receptor (GPCR). Dysregulation of this pathway has been implicated in the development of metabolic disease. Here we demonstrate that GIPR is constitutively trafficked between the plasma membrane and intracellular compartments of both GIP-stimulated and unstimulated adipocytes. GIP induces a downregulation of plasma membrane GIPR by slowing GIPR recycling without affecting internalization kinetics. This transient reduction in the expression of GIPR in the plasma membrane correlates with desensitization to the effects of GIP. A naturally occurring variant of GIPR (E354Q) associated with an increased incidence of insulin resistance, type 2 diabetes, and cardiovascular disease in humans responds to GIP stimulation with an exaggerated downregulation from the plasma membrane and a delayed recovery of GIP sensitivity following cessation of GIP stimulation. This perturbation in the desensitization-resensitization cycle of the GIPR variant, revealed in studies of cultured adipocytes, may contribute to the link of the E354Q variant to metabolic disease.

  3. Decreased TCF7L2 protein levels in type 2 diabetes mellitus correlate with downregulation of GIP- and GLP-1 receptors and impaired beta-cell function

    PubMed Central

    Shu, Luan; Matveyenko, Aleksey V.; Kerr-Conte, Julie; Cho, Jae-Hyoung; McIntosh, Christopher H.S.; Maedler, Kathrin

    2009-01-01

    Recent human genetics studies have revealed that common variants of the TCF7L2 (T-cell factor 7-like 2, formerly known as TCF4) gene are strongly associated with type 2 diabetes mellitus (T2DM). We have shown that TCF7L2 expression in the β-cells is correlated with function and survival of the insulin-producing pancreatic β-cell. In order to understand how variations in TCF7L2 influence diabetes progression, we investigated its mechanism of action in the β-cell. We show robust differences in TCF7L2 expression between healthy controls and models of T2DM. While mRNA levels were approximately 2-fold increased in isolated islets from the diabetic db/db mouse, the Vancouver Diabetic Fatty (VDF) Zucker rat and the high fat/high sucrose diet-treated mouse compared with the non-diabetic controls, protein levels were decreased. A similar decrease was observed in pancreatic sections from patients with T2DM. In parallel, expression of the receptors for glucagon-like peptide 1 (GLP-1R) and glucose-dependent insulinotropic polypeptide (GIP-R) was decreased in islets from humans with T2DM as well as in isolated human islets treated with siRNA to TCF7L2 (siTCF7L2). Also, insulin secretion stimulated by glucose, GLP-1 and GIP, but not KCl or cyclic adenosine monophosphate (cAMP) was impaired in siTCF7L2-treated isolated human islets. Loss of TCF7L2 resulted in decreased GLP-1 and GIP-stimulated AKT phosphorylation, and AKT-mediated Foxo-1 phosphorylation and nuclear exclusion. Our findings suggest that β-cell function and survival are regulated through an interplay between TCF7L2 and GLP-1R/GIP-R expression and signaling in T2DM. PMID:19386626

  4. Regulation of GIP and GLP1 receptor cell surface expression by N-glycosylation and receptor heteromerization.

    PubMed

    Whitaker, Gina M; Lynn, Francis C; McIntosh, Christopher H S; Accili, Eric A

    2012-01-01

    In response to a meal, Glucose-dependent Insulinotropic Polypeptide (GIP) and Glucagon-like Peptide-1 (GLP-1) are released from gut endocrine cells into the circulation and interact with their cognate G-protein coupled receptors (GPCRs). Receptor activation results in tissue-selective pleiotropic responses that include augmentation of glucose-induced insulin secretion from pancreatic beta cells. N-glycosylation and receptor oligomerization are co-translational processes that are thought to regulate the exit of functional GPCRs from the ER and their maintenance at the plasma membrane. Despite the importance of these regulatory processes, their impact on functional expression of GIP and GLP-1 receptors has not been well studied. Like many family B GPCRs, both the GIP and GLP-1 receptors possess a large extracellular N-terminus with multiple consensus sites for Asn-linked (N)-glycosylation. Here, we show that each of these Asn residues is glycosylated when either human receptor is expressed in Chinese hamster ovary cells. N-glycosylation enhances cell surface expression and function in parallel but exerts stronger control over the GIP receptor than the GLP-1 receptor. N-glycosylation mainly lengthens receptor half-life by reducing degradation in the endoplasmic reticulum. N-glycosylation is also required for expression of the GIP receptor at the plasma membrane and efficient GIP potentiation of glucose-induced insulin secretion from the INS-1 pancreatic beta cell line. Functional expression of a GIP receptor mutant lacking N-glycosylation is rescued by co-expressed wild type GLP1 receptor, which, together with data obtained using Bioluminescence Resonance Energy Transfer, suggests formation of a GIP-GLP1 receptor heteromer.

  5. GLP1- and GIP-producing cells rarely overlap and differ by bombesin receptor-2 expression and responsiveness.

    PubMed

    Svendsen, Berit; Pais, Ramona; Engelstoft, Maja S; Milev, Nikolay B; Richards, Paul; Christiansen, Charlotte B; Egerod, Kristoffer L; Jensen, Signe M; Habib, Abdella M; Gribble, Fiona M; Schwartz, Thue W; Reimann, Frank; Holst, Jens J

    2016-01-01

    The incretin hormones glucagon-like peptide-1 (GLP1) and glucose-dependent insulinotropic polypeptide (GIP) are secreted from intestinal endocrine cells, the so-called L- and K-cells. The cells are derived from a common precursor and are highly related, and co-expression of the two hormones in so-called L/K-cells has been reported. To investigate the relationship between the GLP1- and GIP-producing cells more closely, we generated a transgenic mouse model expressing a fluorescent marker in GIP-positive cells. In combination with a mouse strain with fluorescent GLP1 cells, we were able to estimate the overlap between the two cell types. Furthermore, we used primary cultured intestinal cells and isolated perfused mouse intestine to measure the secretion of GIP and GLP1 in response to different stimuli. Overlapping GLP1 and GIP cells were rare (∼5%). KCl, glucose and forskolin+IBMX increased the secretion of both GLP1 and GIP, whereas bombesin/neuromedin C only stimulated GLP1 secretion. Expression analysis showed high expression of the bombesin 2 receptor in GLP1 positive cells, but no expression in GIP-positive cells. These data indicate both expressional and functional differences between the GLP1-producing 'L-cell' and the GIP-producing 'K-cell'.

  6. Postprandial stimulation of insulin release by glucose-dependent insulinotropic polypeptide (GIP). Effect of a specific glucose-dependent insulinotropic polypeptide receptor antagonist in the rat.

    PubMed Central

    Tseng, C C; Kieffer, T J; Jarboe, L A; Usdin, T B; Wolfe, M M

    1996-01-01

    Glucose-dependent insulinotropic polypeptide (GIP) is a 42-amino acid peptide produced by K cells of the mammalian proximal small intestine and is a potent stimulant of insulin release in the presence of hyperglycemia. However, its relative physiological importance as a postprandial insulinotropic agent is unknown. Using LGIPR2 cells stably transfected with rat GIP receptor cDNA, GIP (1-42) stimulation of cyclic adenosine monophosphate (cAMP) production was inhibited in a concentration-dependent manner by GIP (7-30)-NH2. Competition binding assays using stably transfected L293 cells demonstrated an IC50 for GIP receptor binding of 7 nmol/liter for GIP (1-42) and 200 nmol/liter for GIP (7-30)-NH2, whereas glucagonlike peptide-1 (GLP-1) binding to its receptor on ++betaTC3 cells was minimally displaced by GIP (7-30)-NH2. In fasted anesthetized rats, GIP (1-42) stimulated insulin release in a concentration-dependent manner, an effect abolished by the concomitant intraperitoneal administration of GIP (7-30)-NH2 (100 nmol/ kg). In contrast, glucose-, GLP-1-, and arginine-stimulated insulin release were not affected by GIP (7-30)-NH2. In separate experiments, GIP (7-30)-NH2 (100 nmol/kg) reduced postprandial insulin release in conscious rats by 72%. It is concluded that GIP (7-30)-NH2 is a GIP-specific receptor antagonist and that GIP plays a dominant role in mediating postprandial insulin release. PMID:8958204

  7. Understanding interactions of gastric inhibitory polypeptide (GIP) with its G-protein coupled receptor through NMR and molecular modeling.

    PubMed

    Malde, Alpeshkumar K; Srivastava, Sudha S; Coutinho, Evans C

    2007-05-01

    Gastric inhibitory polypeptide (GIP, or glucose-dependent insulinotropic polypeptide) is a 42-amino acid incretin hormone moderating glucose-induced insulin secretion. Antidiabetic therapy based on GIP holds great promise because of the fact that its insulinotropic action is highly dependent on the level of glucose, overcoming the sideeffects of hypoglycemia associated with the current therapy of Type 2 diabetes. The truncated peptide, GIP(1-30)NH2, has the same activity as the full length native peptide. We have studied the structure of GIP(1-30)NH2 and built a model of its G-protein coupled receptor (GPCR). The structure of GIP(1-30)NH2 in DMSO-d6 and H2O has been studied using 2D NMR (total correlation spectroscopy (TOCSY), nuclear overhauser effect spectroscopy (NOESY), double quantum filtered-COSY (DQF-COSY), 13C-heteronuclear single quantum correlation (HSQC) experiments, and its conformation built by MD simulations with the NMR data as constraints. The peptide in DMSO-d6 exhibits an alpha-helix between residues Ile12 and Lys30 with a discontinuity at residues Gln19 and Gln20. In H2O, the alpha-helix starts at Ile7, breaks off at Gln19, and then continues right through to Lys30. GIP(1-30)NH2 has all the structural features of peptides belonging to family B1 GPCRs, which are characterized by a coil at the N-terminal and a long C-terminal alpha-helix with or without a break. A model of the seven transmembrane (TM) helices of the GIP receptor (GIPR) has been built on the principles of comparative protein modeling, using the crystal structure of bovine rhodopsin as a template. The N-terminal domain of GIPR has been constructed from the NMR structure of the N-terminal of corticoptropin releasing factor receptor (CRFR), a family B1 GCPR. The intra and extra cellular loops and the C-terminal have been modeled from fragments retrieved from the PDB. On the basis of the experimental data available for some members of family B1 GPCRs, four pairs of constraints between

  8. Generation of a highly diverse panel of antagonistic chicken monoclonal antibodies against the GIP receptor

    PubMed Central

    Könitzer, Jennifer D.; Pan, Qi; Augustin, Robert; Bandholtz, Sebastian; Harriman, William; Izquierdo, Shelley

    2017-01-01

    ABSTRACT Raising functional antibodies against G protein-coupled receptors (GPCRs) is challenging due to their low density expression, instability in the absence of the cell membrane's lipid bilayer and frequently short extracellular domains that can serve as antigens. In addition, a particular therapeutic concept may require an antibody to not just bind the receptor, but also act as a functional receptor agonist or antagonist. Antagonizing the glucose-dependent insulinotropic polypeptide (GIP) receptor may open up new therapeutic modalities in the treatment of diabetes and obesity. As such, a panel of monoclonal antagonistic antibodies would be a useful tool for in vitro and in vivo proof of concept studies. The receptor is highly conserved between rodents and humans, which has contributed to previous mouse and rat immunization campaigns generating very few usable antibodies. Switching the immunization host to chicken, which is phylogenetically distant from mammals, enabled the generation of a large and diverse panel of monoclonal antibodies containing 172 unique sequences. Three-quarters of all chicken-derived antibodies were functional antagonists, exhibited high-affinities to the receptor extracellular domain and sampled a broad epitope repertoire. For difficult targets, including GPCRs such as GIPR, chickens are emerging as valuable immunization hosts for therapeutic antibody discovery. PMID:28055305

  9. Generation of a highly diverse panel of antagonistic chicken monoclonal antibodies against the GIP receptor.

    PubMed

    Könitzer, Jennifer D; Pramanick, Shreya; Pan, Qi; Augustin, Robert; Bandholtz, Sebastian; Harriman, William; Izquierdo, Shelley

    2017-01-05

    Raising functional antibodies against G protein-coupled receptors (GPCRs) is challenging due to their low density expression, instability in the absence of the cell membrane's lipid bilayer and frequently short extracellular domains that can serve as antigens. In addition, a particular therapeutic concept may require an antibody to not just bind the receptor, but also act as a functional receptor agonist or antagonist. Antagonizing the glucose-dependent insulinotropic polypeptide (GIP) receptor may open up new therapeutic modalities in the treatment of diabetes and obesity. As such, a panel of monoclonal antagonistic antibodies would be a useful tool for in vitro and in vivo proof of concept studies. The receptor is highly conserved between rodents and humans, which has contributed to previous mouse and rat immunization campaigns generating very few usable antibodies. Switching the immunization host to chicken, which is phylogenetically distant from mammals, enabled the generation of a large and diverse panel of monoclonal antibodies containing 172 unique sequences. Three-quarters of all chicken-derived antibodies were functional antagonists, exhibited high-affinities to the receptor extracellular domain and sampled a broad epitope repertoire. For difficult targets, including GPCRs such as GIPR, chickens are emerging as valuable immunization hosts for therapeutic antibody discovery.

  10. Glucose-dependent insulinotropic polypeptide (GIP) receptor deletion leads to reduced bone strength and quality.

    PubMed

    Mieczkowska, Aleksandra; Irwin, Nigel; Flatt, Peter R; Chappard, Daniel; Mabilleau, Guillaume

    2013-10-01

    Bone is permanently remodeled by a complex network of local, hormonal and neuronal factors that affect osteoclast and osteoblast biology. In this context, a role for gastro-intestinal hormones has been proposed based on evidence that bone resorption dramatically falls after a meal. Glucose-dependent insulinotropic polypeptide (GIP) is one of the candidate hormones as its receptor, glucose-dependent insulinotropic polypeptide receptor (GIPR), is expressed in bone. In the present study we investigated bone strength and quality by three-point bending, quantitative x-ray microradiography, microCT, qBEI and FTIR in a GIPR knockout (GIPR KO) mouse model and compared with control wild-type (WT) animals. Animals with a deletion of the GIPR presented with a significant reduction in ultimate load (--11%), stiffness (-16%), total absorbed (-28%) and post-yield energies (-27%) as compared with WT animals. Furthermore, despite no change in bone outer diameter, the bone marrow diameter was significantly increased and as a result cortical thickness was significantly decreased by 20% in GIPR deficient animals. Bone resorption at the endosteal surface was significantly increased whilst bone formation was unchanged in GIPR deficient animals. Deficient animals also presented with a pronounced reduction in the degree of mineralization of bone matrix. Furthermore, the amount of mature cross-links of collagen matrix was significantly reduced in GIPR deficient animals and was associated with lowered intrinsic material properties. Taken together, these data support a positive effect of the GIPR on bone strength and quality.

  11. Immobilization of soluble complement receptor 1 on islets.

    PubMed

    Luan, Nguyen M; Teramura, Yuji; Iwata, Hiroo

    2011-07-01

    Transplantation of pancreatic islets of Langerhans (islets) is a promising method to treat insulin-dependent diabetes mellitus. Control of complement activation is necessary to improve graft survival in alloislet and xenoislet transplantation. In this study, human soluble complement receptor 1 (sCR1) was immobilized on the islet cell surface through poly(ethylene glycol)-conjugated phospholipid (PEG-lipid) without loss of islet cell viability or insulin secretion ability. sCR1 on islets effectively inhibits complement activation and protects islets against attack by xenoreactive antibodies and complement. This method will be an efficient means to control early islet loss in clinical islet transplantation and realize xenoislet transplantation in the future.

  12. Free fatty acid receptor GPR120 is highly expressed in enteroendocrine K cells of the upper small intestine and has a critical role in GIP secretion after fat ingestion.

    PubMed

    Iwasaki, Kanako; Harada, Norio; Sasaki, Kazuki; Yamane, Shunsuke; Iida, Keiko; Suzuki, Kazuyo; Hamasaki, Akihiro; Nasteska, Daniela; Shibue, Kimitaka; Joo, Erina; Harada, Takanari; Hashimoto, Toshihiro; Asakawa, Yoshinori; Hirasawa, Akira; Inagaki, Nobuya

    2015-03-01

    Gastric inhibitory polypeptide (GIP) is an incretin secreted from enteroendocrine K cells in response to meal ingestion. Recently free fatty acid receptor G protein-coupled receptor (GPR) 120 was identified as a lipid sensor involved in glucagon-like peptide-1 secretion. However, Gpr 120 gene expression and its role in K cells remain unclear, partly due to difficulties in separation of K cells from other intestinal epithelial cells. In this study, we purified K cells using GIP-green fluorescent protein (GFP) knock-in mice, in which K cells can be visualized by GFP fluorescence. GFP-positive cells (K cells) were observed in the small intestine but not in the stomach and colon. K cell number and GIP content in K cells were significantly higher in the upper small intestine than those in the lower small intestine. We also examined the expression levels of several free fatty acid receptors in K cells. Among free fatty acid receptors, GPR120 was highly expressed in the K cells of the upper small intestine compared with the lower small intestine. To clarify the role of GPR120 on K cells in vivo, we used GPR120-deficient mice (GPR120(-/-)). GPR120(-/-) exhibited significantly lower GIP secretion (75% reduction, P < .01) after lard oil ingestion compared with that in wild-type mice. Consistently, pharmacological inhibition of GPR120 with grifolic acid methyl ether in wild-type mice significantly attenuated lard oil-induced GIP secretion. In conclusion, GPR120 is expressed abundantly in K cells of the upper small intestine and plays a critical role in lipid-induced GIP secretion.

  13. Xenotransplantation of islets enclosed in agarose microcapsule carrying soluble complement receptor 1.

    PubMed

    Luan, Nguyen Minh; Iwata, Hiroo

    2012-11-01

    Strong immunological reactions remain a major barrier to treating diabetic patients using xenogeneic islets. In a previous study, we developed a method for enclosing islets with agarose microbeads carrying soluble complement receptor 1 (sCR1-Mics), a potent complement inhibitor in both classical and alternative complement activation pathways. This is the follow-up in vivo study to evaluate the protective effect of these sCR1-Mics using a xenotransplantation model (rats to mice). ACI/NSIc rat islets enclosed in sCR1-Mics were transplanted into the intraperitoneal cavity of diabetic C57BL/6 mice without immunosuppression therapy. Transplantation of islets in plain agarose microbeads (Mics) was used as a reference. While islets enclosed in plain Mics were rapidly destroyed (graft survival in recipients of 1000 islets: 11.6±3.8 days), transplantation of islets in sCR1-Mics significantly prolonged graft survival (34.1±3.2 days). Moreover, intraperitoneal glucose tolerance tests revealed that islets in sCR1-Mics normalized blood glucose levels in a similar manner as islets in pancreas of normal mice. In conclusion, sCR1 immobilized onto agarose microbeads exerted some protective effect in xenogeneic islets resulting in prolonged graft survival.

  14. Adverse effect on syngeneic islet transplantation by transgenic coexpression of decoy receptor 3 and heme oxygenase-1 in the islet of NOD mice.

    PubMed

    Huang, S-H; Lin, G-J; Chien, M-W; Chu, C-H; Yu, J-C; Chen, T-W; Hueng, D-Y; Liu, Y-L; Sytwu, H-K

    2013-03-01

    Decoy receptor 3 (DcR3) blocks both Fas ligand- and LIGHT-induced pancreatic β-cell damage in autoimmune diabetes. Heme oxygenase 1 (HO-1) possesses antiapoptotic, anti-inflammatory, and antioxidative effects that protect cells against various forms of attack by the immune system. Previously, we have demonstrated that transgenic islets overexpressing DcR3 or murine HO-1 (mHO-1) exhibit longer survival times than nontransgenic islets in syngeneic islet transplantation. In this study, we evaluated whether DcR3 and mHO-1 double-transgenic islets of NOD mice could provide better protective effects and achieve longer islet graft survival than DcR3 or mHO-1 single-transgenic islets after islet transplantation. We generated DcR3 and mHO-1 double-transgenic NOD mice that specifically overexpress DcR3 and HO-1 in islets. Seven hundred islets isolated from double-transgenic, single-transgenic, or nontransgenic NOD mice were syngeneically transplanted into the kidney capsules of newly diabetic female recipients. Unexpectedly, there was no significant difference in the survival time between double-transgenic or nontransgenic NOD islet grafts, and the survival times of double-transgenic NOD islet grafts were even shorter than those of DcR3 or mHO-1 single-transgenic islets. Our data indicate that transplantation of double-transgenic islets that coexpress HO-1 and DcR3 did not result in a better outcome. On the contrary, this strategy even caused an adverse effect in syngeneic islet transplantation.

  15. The localization of oxytocin receptors in the islets of Langerhans in the rat pancreas.

    PubMed

    Suzuki, Motoaki; Honda, Yuko; Li, Ming-Zi; Masuko, Sadahiko; Murata, Yuzo

    2013-05-10

    In this study, oxytocin receptors (OTRs) in the islets of Langerhans were detected using real-time RT-PCR and immunohistochemical technique. Indeed, OTR mRNA was expressed in the rat pancreas. Double immunohistochemical staining for OTR and either glucagon or insulin demonstrated their co-localization in A-cells or B-cells, respectively. OTR-immunoreactivity in A-cells was stronger than that of B-cells. All A-cells and 94.8% of B-cells were OTR-immunoreactive. We reveal the statistically significant relations of OTR with A-cells and B-cells in the islets of Langerhans. This is the first demonstration of the OTR localization in the islets of Langerhans immunohistochemically. It suggests that oxytocin (OT) is involved in the release of insulin and glucagon.

  16. Layer-by-layer co-immobilization of soluble complement receptor 1 and heparin on islets.

    PubMed

    Luan, Nguyen Minh; Teramura, Yuji; Iwata, Hiroo

    2011-09-01

    Early graft loss due to instant blood-mediated inflammatory reactions (IBMIRs) is a major obstacle of clinical islet transplantation; inhibition of blood coagulation and complement activation is necessary to inhibit IBMIRs. Here, human soluble form complement receptor 1 (sCR1) and heparin were co-immobilized onto the surfaces of islet cells. sCR1 molecules carrying thiol groups were immobilized through maleimide-poly(ethylene glycol)-phospholipids anchored in the lipid bilayers of islet cells. Heparin was immobilized on the sCR1 layer via the affinity between sCR1 and heparin, and additional layers of sCR1 and heparin were formed layer-by-layer. The sCR1 and heparin molecules in these layers maintained anti-complement activation and anti-coagulation activities, respectively. This promising method could be employed to reduce the number of islet cells required to reverse hyperglycemia and prolong graft survival in both allo- and xeno-islet transplantation.

  17. Leptin modulates β cell expression of IL-1 receptor antagonist and release of IL-1β in human islets

    PubMed Central

    Maedler, Kathrin; Sergeev, Pavel; Ehses, Jan A.; Mathe, Zoltan; Bosco, Domenico; Berney, Thierry; Dayer, Jean-Michel; Reinecke, Manfred; Halban, Philippe A.; Donath, Marc Y.

    2004-01-01

    High concentrations of glucose induce β cell production of IL-1β, leading to impaired β cell function and apoptosis in human pancreatic islets. IL-1 receptor antagonist (IL-1Ra) is a naturally occurring antagonist of IL-1β and protects cultured human islets from glucotoxicity. Therefore, the balance of IL-1β and IL-1Ra may play a crucial role in the pathogenesis of diabetes. In the present study, we observed expression of IL-1Ra in human pancreatic β cells of nondiabetic individuals, which was decreased in tissue sections of type 2 diabetic patients. In vitro, chronic exposure of human islets to leptin, a hormone secreted by adipocytes, decreased β cell production of IL-1Ra and induced IL-1β release from the islet preparation, leading to impaired β cell function, caspase-3 activation, and apoptosis. Exogenous addition of IL-1Ra protected cultured human islets from the deleterious effects of leptin. Antagonizing IL-1Ra by introduction of small interfering RNA to IL-1Ra into human islets led to caspase-3 activation, DNA fragmentation, and impaired β cell function. Moreover, siIL-1Ra enhanced glucose-induced β cell apoptosis. These findings demonstrate expression of IL-1Ra in the human β cell, providing localized protection against leptin- and glucose-induced islet IL-1β. PMID:15141093

  18. Demonstration of. cap alpha. /sub 2/-adrenergic receptors in rat pancreatic islets using radioligand binding

    SciTech Connect

    Cherksey, B.; Mendelsohn, S.; Zadunaisky, J.; Altszuler, N.

    1982-11-01

    The type of the ..cap alpha..-adrenergic receptors on rat pancreatic islet cells was characterized directly using specific radioligands and displacement agonists and antagonists. Scatchard plots for binding of (/sup 3/H)clonidine (..cap alpha../sub 2/-agonist) revealed a dissociation constant, K/sub d/ of 0.542 +/- 0.1 nM and density of binding sites (B/sub max/) of 50.4 +/- 3.6 fmole/mg protein. Similar values were obtained with (/sup 3/H)dihydroergocryptine (antagonist). The various agonists displaced (/sup 3/H)clonidine with the following order of potency: clonidine > epinephrine approx. = norepinephrine > isoproterenol. Yohimbine, the ..cap alpha../sub 2/-antagonist, was very effective in displacing (/sup 3/H)clonidine, whereas the ..cap alpha../sub 1/-antagonist, prazosin, was much less effective. The data indicate that the ..cap alpha..-adrenergic receptors on rat pancreatic islets are of the ..cap alpha../sub 2/ subtype.

  19. Impaired muscarinic type 3 (M3) receptor/PKC and PKA pathways in islets from MSG-obese rats.

    PubMed

    Ribeiro, Rosane Aparecida; Balbo, Sandra Lucinei; Roma, Letícia Prates; Camargo, Rafael Ludemann; Barella, Luiz Felipe; Vanzela, Emerielle Cristine; de Freitas Mathias, Paulo Cesar; Carneiro, Everardo Magalhães; Boschero, Antonio Carlos; Bonfleur, Maria Lúcia

    2013-07-01

    Monosodium glutamate-obese rats are glucose intolerant and insulin resistant. Their pancreatic islets secrete more insulin at increasing glucose concentrations, despite the possible imbalance in the autonomic nervous system of these rats. Here, we investigate the involvement of the cholinergic/protein kinase (PK)-C and PKA pathways in MSG β-cell function. Male newborn Wistar rats received a subcutaneous injection of MSG (4 g/kg body weight (BW)) or hyperosmotic saline solution during the first 5 days of life. At 90 days of life, plasma parameters, islet static insulin secretion and protein expression were analyzed. Monosodium glutamate rats presented lower body weight and decreased nasoanal length, but had higher body fat depots, glucose intolerance, hyperinsulinemia and hypertrigliceridemia. Their pancreatic islets secreted more insulin in the presence of increasing glucose concentrations with no modifications in the islet-protein content of the glucose-sensing proteins: the glucose transporter (GLUT)-2 and glycokinase. However, MSG islets presented a lower secretory capacity at 40 mM K(+) (P < 0.05). The MSG group also released less insulin in response to 100 μM carbachol, 10 μM forskolin and 1 mM 3-isobutyl-1-methyl-xantine (P < 0.05, P < 0.0001 and P < 0.01). These effects may be associated with a the decrease of 46 % in the acetylcholine muscarinic type 3 (M3) receptor, and a reduction of 64 % in PKCα and 36 % in PKAα protein expressions in MSG islets. Our data suggest that MSG islets, whilst showing a compensatory increase in glucose-induced insulin release, demonstrate decreased islet M3/PKC and adenylate cyclase/PKA activation, possibly predisposing these prediabetic rodents to the early development of β-cell dysfunction.

  20. Melatonin receptors in pancreatic islets: good morning to a novel type 2 diabetes gene.

    PubMed

    Mulder, H; Nagorny, C L F; Lyssenko, V; Groop, L

    2009-07-01

    Melatonin is a circulating hormone that is primarily released from the pineal gland. It is best known as a regulator of seasonal and circadian rhythms; its levels are high during the night and low during the day. Interestingly, insulin levels also exhibit a nocturnal drop, which has previously been suggested to be controlled, at least in part, by melatonin. This regulation can be explained by the proposed inhibitory action of melatonin on insulin release. Indeed, both melatonin receptor 1A (MTNR1A) and MTNR1B are expressed in pancreatic islets. The role of melatonin in the regulation of glucose homeostasis has been highlighted by three independent publications based on genome-wide association studies of traits connected with type 2 diabetes, such as elevated fasting glucose, and, subsequently, of the disease itself. The studies demonstrate a link between variations in the MTNR1B gene, hyperglycaemia, impaired early phase insulin secretion and beta cell function. The risk genotype predicts the future development of type 2 diabetes. Carriers of the risk genotype exhibit increased expression of MTNR1B in islets. This suggests that these individuals may be more sensitive to the actions of melatonin, leading to impaired insulin secretion. Blocking the inhibition of insulin secretion by melatonin may be a novel therapeutic avenue for type 2 diabetes.

  1. 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

  2. Novel Small Molecule Glucagon-Like Peptide-1 Receptor Agonist Stimulates Insulin Secretion in Rodents and From Human Islets

    PubMed Central

    Sloop, Kyle W.; Willard, Francis S.; Brenner, Martin B.; Ficorilli, James; Valasek, Kathleen; Showalter, Aaron D.; Farb, Thomas B.; Cao, Julia X.C.; Cox, Amy L.; Michael, M. Dodson; Gutierrez Sanfeliciano, Sonia Maria; Tebbe, Mark J.; Coghlan, Michael J.

    2010-01-01

    OBJECTIVE The clinical effectiveness of parenterally-administered glucagon-like peptide-1 (GLP-1) mimetics to improve glucose control in patients suffering from type 2 diabetes strongly supports discovery pursuits aimed at identifying and developing orally active, small molecule GLP-1 receptor agonists. The purpose of these studies was to identify and characterize novel nonpeptide agonists of the GLP-1 receptor. RESEARCH DESIGN AND METHODS Screening using cells expressing the GLP-1 receptor and insulin secretion assays with rodent and human islets were used to identify novel molecules. The intravenous glucose tolerance test (IVGTT) and hyperglycemic clamp characterized the insulinotropic effects of compounds in vivo. RESULTS Novel low molecular weight pyrimidine-based compounds that activate the GLP-1 receptor and stimulate glucose-dependent insulin secretion are described. These molecules induce GLP-1 receptor-mediated cAMP signaling in HEK293 cells expressing the GLP-1 receptor and increase insulin secretion from rodent islets in a dose-dependent manner. The compounds activate GLP-1 receptor signaling, both alone or in an additive fashion when combined with the endogenous GLP-1 peptide; however, these agonists do not compete with radiolabeled GLP-1 in receptor-binding assays. In vivo studies using the IVGTT and the hyperglycemic clamp in Sprague Dawley rats demonstrate increased insulin secretion in compound-treated animals. Further, perifusion assays with human islets isolated from a donor with type 2 diabetes show near-normalization of insulin secretion upon compound treatment. CONCLUSIONS These studies characterize the insulinotropic effects of an early-stage, small molecule GLP-1 receptor agonist and provide compelling evidence to support pharmaceutical optimization. PMID:20823098

  3. A novel insulinotropic mechanism of whole grain-derived γ-oryzanol via the suppression of local dopamine D2 receptor signalling in mouse islet

    PubMed Central

    Kozuka, Chisayo; Sunagawa, Sumito; Ueda, Rei; Higa, Moritake; Ohshiro, Yuzuru; Tanaka, Hideaki; Shimizu-Okabe, Chigusa; Takayama, Chitoshi; Matsushita, Masayuki; Tsutsui, Masato; Ishiuchi, Shogo; Nakata, Masanori; Yada, Toshihiko; Miyazaki, Jun-ichi; Oyadomari, Seiichi; Shimabukuro, Michio; Masuzaki, Hiroaki

    2015-01-01

    Background and Purpose γ-Oryzanol, derived from unrefined rice, attenuated the preference for dietary fat in mice, by decreasing hypothalamic endoplasmic reticulum stress. However, no peripheral mechanisms, whereby γ-oryzanol could ameliorate glucose dyshomeostasis were explored. Dopamine D2 receptor signalling locally attenuates insulin secretion in pancreatic islets, presumably via decreased levels of intracellular cAMP. We therefore hypothesized that γ-oryzanol would improve high-fat diet (HFD)-induced dysfunction of islets through the suppression of local D2 receptor signalling. Experimental Approach Glucose metabolism and regulation of molecules involved in D2 receptor signalling in pancreatic islets were investigated in male C57BL/6J mice, fed HFD and treated with γ-oryzanol. In isolated murine islets and the beta cell line, MIN6, the effects of γ-oryzanol on glucose-stimulated insulin secretion (GSIS) was analysed using siRNA for D2 receptors and a variety of compounds which alter D2 receptor signalling. Key Results In islets, γ-oryzanol enhanced GSIS via the activation of the cAMP/PKA pathway. Expression of molecules involved in D2 receptor signalling was increased in islets from HFD-fed mice, which were reciprocally decreased by γ-oryzanol. Experiments with siRNA for D2 receptors and D2 receptor ligands in vitro suggest that γ-oryzanol suppressed D2 receptor signalling and augmented GSIS. Conclusions and Implications γ-Oryzanol exhibited unique anti-diabetic properties. The unexpected effects of γ-oryzanol on D2 receptor signalling in islets may provide a novel; natural food-based, approach to anti-diabetic therapy. PMID:26140534

  4. Distinct effects of dipeptidyl peptidase-4 inhibitor and glucagon-like peptide-1 receptor agonist on islet morphology and function.

    PubMed

    Morita, Asuka; Mukai, Eri; Hiratsuka, Ayano; Takatani, Tomozumi; Iwanaga, Toshihiko; Lee, Eun Young; Miki, Takashi

    2016-03-01

    Although the two anti-diabetic drugs, dipeptidyl peptidase-4 inhibitors (DPP4is) and glucagon-like peptide-1 (GLP-1) receptor agonists (GLP1RAs), have distinct effects on the dynamics of circulating incretins, little is known of the difference in their consequences on morphology and function of pancreatic islets. We examined these in a mouse model of β cell injury/regeneration. The model mice were generated so as to express diphtheria toxin (DT) receptor and a fluorescent protein (Tomato) specifically in β cells. The mice were treated with a DPP4i (MK-0626) and a GLP1RA (liraglutide), singly or doubly, and the morphology and function of the islets were compared. Prior administration of MK-0626 and/or liraglutide similarly protected β cells from DT-induced cell death, indicating that enhanced GLP-1 signaling can account for the cytoprotection. However, 2-week intervention of MK-0626 and/or liraglutide in DT-injected mice resulted in different islet morphology and function: β cell proliferation and glucose-stimulated insulin secretion (GSIS) were increased by MK-0626 but not by liraglutide; α cell mass was decreased by liraglutide but not by MK-0626. Although liraglutide administration nullified MK-0626-induced β cell proliferation, their co-administration resulted in increased GSIS, decreased α cell mass, and improved glucose tolerance. The pro-proliferative effect of MK-0626 was lost by co-administration of the GLP-1 receptor antagonist exendin-(9-39), indicating that GLP-1 signaling is required for this effect. Comparison of the effects of DPP4is and/or GLP1RAs treatment in a single mouse model shows that the two anti-diabetic drugs have distinct consequences on islet morphology and function.

  5. Cannabinoids inhibit insulin secretion and cytosolic Ca2+ oscillation in islet beta-cells via CB1 receptors.

    PubMed

    Nakata, Masanori; Yada, Toshihiko

    2008-01-10

    Obesity is the main risk factor for the development of metabolic syndrome. Endogenous cannabinoids act on the cannabinoid type 1 (CB1) receptor, a GPCR, and stimulate appetite via central and peripheral actions, while blockade of CB1 receptor reduces body weight in humans. In this study, we aimed to explore a role of the peripheral endocannabinoid system in insulin secretion, which could be important in the metabolic effects of the cannabinoid-CB1 system. We found that mRNA for CB1 receptor, but not CB2 receptor, was expressed in mouse pancreatic islets using RT-PCR. Immunohistochemical study revealed that CB1 receptor was expressed in beta-cells. Furthermore, anandamide and a CB1 agonist, arachidonylcyclopropylamide (ACPA), inhibited glucose-induced insulin secretion from mouse pancreatic islets. Both anandamide and ACPA inhibited glucose-induced cytosolic Ca(2+) oscillation in mouse pancreatic beta-cells. These results demonstrate a novel peripheral action of cannabinoids to inhibit insulin secretion via CB1 receptors.

  6. Heterotrimeric G-protein shuttling via Gip1 extends the dynamic range of eukaryotic chemotaxis

    PubMed Central

    Kamimura, Yoichiro; Miyanaga, Yukihiro; Ueda, Masahiro

    2016-01-01

    Chemotactic eukaryote cells can sense chemical gradients over a wide range of concentrations via heterotrimeric G-protein signaling; however, the underlying wide-range sensing mechanisms are only partially understood. Here we report that a novel regulator of G proteins, G protein-interacting protein 1 (Gip1), is essential for extending the chemotactic range of Dictyostelium cells. Genetic disruption of Gip1 caused severe defects in gradient sensing and directed cell migration at high but not low concentrations of chemoattractant. Also, Gip1 was found to bind and sequester G proteins in cytosolic pools. Receptor activation induced G-protein translocation to the plasma membrane from the cytosol in a Gip1-dependent manner, causing a biased redistribution of G protein on the membrane along a chemoattractant gradient. These findings suggest that Gip1 regulates G-protein shuttling between the cytosol and the membrane to ensure the availability and biased redistribution of G protein on the membrane for receptor-mediated chemotactic signaling. This mechanism offers an explanation for the wide-range sensing seen in eukaryotic chemotaxis. PMID:27044073

  7. Restoration of insulin secretion in pancreatic islets of protein-deficient rats by reduced expression of insulin receptor substrate (IRS)-1 and IRS-2.

    PubMed

    Araujo, E P; Amaral, M E C; Filiputti, E; De Souza, C T; Laurito, T L; Augusto, V D; Saad, M J A; Boschero, A C; Velloso, L A; Carneiro, E M

    2004-04-01

    Autocrine and paracrine insulin signaling may participate in the fine control of insulin secretion. In the present study, tissue distribution and protein amounts of the insulin receptor and its major substrates, insulin receptor substrate (IRS)-1 and IRS-2, were evaluated in a model of impaired glucose-induced insulin secretion, the protein-deficient rat. Immunoblot and RT-PCR studies showed that the insulin receptor and IRS-2 expression are increased, whilst IRS-1 protein and mRNA contents are decreased in pancreatic islets of protein-deficient rats. Immunohistochemical studies revealed that the insulin receptor and IRS-1 and -2 are present in the great majority of islet cells; however, the greatest staining was localized at the periphery, suggesting a co-localization with non-insulin-secreting cells. Exogenous insulin stimulation of isolated islets promoted higher insulin receptor and IRS-1 and -2 tyrosine phosphorylation in islets from protein-deficient rats, as compared with controls. Moreover, insulin-induced IRS-1- and IRS-2-associated phosphatidylinositol 3-kinase activity are increased in islets of protein-deficient rats. The reduction of IRS-1 and IRS-2 protein expression in islets isolated from protein-deficient rats by the use of antisense IRS-1 or IRS-2 phosphorthioate-modified oligonucleotides partially restored glucose-induced insulin secretion. Thus, the impairment of insulin cell signaling through members of the IRS family of proteins in isolated rat pancreatic islets improves glucose-induced insulin secretion. The present data reinforced the role of insulin paracrine and autocrine signaling in the control of its own secretion.

  8. Coexpression of the platelet-derived growth factor (PDGF) B chain and the PDGF. beta. receptor in isolated pancreatic islet cells stimulates DNA synthesis

    SciTech Connect

    Welsh, M.; Hallberg, A.; Welsh, N.; Arkhammar, P.; Nilsson, T.; Berggren, P.O. ); Claesson-Welsh, L.; Heldin, C.H. ); Betsholtz, C. ); Berggren, P.O. )

    1990-08-01

    Suspensions rich in pancreatic {beta} cells were transfected by means of electroporation or by using the liposome technique with DNA constructs coding for the {beta} chain of platelet-derived growth factor (PDGF) and the PDGF {alpha} and {beta} receptors to induce a mitotic response in this slowly replicating cell type. Transfection with the B-chain construct induced synthesis of the PDGF B-chain homodimer (PDGF-BB) as assessed by the presence of {sup 125}I-labeled PDGF-BB competing activity in the conditioned medium of the transfected islet cells. Moreover, islet cells transfected with the PDGF {beta}-receptor construct exhibited increased immunofluorescence staining with a PDGF {beta}-receptor antibody. These cells also displayed increased {sup 125}I-labeled PDGF-BB binding compared with control transfected cells. The {beta} cells exhibited elevated levels of ({sup 3}H)inositol trisphosphate after transfection with the B-chain and {beta}-receptor constructs, indicating activation of phospholipase C. Islet cells transfected with the different receptor constructs exhibited different patterns of tyrosine phosphorylation upon ligand activation. The results demonstrate that pancreatic islet cells can be stimulated to increase DNA synthesis by transfection with the PDGF {beta}-receptor gene, whereas cotransfection with the {alpha}-receptor gene may attenuate the growth response.

  9. Melatonin-receptor-1-deficiency affects neurogenic differentiation factor immunoreaction in pancreatic islets and enteroendocrine cells of mice.

    PubMed

    Shalabi, Andree; Fischer, Claudia; Korf, Horst-Werner; von Gall, Charlotte

    2013-09-01

    Neurogenic differentiation factor (NeuroD) is a transcription factor involved in the differentiation of neurons and in the control of energy balance and metabolism. It plays a key role in type 1 and type 2 diabetes. Melatonin is an important rhythmic endocrine signal within the circadian system of mammals and modulates insulin secretion and glucose metabolism. In the mouse pars tuberalis, NeuroD mRNA levels show day/night variation, which is independent of the molecular clock gene mPER1 but depends on the functional melatonin receptor 1 (MT1). So far, little is known about the effect of melatonin on NeuroD synthesis in the gastrointestinal tract. Thus, NeuroD protein levels and cellular localization were analyzed by immunohistochemistry in pancreatic islets and duodenal enteroendocrine cells of MT1- and mPER1-deficienct mice. In addition, the localization of NeuroD-positive cells was analyzed by double-immunofluorescence and confocal laser microscopy. In duodenal enteroendocrine cells and pancreatic islets of WT and PER1-deficient mice, NeuroD immunoreaction showed a peak during the early subjective night. In contrast, this peak was absent in MT1-deficent mice. These data suggest that melatonin, by acting on MT1 receptors, affects NeuroD expression in the gastrointestinal tract and thus might contribute to circadian regulation in metabolic functions.

  10. Vitamin D3 restores altered cholinergic and insulin receptor expression in the cerebral cortex and muscarinic M3 receptor expression in pancreatic islets of streptozotocin induced diabetic rats.

    PubMed

    Kumar, Peeyush T; Antony, Sherin; Nandhu, Mohan S; Sadanandan, Jayanarayanan; Naijil, George; Paulose, Chiramadathikudiyil S

    2011-05-01

    Nutritional therapy is a challenging but necessary dimension in the management of diabetes and neurodegenerative changes associated with it. The study evaluates the effect of vitamin D(3) in preventing the altered function of cholinergic, insulin receptors and GLUT3 in the cerebral cortex of diabetic rats. Muscarinic M3 acetylcholine receptors in pancreas control insulin secretion. Vitamin D(3) treatment in M3 receptor regulation in the pancreatic islets was also studied. Radioreceptor binding assays and gene expression was done in the cerebral cortex of male Wistar rats. Immunocytochemistry of muscarinic M3 receptor was studied in the pancreatic islets using specific antibodies. Y-maze was used to evaluate the exploratory and spatial memory. Diabetes induced a decrease in muscarinic M1, insulin and vitamin D receptor expression and an increase in muscarinic M3, α7 nicotinic acetylcholine receptor, acetylcholine esterase and GLUT3 expression. Vitamin D(3) and insulin treatment reversed diabetes-induced alterations to near control. Diabetic rats showed a decreased Y-maze performance while vitamin D(3) supplementation improved the behavioural deficit. In conclusion, vitamin D(3) shows a potential therapeutic effect in normalizing diabetes-induced alterations in cholinergic, insulin and vitamin D receptor and maintains a normal glucose transport and utilisation in the cortex. In addition vitamin D(3) modulated muscarinic M3 receptors activity in pancreas and plays a pivotal role in controlling insulin secretion. Hence our findings proved, vitamin D(3) supplementation as a potential nutritional therapy in ameliorating diabetes mediated cortical dysfunctions and suggest an interaction between vitamin D(3) and muscarinic M3 receptors in regulating insulin secretion from pancreas.

  11. Glucose-Dependent Insulin Secretion in Pancreatic β-Cell Islets from Male Rats Requires Ca2+ Release via ROS-Stimulated Ryanodine Receptors

    PubMed Central

    Llanos, Paola; Contreras-Ferrat, Ariel; Barrientos, Genaro; Valencia, Marco; Mears, David; Hidalgo, Cecilia

    2015-01-01

    Glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells requires an increase in intracellular free Ca2+ concentration ([Ca2+]). Glucose uptake into β-cells promotes Ca2+ influx and reactive oxygen species (ROS) generation. In other cell types, Ca2+ and ROS jointly induce Ca2+ release mediated by ryanodine receptor (RyR) channels. Therefore, we explored here if RyR-mediated Ca2+ release contributes to GSIS in β-cell islets isolated from male rats. Stimulatory glucose increased islet insulin secretion, and promoted ROS generation in islets and dissociated β-cells. Conventional PCR assays and immunostaining confirmed that β-cells express RyR2, the cardiac RyR isoform. Extended incubation of β-cell islets with inhibitory ryanodine suppressed GSIS; so did the antioxidant N-acetyl cysteine (NAC), which also decreased insulin secretion induced by glucose plus caffeine. Inhibitory ryanodine or NAC did not affect insulin secretion induced by glucose plus carbachol, which engages inositol 1,4,5-trisphosphate receptors. Incubation of islets with H2O2 in basal glucose increased insulin secretion 2-fold. Inhibitory ryanodine significantly decreased H2O2-stimulated insulin secretion and prevented the 4.5-fold increase of cytoplasmic [Ca2+] produced by incubation of dissociated β-cells with H2O2. Addition of stimulatory glucose or H2O2 (in basal glucose) to β-cells disaggregated from islets increased RyR2 S-glutathionylation to similar levels, measured by a proximity ligation assay; in contrast, NAC significantly reduced the RyR2 S-glutathionylation increase produced by stimulatory glucose. We propose that RyR2-mediated Ca2+ release, induced by the concomitant increases in [Ca2+] and ROS produced by stimulatory glucose, is an essential step in GSIS. PMID:26046640

  12. Effects of endogenous GLP-1 and GIP on glucose tolerance after Roux-en-Y gastric bypass surgery.

    PubMed

    Svane, Maria S; Bojsen-Møller, Kirstine N; Nielsen, Signe; Jørgensen, Nils B; Dirksen, Carsten; Bendtsen, Flemming; Kristiansen, Viggo B; Hartmann, Bolette; Holst, Jens J; Madsbad, Sten

    2016-04-01

    Exaggerated secretion of glucagon-like peptide 1 (GLP-1) is important for postprandial glucose tolerance after Roux-en-Y gastric bypass (RYGB), whereas the role of glucose-dependent insulinotropic polypeptide (GIP) remains to be resolved. We aimed to explore the relative importance of endogenously secreted GLP-1 and GIP on glucose tolerance and β-cell function after RYGB. We used DPP-4 inhibition to enhance concentrations of intact GIP and GLP-1 and the GLP-1 receptor antagonist exendin-(9-39) (Ex-9) for specific blockage of GLP-1 actions. Twelve glucose-tolerant patients were studied after RYGB in a randomized, placebo-controlled, 4-day crossover study with standard mixed-meal tests and concurrent administration of placebo, oral sitagliptin, Ex-9 infusion, or combined Ex-9-sitagliptin. GLP-1 receptor antagonism increased glucose excursions, clearly attenuated β-cell function, and aggravated postprandial hyperglucagonemia compared with placebo, whereas sitagliptin had no effect despite two- to threefold increased concentrations of intact GLP-1 and GIP. Similarly, sitagliptin did not affect glucose tolerance or β-cell function during GLP-1R blockage. This study confirms the importance of GLP-1 for glucose tolerance after RYGB via increased insulin and attenuated glucagon secretion in the postprandial state, whereas amplification of the GIP signal (or other DPP-4-sensitive glucose-lowering mechanisms) did not appear to contribute to the improved glucose tolerance seen after RYGB.

  13. The effect of the putative endogenous imidazoline receptor ligand, clonidine-displacing substance, on insulin secretion from rat and human islets of Langerhans

    PubMed Central

    Chan, Susan L F; Atlas, Daphne; James, Roger F L; Morgan, Noel G

    1997-01-01

    The effects of a rat brain extract containing clonidine-displacing substance (CDS), a putative endogenous imidazoline receptor ligand, on insulin release from rat and human isolated islets of Langerhans were investigated.CDS was able to potentiate the insulin secretory response of rat islets incubated at 6 mM glucose, in a dose-dependent manner. The magnitude of this effect was similar to that in response to the well-characterized imidazoline secretagogue, efaroxan.CDS, like other imidazoline secretagogues, was also able to reverse the inhibitory action of diazoxide on glucose-induced insulin release, in both rat and human islets.These effects of CDS on secretion were reversed by the imidazoline secretagogue antagonists, RX801080 and the newly defined KU14R, providing the first evidence that imidazoline-mediated actions of CDS can be blocked by specific imidazoline antagonists.The effects of CDS on insulin secretion were unaffected when the method of preparation involved centri-filtration through a 3,000 Da cut-off membrane or when the extract was treated with protease. These results confirm that the active principle is of low molecular weight and is not a peptide.Overall, the data suggest that CDS behaves as a potent endogenous insulin secretagogue acting at the islet imidazoline receptor. PMID:9138700

  14. Upregulated insulin secretion in insulin-resistant mice: evidence of increased islet GLP1 receptor levels and GPR119-activated GLP1 secretion.

    PubMed

    Ahlkvist, L; Brown, K; Ahrén, B

    2013-06-01

    We previously demonstrated that the overall incretin effect and the β-cell responsiveness to glucagon-like peptide-1 (GLP1) are increased in insulin-resistant mice and may contribute to the upregulated β-cell function. Now we examined whether this could, first, be explained by increased islet GLP1 receptor (GLP1R) protein levels and, secondly, be leveraged by G-protein-coupled receptor 119 (GPR119) activation, which stimulates GLP1 secretion. Female C57BL/6J mice, fed a control (CD, 10% fat) or high-fat (HFD, 60% fat) diet for 8 weeks, were anesthetized and orally given a GPR119 receptor agonist (GSK706A; 10 mg/kg) or vehicle, followed after 10 min with gavage with a liquid mixed meal (0.285 kcal). Blood was sampled for determination of glucose, insulin, intact GLP1, and glucagon, and islets were isolated for studies on insulin and glucagon secretion and GLP1R protein levels. In HFD vs CD mice, GPR119 activation augmented the meal-induced increase in the release of both GLP1 (AUCGLP1 81±9.6 vs 37±6.9 pM×min, P=0.002) and insulin (AUCINS 253±29 vs 112±19 nM×min, P<0.001). GPR119 activation also significantly increased glucagon levels in both groups (P<0.01) with, however, no difference between the groups. By contrast, GPR119 activation did not affect islet hormone secretion from isolated islets. Glucose elimination after meal ingestion was significantly increased by GPR119 activation in HFD mice (0.57±0.04 vs 0.43±0.03% per min, P=0.014) but not in control mice. Islet GLP1R protein levels was higher in HFD vs CD mice (0.8±0.1 vs 0.5±0.1, P=0.035). In conclusion, insulin-resistant mice display increased islet GLP1R protein levels and augmented meal-induced GLP1 and insulin responses to GPR119 activation, which results in increased glucose elimination. We suggest that the increased islet GLP1R protein levels together with the increased GLP1 release may contribute to the upregulated β-cell function in insulin resistance.

  15. The Islet Estrogen Receptor-α Is Induced by Hyperglycemia and Protects Against Oxidative Stress-Induced Insulin-Deficient Diabetes

    PubMed Central

    Kilic, Gamze; Alvarez-Mercado, Ana I.; Zarrouki, Bader; Opland, Darren; Liew, Chong Wee; Alonso, Laura C.; Myers, Martin G.; Jonas, Jean-Christophe; Poitout, Vincent; Kulkarni, Rohit N.; Mauvais-Jarvis, Franck

    2014-01-01

    The female steroid, 17β-estradiol (E2), is important for pancreatic β-cell function and acts via at least three estrogen receptors (ER), ERα, ERβ, and the G-protein coupled ER (GPER). Using a pancreas-specific ERα knockout mouse generated using the Cre-lox-P system and a Pdx1-Cre transgenic line (PERαKO−/−), we previously reported that islet ERα suppresses islet glucolipotoxicity and prevents β-cell dysfunction induced by high fat feeding. We also showed that E2 acts via ERα to prevent β-cell apoptosis in vivo. However, the contribution of the islet ERα to β-cell survival in vivo, without the contribution of ERα in other tissues is still unclear. Using the PERαKO−/− mouse, we show that ERα mRNA expression is only decreased by 20% in the arcuate nucleus of the hypothalamus, without a parallel decrease in the VMH, making it a reliable model of pancreas-specific ERα elimination. Following exposure to alloxan-induced oxidative stress in vivo, female and male PERαKO−/− mice exhibited a predisposition to β-cell destruction and insulin deficient diabetes. In male PERαKO−/− mice, exposure to E2 partially prevented alloxan-induced β-cell destruction and diabetes. ERα mRNA expression was induced by hyperglycemia in vivo in islets from young mice as well as in cultured rat islets. The induction of ERα mRNA by hyperglycemia was retained in insulin receptor-deficient β-cells, demonstrating independence from direct insulin regulation. These findings suggest that induction of ERα expression acts to naturally protect β-cells against oxidative injury. PMID:24498408

  16. Regulation of the pro-inflammatory cytokine osteopontin by GIP in adipocytes - A role for the transcription factor NFAT and phosphodiesterase 3B

    SciTech Connect

    Omar, Bilal; Banke, Elin; Guirguis, Emilia; Aakesson, Lina; Manganiello, Vincent; Lyssenko, Valeriya; Groop, Leif; Gomez, Maria F.; Degerman, Eva

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer GIP stimulates lipogenesis and osteopontin expression in primary adipocytes. Black-Right-Pointing-Pointer GIP-induced osteopontin expression is NFAT-dependent. Black-Right-Pointing-Pointer Osteopontin expression is PDE3-dependent. Black-Right-Pointing-Pointer Osteopontin expression is increased in PDE3B KO mice. -- Abstract: The incretin - glucose-dependent insulinotropic polypeptide (GIP) - and the pro-inflammatory cytokine osteopontin are known to have important roles in the regulation of adipose tissue functions. In this work we show that GIP stimulates lipogenesis and osteopontin expression in primary adipocytes. The GIP-induced increase in osteopontin expression was inhibited by the NFAT (the transcription factor nuclear factor of activated T-cells) inhibitor A-285222. Also, the NFAT kinase glycogen synthase kinase (GSK) 3 was upregulated by GIP. To test whether cAMP might be involved in GIP-mediated effects on osteopontin a number of strategies were used. Thus, the {beta}3-adrenergic receptor agonist CL316,243 stimulated osteopontin expression, an effects which was mimicked by OPC3911, a specific inhibitor of phosphodiesterase 3. Furthermore, treatment of phosphodiesterase 3B knock-out mice with CL316,243 resulted in a dramatic upregulation of osteopontin in adipose tissue which was not the case in wild-type mice. In summary, we delineate mechanisms by which GIP stimulates osteopontin in adipocytes. Given the established link between osteopontin and insulin resistance, our data suggest that GIP by stimulating osteopontin expression, also could promote insulin resistance in adipocytes.

  17. Expression of Receptors for Tetanus Toxin and Monoclonal Antibody A2B5 by Pancreatic Islet Cells

    NASA Astrophysics Data System (ADS)

    Eisenbarth, G. S.; Shimizu, K.; Bowring, M. A.; Wells, S.

    1982-08-01

    Studies of the reaction of antibody A2B5 and tetanus toxin with pancreatic islet cells, islet cell tumors, and other human amine precursor uptake and decarboxylation (APUD) tumors are described. By indirect immunofluorescence, antibody A2B5 and tetanus toxin were shown to specifically bind to the plasma membrane of human, rat, chicken, and mouse islet cells. The binding of antibody A2B5 to the cell surface of living islet cells has allowed isolation of these cells from a suspension of pancreatic cells by using a fluorescence-activated cell sorter. In studies designed to determine whether tetanus toxin and antibody A2B5 bound to the same surface antigen, A2B5 and tetanus toxin did not compete for binding to normal islet cells, a human islet cell tumor, or a rat islet cell tumor. In addition to binding to islet cell tumors, antibody A2B5 reacts with frozen sections, isolated cells, and cell lines of neural, neural crest, and APUD origin.

  18. Overexpression of leptin receptors in pancreatic islets of Zucker diabetic fatty rats restores GLUT-2, glucokinase, and glucose-stimulated insulin secretion

    PubMed Central

    Wang, May-Yun; Koyama, Kazunori; Shimabukuro, Michio; Mangelsdorf, David; Newgard, Christopher B.; Unger, Roger H.

    1998-01-01

    The high-Km glucose transporter, GLUT-2, and the high-Km hexokinase of β cells, glucokinase (GK), are required for glucose-stimulated insulin secretion (GSIS). GLUT-2 expression in β cells of Zucker diabetic fatty (ZDF) rats is profoundly reduced at the onset of β-cell dysfunction of diabetes. Because ZDF rats are homozygous for a mutation in their leptin receptor (OB-R) gene and are therefore leptin-insensitive, we expressed the wild-type OB-R gene in diabetic islets by infusing a recombinant adenovirus (AdCMV-OB-Rb) to determine whether this reversed the abnormalities. Leptin induced a rise in phosphorylated STAT3, indicating that the transferred wild-type OB-R was functional. GLUT-2 protein rose 17-fold in AdCMV-OB-Rb-treated ZDF islets without leptin, and leptin caused no further rise. GK protein rose 7-fold without and 12-fold with leptin. Preproinsulin mRNA increased 64% without leptin and rose no further with leptin, but leptin was required to restore GSIS. Clofibrate and 9-cis-retinoic acid, the partner ligands for binding to peroxisome proliferator-activator receptor α (PPARα) and retinoid X receptor, up-regulated GLUT-2 expression in islets of normal rats, but not in ZDF rats, in which PPARα is very low. Because the fat content of islets of diabetic ZDF rats remains high unless they are treated with leptin, it appears that restoration of GSIS requires normalization of intracellular nutrient homeostasis, whereas up-regulation of GLUT-2 and GK is leptin-independent, requiring only high expression of OB-Rb. PMID:9751766

  19. Bone marrow mesenchymal stem cells express a restricted set of functionally active chemokine receptors capable of promoting migration to pancreatic islets.

    PubMed

    Sordi, Valeria; Malosio, Maria Luisa; Marchesi, Federica; Mercalli, Alessia; Melzi, Raffaella; Giordano, Tiziana; Belmonte, Nathalie; Ferrari, Giuliana; Leone, Biagio Eugenio; Bertuzzi, Federico; Zerbini, Gianpaolo; Allavena, Paola; Bonifacio, Ezio; Piemonti, Lorenzo

    2005-07-15

    Bone marrow-derived mesenchymal stem cells (BM-MSCs) are stromal cells with the ability to proliferate and differentiate into many tissues. Although they represent powerful tools for several therapeutic settings, mechanisms regulating their migration to peripheral tissues are still unknown. Here, we report chemokine receptor expression on human BM-MSCs and their role in mediating migration to tissues. A minority of BM-MSCs (2% to 25%) expressed a restricted set of chemokine receptors (CXC receptor 4 [CXCR4], CX3C receptor 1 [CX3CR1], CXCR6, CC chemokine receptor 1 [CCR1], CCR7) and, accordingly, showed appreciable chemotactic migration in response to the chemokines CXC ligand 12 (CXCL12), CX3CL1, CXCL16, CC chemokine ligand 3 (CCL3), and CCL19. Using human pancreatic islets as an in vitro model of peripheral tissue, we showed that islet supernatants released factors able to attract BM-MSCs in vitro, and this attraction was principally mediated by CX3CL1 and CXCL12. Moreover, cells with features of BM-MSCs were detected within the pancreatic islets of mice injected with green fluorescent protein (GFP)-positive BM. A population of bona fide MSCs that also expressed CXCR4, CXCR6, CCR1, and CCR7 could be isolated from normal adult human pancreas. This study defines the chemokine receptor repertoire of human BM-MSCs that determines their migratory activity. Modulation of homing capacity may be instrumental for harnessing the therapeutic potential of BM-MSCs.

  20. The GIP/GIPR axis is functionally linked to GH-secretion increase in a significant proportion of gsp(-) somatotropinomas.

    PubMed

    Regazzo, Daniela; Losa, Marco; Albiger, Nora; Terreni, Maria Rosa; Vazza, Giovanni; Ceccato, Filippo; Emanuelli, Enzo; Denaro, Luca; Scaroni, Carla; Occhi, Gianluca

    2017-02-08

    Objective: Glucose-dependent insulinotropic polypeptide receptor (GIPR) overexpression has been recently described in a proportion of gsp(-) somatotropinomas and suggested to be associated with the paradoxical increase of GH (GH-PI) during an oral glucose load. Design and Methods: This study was aimed at linking the GIP/GIPR pathway to GH secretion in 25 somatotropinomas-derived primary cultures and correlating molecular with clinical features in acromegalic patients. Given the impairment of the GIPR/GIPR axis in acromegaly, an additional aim was to assess the effect of GH/IGF-1 stimulation on GIP expression in the enteroendocrine cell line STC-1. Results: nearly 80% of GIPR-expressing somatotropinomas, all of them negative for gsp mutations, show increased GH secretion upon GIP stimulation, higher sensitivity to forskolin but not to somatostatin analogues. Beside increased frequency of GH-PI, GIPR overexpression does not appear to affect acromegalic patients' clinical features. In STC-1 cells transfected with GIP promoter-driven luciferase vector, IGF-1 but not GH induced dose-dependent increase in luciferase activity. Conclusions: we demonstrate that GIPR mediates the GH-PI in a significant proportion of gsp(-) acromegalic patients. In these cases the stimulatory effect of IGF-I on GIP promoter support the hypothesis of a functional GH-IGF1-GIP axis. Further studies based on larger cohorts and the development of a stable transgenic model with inducible GIPR overexpression targeted to pituitary somatotroph lineage will be mandatory to establish the real role of GIPR in the pathogenesis of somatotropinomas.

  1. Regulatory development of geriatric medicines: To GIP or not to GIP?

    PubMed

    De Spiegeleer, Bart; Wynendaele, Evelien; Bracke, Nathalie; Veryser, Lieselotte; Taevernier, Lien; Degroote, Agnes; Stalmans, Sofie

    2016-05-01

    Geriatric patients represent the main users of medicines, but are historically often minimally included in clinical trials, resulting in a gap in the knowledge of the benefit/risk balance of medicines in this heterogeneous population. As the worldwide population is aging, the need for safe and effective medicines for older patients is proportionally increasing. The aim of this review is to provide an overview of the current regulatory status of the development of geriatric medicines, the encountered challenges and the view of the involved stakeholders, coming to the conclusion whether it is necessary or not to implement a Geriatric Investigation Plan (GIP), by analogy with pediatrics.

  2. Direct effects of glucose, insulin, GLP-1, and GIP on bulbospinal neurons in the rostral ventrolateral medulla in neonatal wistar rats.

    PubMed

    Oshima, Naoki; Onimaru, Hiroshi; Matsubara, Hidehito; Uchida, Takahiro; Watanabe, Atsushi; Imakiire, Toshihiko; Nishida, Yasuhiro; Kumagai, Hiroo

    2017-03-06

    Although patients with diabetes mellitus (DM) often exhibit hypertension, the mechanisms responsible for this correlation are not well known. We hypothesized that the bulbospinal neurons in the rostral ventrolateral medulla (RVLM) are affected by the levels of glucose, insulin, or incretins (glucagon like peptide-1 [GLP-1] or glucose-dependent insulinotropic peptide [GIP]) in patients with DM. To investigate whether RVLM neurons are activated by glucose, insulin, GLP-1, or GIP, we examined changes in the membrane potentials of bulbospinal RVLM neurons using whole-cell patch-clamp technique during superfusion with various levels of glucose or these hormones in neonatal Wistar rats. A brainstem-spinal cord preparation was used for the experiments. A low level of glucose stimulated bulbospinal RVLM neurons. During insulin superfusion, almost all the RVLM neurons were depolarized, while during GLP-1 or GIP superfusion, almost all the RVLM neurons were hyperpolarized. Next, histological examinations were performed to examine transporters for glucose and receptors for insulin, GLP-1, and GIP on RVLM neurons. Low-level glucose-depolarized RVLM neurons exhibited the presence of glucose transporter 3 (GLUT3). Meanwhile, insulin-depolarized, GLP-1-hyperpolarized, and GIP-hyperpolarized RVLM neurons showed each of the respective specific receptor. These results indicate that a low level of glucose stimulates bulbospinal RVLM neurons via specific transporters on these neurons, inducing hypertension. Furthermore, an increase in insulin or a reduction in incretins may also activate the sympathetic nervous system and induce hypertension by activating RVLM neurons via their own receptors.

  3. Loss of Free Fatty Acid Receptor 2 leads to impaired islet mass and beta cell survival

    PubMed Central

    Villa, Stephanie R.; Priyadarshini, Medha; Fuller, Miles H.; Bhardwaj, Tanya; Brodsky, Michael R.; Angueira, Anthony R.; Mosser, Rockann E.; Carboneau, Bethany A.; Tersey, Sarah A.; Mancebo, Helena; Gilchrist, Annette; Mirmira, Raghavendra G.; Gannon, Maureen; Layden, Brian T.

    2016-01-01

    The regulation of pancreatic β cell mass is a critical factor to help maintain normoglycemia during insulin resistance. Nutrient-sensing G protein-coupled receptors (GPCR) contribute to aspects of β cell function, including regulation of β cell mass. Nutrients such as free fatty acids (FFAs) contribute to precise regulation of β cell mass by signaling through cognate GPCRs, and considerable evidence suggests that circulating FFAs promote β cell expansion by direct and indirect mechanisms. Free Fatty Acid Receptor 2 (FFA2) is a β cell-expressed GPCR that is activated by short chain fatty acids, particularly acetate. Recent studies of FFA2 suggest that it may act as a regulator of β cell function. Here, we set out to explore what role FFA2 may play in regulation of β cell mass. Interestingly, Ffar2−/− mice exhibit diminished β cell mass at birth and throughout adulthood, and increased β cell death at adolescent time points, suggesting a role for FFA2 in establishment and maintenance of β cell mass. Additionally, activation of FFA2 with Gαq/11-biased agonists substantially increased β cell proliferation in in vitro and ex vivo proliferation assays. Collectively, these data suggest that FFA2 may be a novel therapeutic target to stimulate β cell growth and proliferation. PMID:27324831

  4. Nkx6.1 regulates islet β-cell proliferation via Nr4a1 and Nr4a3 nuclear receptors.

    PubMed

    Tessem, Jeffery S; Moss, Larry G; Chao, Lily C; Arlotto, Michelle; Lu, Danhong; Jensen, Mette V; Stephens, Samuel B; Tontonoz, Peter; Hohmeier, Hans E; Newgard, Christopher B

    2014-04-08

    Loss of functional β-cell mass is a hallmark of type 1 and type 2 diabetes, and methods for restoring these cells are needed. We have previously reported that overexpression of the homeodomain transcription factor NK6 homeobox 1 (Nkx6.1) in rat pancreatic islets induces β-cell proliferation and enhances glucose-stimulated insulin secretion, but the pathway by which Nkx6.1 activates β-cell expansion has not been defined. Here, we demonstrate that Nkx6.1 induces expression of the nuclear receptor subfamily 4, group A, members 1 and 3 (Nr4a1 and Nr4a3) orphan nuclear receptors, and that these factors are both necessary and sufficient for Nkx6.1-mediated β-cell proliferation. Consistent with this finding, global knockout of Nr4a1 results in a decrease in β-cell area in neonatal and young mice. Overexpression of Nkx6.1 and the Nr4a receptors results in increased expression of key cell cycle inducers E2F transcription factor 1 and cyclin E1. Furthermore, Nkx6.1 and Nr4a receptors induce components of the anaphase-promoting complex, including ubiquitin-conjugating enzyme E2C, resulting in degradation of the cell cycle inhibitor p21. These studies identify a unique bipartite pathway for activation of β-cell proliferation, suggesting several unique targets for expansion of functional β-cell mass.

  5. A new stable GIP-Oxyntomodulin hybrid peptide improved bone strength both at the organ and tissue levels in genetically-inherited type 2 diabetes mellitus.

    PubMed

    Mansur, Sity Aishah; Mieczkowska, Aleksandra; Flatt, Peter R; Bouvard, Beatrice; Chappard, Daniel; Irwin, Nigel; Mabilleau, Guillaume

    2016-06-01

    Obesity and type 2 diabetes mellitus (T2DM) progress worldwide with detrimental effects on several physiological systems including bone tissue mainly by affecting bone quality. Several gut hormones analogues have been proven potent in ameliorating bone quality. In the present study, we used the leptin receptor-deficient db/db mice as a model of obesity and severe T2DM to assess the extent of bone quality alterations at the organ and tissue levels. We also examined the beneficial effects of gut hormone therapy in this model by using a new triple agonist ([d-Ala(2)]GIP-Oxm) active at the GIP, GLP-1 and glucagon receptors. As expected, db/db mice presented with dramatic alterations of bone strength at the organ level associated with deterioration of trabecular and cortical microarchitectures and an augmentation in osteoclast numbers. At the tissue level, these animals presented also with alterations of bone strength (reduced hardness, indentation modulus and dissipated energy) with modifications of tissue mineral distribution, collagen glycation and collagen maturity. The use of [d-Ala(2)]GIP-Oxm considerably improved bone strength at the organ level with modest effects on trabecular microarchitecture. At the tissue level, [d-Ala(2)]GIP-Oxm ameliorated bone strength reductions with positive effects on collagen glycation and collagen maturity. This study provides support for including gut hormone analogues as possible new therapeutic strategies for improving bone quality in bone complications associated to T2DM.

  6. Oral administration of corn zein hydrolysate stimulates GLP-1 and GIP secretion and improves glucose tolerance in male normal rats and Goto-Kakizaki rats.

    PubMed

    Higuchi, Noriyuki; Hira, Tohru; Yamada, Nao; Hara, Hiroshi

    2013-09-01

    We have previously demonstrated that ileal administration of the dietary protein hydrolysate prepared from corn zein (ZeinH) stimulated glucagon-like peptide-1 (GLP-1) secretion and attenuated hyperglycemia in rats. In this study, to examine whether oral administration of ZeinH improves glucose tolerance by stimulating GLP-1 and glucose-dependent insulinotropic polypeptide (GIP) secretion, glucose tolerance tests were performed in normal Sprague-Dawley male rats and diabetic Goto-Kakizaki (GK) male rats. The test solution was gavaged before ip glucose injection in normal rats or gavaged together with glucose in GK rats. Blood samples were collected from the tail vein or by using the jugular catheter to measure glucose, insulin, GLP-1, and GIP levels. In the ip glucose tolerance test, oral administration of ZeinH (2 g/kg) significantly suppressed the glycemic response accompanied by an immediate increase in plasma GLP-1 and GIP levels in normal rats. In contrast, oral administration of another dietary peptide, meat hydrolysate, did not elicit a similar effect. The glucose-lowering effect of ZeinH was attenuated by a GLP-1 receptor antagonist or by a GIP receptor antagonist. Furthermore, oral ZeinH induced GLP-1 secretion and reduced glycemic response in GK rats under the oral glucose tolerance test. These results indicate that the oral administration of the dietary peptide ZeinH improves glucose tolerance in normal and diabetic rats by its incretin-releasing activity, namely, the incretinotropic effect.

  7. High-Fat Diet During Mouse Pregnancy and Lactation Targets GIP-Regulated Metabolic Pathways in Adult Male Offspring.

    PubMed

    Kruse, Michael; Keyhani-Nejad, Farnaz; Isken, Frank; Nitz, Barbara; Kretschmer, Anja; Reischl, Eva; de las Heras Gala, Tonia; Osterhoff, Martin A; Grallert, Harald; Pfeiffer, Andreas F H

    2016-03-01

    Maternal obesity is a worldwide problem associated with increased risk of metabolic diseases in the offspring. Genetic deletion of the gastric inhibitory polypeptide (GIP) receptor (GIPR) prevents high-fat diet (HFD)-induced obesity in mice due to specific changes in energy and fat cell metabolism. We investigated whether GIP-associated pathways may be targeted by fetal programming and mimicked the situation by exposing pregnant mice to control or HFD during pregnancy (intrauterine [IU]) and lactation (L). Male wild-type (WT) and Gipr(-/-) offspring received control chow until 25 weeks of age followed by 20 weeks of HFD. Gipr(-/-) offspring of mice exposed to HFD during IU/L became insulin resistant and obese and exhibited increased adipose tissue inflammation and decreased peripheral tissue substrate utilization after being reintroduced to HFD, similar to WT mice on regular chow during IU/L. They showed decreased hypothalamic insulin sensitivity compared with Gipr(-/-) mice on control diet during IU/L. DNA methylation analysis revealed increased methylation of CpG dinucleotides and differential transcription factor binding of promoter regions of genes involved in lipid oxidation in the muscle of Gipr(-/-) offspring on HFD during IU/L, which were inversely correlated with gene expression levels. Our data identify GIP-regulated metabolic pathways that are targeted by fetal programming.

  8. Pancreatic Islet Transplantation

    MedlinePlus

    ... allo-transplantation?" For each pancreatic islet allo-transplant infusion, researchers use specialized enzymes to remove islets from ... in a lab. Transplant patients typically receive two infusions with an average of 400,000 to 500, ...

  9. The Langerhans islet cells of female rabbits are differentially affected by hypothyroidism depending on the islet size.

    PubMed

    Rodríguez-Castelán, J; Nicolás, L; Morimoto, S; Cuevas, E

    2015-04-01

    Effects of hypothyroidism on the glucose and insulin levels are controversial, and its impact on the Langerhans islet morphology of adult subjects has been poorly addressed. In spite of hypothyroidism and diabetes mellitus are more frequent in females than in males, most studies using animal models have been done in males. The effect of hypothyroidism on the immunolabeling of thyroid hormone receptors (TRs) and thyrotropin receptor (TSHR) of islet cells is unknown. The aim of this study was to determine the effect of hypothyroidism on the glucose and insulin concentrations, morphometry of islets, and immunostaining of TRs α1-2 and β1 and TSHR of islet cells in female rabbits. Control and hypothyroid (0.02% of methimazole for 30 days) animals were used to quantify blood levels of glucose and insulin, density of islets, cross-sectional area (CSA) of islets, number of cells per islet, cell proliferation, and the immunolabeling of TRs α1-2, TRβ1, and TSHR. Student's t or Mann-Whitney-U tests, two-way ANOVAs, and Fischer's tests were applied. Concentrations of glucose and insulin, as well as the insulin resistance were similar between groups. Hypothyroidism did not affect the density or the CSA of islets. The analysis of islets by size showed that hypothyroidism reduced the cell number in large and medium islets, but not in small ones. In small islets, cell proliferation was increased. The immunoreactivity of TRα1-2, TRβ1, and TSHR was increased by hypothyroidism in all islet sizes. Our results show that hypothyroidism affects differentially the islet cells depending on the size of islets.

  10. Receptor-mediated inhibition of adenylate cyclase and stimulation of arachidonic acid release in 3T3 fibroblasts. Selective susceptibility to islet-activating protein, pertussis toxin

    SciTech Connect

    Murayama, T.; Ui, M.

    1985-06-25

    Thrombin exhibited diverse effects on mouse 3T3 fibroblasts. It (a) decreased cAMP in the cell suspension, (b) inhibited adenylate cyclase in the Lubrol-permeabilized cell suspension in a GTP-dependent manner, increased releases of (c) arachidonic acid and (d) inositol from the cell monolayer prelabeled with these labeled compounds, (e) increased /sup 45/Ca/sup 2 +/ uptake into the cell monolayer, and (f) increased /sup 86/Rb/sup +/ uptake into the cell monolayer in a ouabain-sensitive manner. Most of the effects were reproduced by bradykinin, platelet-activating factor, and angiotensin II. The receptors for these agonists are thus likely to be linked to three separate effector systems: the adenylate cyclase inhibition, the phosphoinositide breakdown leading to Ca/sup 2 +/ mobilization and phospholipase A2 activation, and the Na,K-ATPase activation. Among the effects of these agonists, (a), (b), (c), and (e) were abolished, but (d) and (f) were not, by prior treatment of the cells with islet-activating protein (IAP), pertussis toxin, which ADP-ribosylates the Mr = 41,000 protein, the alpha-subunit of the inhibitory guanine nucleotide regulatory protein (Ni), thereby abolishing receptor-mediated inhibition of adenylate cyclase. The effects (a), (c), (d), and (e) of thrombin, but not (b), were mimicked by A23187, a calcium ionophore. The effects of A23187, in contrast to those of receptor agonists, were not affected by the treatment of cells with IAP. Thus, the IAP substrate, the alpha-subunit of Ni, or the protein alike, may play an additional role in signal transduction arising from the Ca/sup 2 +/-mobilizing receptors, probably mediating process(es) distal to phosphoinositide breakdown and proximal to Ca/sup 2 +/ gating.

  11. Islet Cell Transplantation

    MedlinePlus

    ... person who has type 1 diabetes must take insulin daily to live. Transplanted islet cells, however, can take over the work of the destroyed cells. The beta cells in these islets will begin to make and release insulin. Researchers hope islet transplantation will help people with ...

  12. Inhibition by islet-activating protein, pertussis toxin, of P2-purinergic receptor-mediated iodide efflux and phosphoinositide turnover in FRTL-5 cells

    SciTech Connect

    Okajima, F.; Sho, K.; Kondo, Y.

    1988-08-01

    Exposure of FRTL-5 thyroid cells to ATP (1 microM to 1 mM) resulted in the stimulation of I- efflux in association with the induction of inositol trisphosphate production and intracellular Ca2+ mobilization. Nonhydrolyzable ATP derivatives, ADP and GTP, were also as effective in magnitude as ATP, whereas neither AMP nor adenosine exerted significant effect on I- efflux, suggesting a P2-purinergic receptor-mediated activation of I- efflux. Treatment of the cells with the islet-activating protein (IAP) pertussis toxin, which ADP-ribosylated a 41,000 mol wt membrane protein, effectively suppressed the phosphoinositide response to ATP in addition to ATP-dependent I- efflux at agonist concentrations below 10 microM. In contrast, the I- efflux stimulated by TSH, A23187, or phorbol myristate acetate was insusceptible to IAP. The IAP substrate, probably GTP-binding protein, is hence proposed to mediate the activation of P2-purinergic receptor-linked phospholipase-C in FRTL-5 cells. However, the responses to ATP, its nonhydrolyzable derivatives, or ADP at the higher agonist concentrations, especially above 100 microM, were only partially inhibited by IAP, even though the IAP substrate was totally ADP ribosylated by the toxin. The responses to GTP in the whole concentration range tested were not influenced by IAP treatment. Thus, signals arising from the P2-receptor might be transduced to phospholipase-C by two different pathways, i.e. IAP-sensitive and insensitive ones, and result in the stimulation of I- efflux.

  13. Double incretin receptor knockout (DIRKO) mice reveal an essential role for the enteroinsular axis in transducing the glucoregulatory actions of DPP-IV inhibitors.

    PubMed

    Hansotia, Tanya; Baggio, Laurie L; Delmeire, Dominique; Hinke, Simon A; Yamada, Yuichiro; Tsukiyama, Katsushi; Seino, Yutaka; Holst, Jens J; Schuit, Frans; Drucker, D J

    2004-05-01

    Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1) are gut-derived incretins that potentiate glucose clearance following nutrient ingestion. Elimination of incretin receptor action in GIPR(-/-) or GLP-1R(-/-) mice produces only modest impairment in glucose homeostasis, perhaps due to compensatory upregulation of the remaining incretin. We have now studied glucose homeostasis in double incretin receptor knockout (DIRKO) mice. DIRKO mice exhibit normal body weight and fail to exhibit an improved glycemic response after exogenous administration of GIP or the GLP-1R agonist exendin-4. Plasma glucagon and the hypoglycemic response to exogenous insulin were normal in DIRKO mice. Glycemic excursion was abnormally increased and levels of glucose-stimulated insulin secretion were decreased following oral but not intraperitoneal glucose challenge in DIRKO compared with GIPR(-/-) or GLP-1R(-/-) mice. Similarly, glucose-stimulated insulin secretion and the response to forskolin were well preserved in perifused DIRKO islets. Although the dipeptidyl peptidase-IV (DPP-IV) inhibitors valine pyrrolidide (Val-Pyr) and SYR106124 lowered glucose and increased plasma insulin in wild-type and single incretin receptor knockout mice, the glucose-lowering actions of DPP-IV inhibitors were eliminated in DIRKO mice. These findings demonstrate that glucose-stimulated insulin secretion is maintained despite complete absence of both incretin receptors, and they delineate a critical role for incretin receptors as essential downstream targets for the acute glucoregulatory actions of DPP-IV inhibitors.

  14. Neurotransmitters act as paracrine signals to regulate insulin secretion from the human pancreatic islet.

    PubMed

    Rodriguez-Diaz, Rayner; Menegaz, Danusa; Caicedo, Alejandro

    2014-08-15

    In this symposium review we discuss the role of neurotransmitters as paracrine signals that regulate pancreatic islet function. A large number of neurotransmitters and their receptors has been identified in the islet, but relatively little is known about their involvement in islet biology. Interestingly, neurotransmitters initially thought to be present in autonomic axons innervating the islet are also present in endocrine cells of the human islet. These neurotransmitters can thus be released as paracrine signals to help control hormone release. Here we propose that the role of neurotransmitters may extend beyond controlling endocrine cell function to work as signals modulating vascular flow and immune responses within the islet.

  15. Incretin hormone receptors are required for normal beta cell development and function in female mice.

    PubMed

    Omar, Bilal; Ahlkvist, Linda; Yamada, Yuchiro; Seino, Yutaka; Ahrén, Bo

    2016-05-01

    The incretin hormones, glucose dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1), potentiate insulin secretion and are responsible for the majority of insulin secretion that occurs after a meal. They may also, however, have a fundamental role in pancreatic beta cell development and function, independently of their role in potentiating insulin secretion after a meal. This has led to observations that a loss of GIP or GLP-1 action affects normal beta cell function, however each one of the incretin hormones may compensate when the action of the other is lost and therefore the overall impact of the incretin hormones on beta cell function is not known. We therefore utilized a mouse line deficient in both the GLP-1 and GIP receptor genes, the double incretin receptor knockout (DIRKO), to determine the consequences of a lifelong, complete lack of incretin hormone action on beta cell function, in vivo, in intact animals. We found that DIRKO mice displayed impaired glucose tolerance and insulin secretion in response to both oral glucose and mixed meal tolerance tests compared to wild-type mice. Assessment of beta cell function using the hyperglycemic clamp technique revealed an 80% decrease in first phase insulin response in DIRKO mice, but a normal second phase insulin secretion. A similar decline was seen when wild-type mice were given acute intravenous injection of glucose together with the GLP-1 receptor antagonist Ex9-39. Ex vivo assessments of the pancreas revealed significantly fewer islets in the pancreata of DIRKO mice despite no differences in total pancreatic mass. Insulin secretion from isolated islets of DIRKO mice was impaired to a similar extent to that seen during the hyperglycemic clamp. Insulin secretion in wild-type islets was impaired by acute treatment with Ex9-39 to a similar extent as the in vivo intravenous glucose tolerance tests. In conclusion, a loss of the action of both incretin hormones results in direct impairment

  16. CD8+ T cells specific for the islet autoantigen IGRP are restricted in their T cell receptor chain usage

    PubMed Central

    Fuchs, Yannick F.; Eugster, Anne; Dietz, Sevina; Sebelefsky, Christian; Kühn, Denise; Wilhelm, Carmen; Lindner, Annett; Gavrisan, Anita; Knoop, Jan; Dahl, Andreas; Ziegler, Anette-G.; Bonifacio, Ezio

    2017-01-01

    CD8+ T cells directed against beta cell autoantigens are considered relevant for the pathogenesis of type 1 diabetes. Using single cell T cell receptor sequencing of CD8+ T cells specific for the IGRP265-273 epitope, we examined whether there was expansion of clonotypes and sharing of T cell receptor chains in autoreactive CD8+ T cell repertoires. HLA-A*0201 positive type 1 diabetes patients (n = 19) and controls (n = 18) were analysed. TCR α- and β-chain sequences of 418 patient-derived IGRP265-273-multimer+ CD8+ T cells representing 48 clonotypes were obtained. Expanded populations of IGRP265-273-specific CD8+ T cells with dominant clonotypes that had TCR α-chains shared across patients were observed. The SGGSNYKLTF motif corresponding to TRAJ53 was contained in 384 (91.9%) cells, and in 20 (41.7%) patient-derived clonotypes. TRAJ53 together with TRAV29/DV5 was found in 15 (31.3%) clonotypes. Using next generation TCR α-chain sequencing, we found enrichment of one of these TCR α-chains in the memory CD8+ T cells of patients as compared to healthy controls. CD8+ T cell clones bearing the enriched motifs mediated antigen-specific target cell lysis. We provide the first evidence for restriction of T cell receptor motifs in the alpha chain of human CD8+ T cells with specificity to a beta cell antigen. PMID:28300170

  17. Transcriptional Regulation of the Pancreatic Islet: Implications for Islet Function

    PubMed Central

    Stitzel, Michael L.; Kycia, Ina; Kursawe, Romy; Ucar, Duygu

    2015-01-01

    Islets of Langerhans contain multiple hormone-producing endocrine cells controlling glucose homeostasis. Transcription establishes and maintains islet cellular fates and identities. Genetic and environmental disruption of islet transcription triggers cellular dysfunction and disease. Early transcriptional regulation studies of specific islet genes, including insulin (INS) and the transcription factor PDX1, identified the first cis-regulatory DNA sequences and trans-acting factors governing islet function. Here, we review how human islet “omics” studies are reshaping our understanding of transcriptional regulation in islet (dys)function and diabetes. First, we highlight the expansion of islet transcript number, form, and function and of DNA transcriptional regulatory elements controlling their production. Next, we cover islet transcriptional effects of genetic and environmental perturbation. Finally, we discuss how these studies’ emerging insights should empower our diabetes research community to build mechanistic understanding of diabetes pathophysiology and to equip clinicians with tailored, precision medicine options to prevent and treat islet dysfunction and diabetes. PMID:26272056

  18. Conjugated Linoleic Acids Mediate Insulin Release through Islet G Protein-coupled Receptor FFA1/GPR40*

    PubMed Central

    Schmidt, Johannes; Liebscher, Kathrin; Merten, Nicole; Grundmann, Manuel; Mielenz, Manfred; Sauerwein, Helga; Christiansen, Elisabeth; Due-Hansen, Maria E.; Ulven, Trond; Ullrich, Susanne; Gomeza, Jesús; Drewke, Christel; Kostenis, Evi

    2011-01-01

    Among dietary components, conjugated linoleic acids (CLAs) have attracted considerable attention as weight loss supplements in the Western world because they reduce fat stores and increase muscle mass. However, a number of adverse effects are also ascribed to the intake of CLAs such as aggravation of insulin resistance and the risk of developing diabetes. However, the mechanisms accounting for the effects of CLAs on glucose homeostasis are incompletely understood. Herein we provide evidence that CLAs specifically activate the cell surface receptor FFA1, an emerging therapeutic target to treat type 2 diabetes. Using different recombinant cellular systems engineered to stably express FFA1 and a set of diverse functional assays including the novel, label-free non-invasive dynamic mass redistribution technology (Corning® Epic® biosensor), both CLA isomers cis-9, trans-11-CLA and trans-10, cis-12-CLA were found to activate FFA1 in vitro at concentrations sufficient to also account for FFA1 activation in vivo. Each CLA isomer markedly increased glucose-stimulated insulin secretion in insulin-producing INS-1E cells that endogenously express FFA1 and in primary pancreatic β-cells of wild type but not FFA1−/− knock-out mice. Our findings establish a clear mechanistic link between CLAs and insulin production and identify the cell surface receptor FFA1 as a molecular target for CLAs, explaining their acute stimulatory effects on insulin secretion in vivo. CLAs are also revealed as insulinotropic components in widely used nutraceuticals, a finding with significant implication for development of FFA1 modulators to treat type 2 diabetes. PMID:21339298

  19. Islet transplantation: immunological perspectives.

    PubMed

    Inverardi, Luca; Kenyon, Norma S; Ricordi, Camillo

    2003-10-01

    Clinical trials of islet transplantation are showing remarkable success, but they require administration of chronic immunosuppression, and are underscoring the large gap that exists between the number of human donors available and the number of patients that could benefit from the procedure. Recent progress has been made in the definition of key immunological mechanisms that are involved in determining islet transplant outcome. Clinical and preclinical studies, and studies in small animal model systems, will all eventually contribute to the definition of efficient and safe protocols for islet transplantation. If the use of xenografts is successful, it might represent a solution to the shortage of human organs.

  20. Pancreatic islet cell tumor

    MedlinePlus

    ... functions. These include blood sugar level and the production of stomach acid. Tumors that arise from islet ... try and shrink the tumors. If the abnormal production of hormones is causing symptoms, you may receive ...

  1. The GIP gamma-tubulin complex-associated proteins are involved in nuclear architecture in Arabidopsis thaliana

    PubMed Central

    Batzenschlager, Morgane; Masoud, Kinda; Janski, Natacha; Houlné, Guy; Herzog, Etienne; Evrard, Jean-Luc; Baumberger, Nicolas; Erhardt, Mathieu; Nominé, Yves; Kieffer, Bruno; Schmit, Anne-Catherine; Chabouté, Marie-Edith

    2013-01-01

    During interphase, the microtubular cytoskeleton of cycling plant cells is organized in both cortical and perinuclear arrays. Perinuclear microtubules (MTs) are nucleated from γ-Tubulin Complexes (γ-TuCs) located at the surface of the nucleus. The molecular mechanisms of γ-TuC association to the nuclear envelope (NE) are currently unknown. The γ-TuC Protein 3 (GCP3)-Interacting Protein 1 (GIP1) is the smallest γ-TuC component identified so far. AtGIP1 and its homologous protein AtGIP2 participate in the localization of active γ-TuCs at interphasic and mitotic MT nucleation sites. Arabidopsis gip1gip2 mutants are impaired in establishing a fully functional mitotic spindle and exhibit severe developmental defects. In this study, gip1gip2 knock down mutants were further characterized at the cellular level. In addition to defects in both the localization of γ-TuC core proteins and MT fiber robustness, gip1gip2 mutants exhibited a severe alteration of the nuclear shape associated with an abnormal distribution of the nuclear pore complexes. Simultaneously, they showed a misorganization of the inner nuclear membrane protein AtSUN1. Furthermore, AtGIP1 was identified as an interacting partner of AtTSA1 which was detected, like the AtGIP proteins, at the NE. These results provide the first evidence for the involvement of a γ-TuC component in both nuclear shaping and NE organization. Functional hypotheses are discussed in order to propose a model for a GIP-dependent nucleo-cytoplasmic continuum. PMID:24348487

  2. Inflammatory Response in Islet Transplantation

    PubMed Central

    Kanak, Mazhar A.; Kunnathodi, Faisal; Lawrence, Michael C.; Levy, Marlon F.

    2014-01-01

    Islet cell transplantation is a promising beta cell replacement therapy for patients with brittle type 1 diabetes as well as refractory chronic pancreatitis. Despite the vast advancements made in this field, challenges still remain in achieving high frequency and long-term successful transplant outcomes. Here we review recent advances in understanding the role of inflammation in islet transplantation and development of strategies to prevent damage to islets from inflammation. The inflammatory response associated with islets has been recognized as the primary cause of early damage to islets and graft loss after transplantation. Details on cell signaling pathways in islets triggered by cytokines and harmful inflammatory events during pancreas procurement, pancreas preservation, islet isolation, and islet infusion are presented. Robust control of pre- and peritransplant islet inflammation could improve posttransplant islet survival and in turn enhance the benefits of islet cell transplantation for patients who are insulin dependent. We discuss several potent anti-inflammatory strategies that show promise for improving islet engraftment. Further understanding of molecular mechanisms involved in the inflammatory response will provide the basis for developing potent therapeutic strategies for enhancing the quality and success of islet transplantation. PMID:24883060

  3. Application of the SQUG-GIP to the seismic upgrade program of the Savannah River reactors

    SciTech Connect

    Antaki, G.A.

    1991-12-31

    In August 1991, the Savannah River Site (SRS) seismic evaluation program using the Generic Implementation Procedure (GIP) celebrated its third anniversary -- a respectable age for such a new methodology. During these three years, the GIP, developed for the commercial nuclear industry`s Seismic Qualification Utility Group (SQUG), had evolved through Revision 0, Revision 1, Revision 2 and a Revision 2 ``update`` which is currently in the works. This evolution is not surprising for such an important, and in many ways pioneering, document. The various revisions were anticipated at SRS, and the program adjusted accordingly. The verification of seismic adequacy of equipment at the SRS nuclear reactors has been outlined in previous publications. The purpose of this paper is to relate the more practical and managerial aspects of our relatively mature SQUG-GIP implementation program, which will hopefully prove useful to future users of the GIP. This report is divided into four sections, which follow the normal flow of work under GIP: (1) Program Prerequisites (2) Definition of Scope (3) Equipment Evaluations, and (4) Resolution of Outliers.

  4. Application of the SQUG-GIP to the seismic upgrade program of the Savannah River reactors

    SciTech Connect

    Antaki, G.A.

    1991-01-01

    In August 1991, the Savannah River Site (SRS) seismic evaluation program using the Generic Implementation Procedure (GIP) celebrated its third anniversary -- a respectable age for such a new methodology. During these three years, the GIP, developed for the commercial nuclear industry's Seismic Qualification Utility Group (SQUG), had evolved through Revision 0, Revision 1, Revision 2 and a Revision 2 update'' which is currently in the works. This evolution is not surprising for such an important, and in many ways pioneering, document. The various revisions were anticipated at SRS, and the program adjusted accordingly. The verification of seismic adequacy of equipment at the SRS nuclear reactors has been outlined in previous publications. The purpose of this paper is to relate the more practical and managerial aspects of our relatively mature SQUG-GIP implementation program, which will hopefully prove useful to future users of the GIP. This report is divided into four sections, which follow the normal flow of work under GIP: (1) Program Prerequisites (2) Definition of Scope (3) Equipment Evaluations, and (4) Resolution of Outliers.

  5. Automated separation of merged Langerhans islets

    NASA Astrophysics Data System (ADS)

    Švihlík, Jan; Kybic, Jan; Habart, David

    2016-03-01

    This paper deals with separation of merged Langerhans islets in segmentations in order to evaluate correct histogram of islet diameters. A distribution of islet diameters is useful for determining the feasibility of islet transplantation in diabetes. First, the merged islets at training segmentations are manually separated by medical experts. Based on the single islets, the merged islets are identified and the SVM classifier is trained on both classes (merged/single islets). The testing segmentations were over-segmented using watershed transform and the most probable back merging of islets were found using trained SVM classifier. Finally, the optimized segmentation is compared with ground truth segmentation (correctly separated islets).

  6. Glucose-dependent insulinotropic polypeptide receptor knockout mice have altered bone turnover.

    PubMed

    Xie, Ding; Cheng, Hua; Hamrick, Mark; Zhong, Qing; Ding, Ke-Hong; Correa, Daniel; Williams, Sandra; Mulloy, Anthony; Bollag, Wendy; Bollag, Roni J; Runner, Royce R; McPherson, James C; Insogna, Karl; Isales, Carlos M

    2005-12-01

    Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone, which is secreted from endocrine cells in the small intestine after meal ingestion. GIP has been shown to affect osteoblastic function in vitro; however, the in vivo effects of GIP on bone remodeling remain unclear. In the present study, we investigated the role of GIP in modulating bone turnover, by evaluating serum markers of bone turnover, bone density, bone morphology, and changes in biomechanical bone strength over time (one to five months) in GIP receptor knockout mice (GIPR-/- mice). The GIPR-/- mice showed a decreased bone size, lower bone mass, altered bone microarchitecture and biomechanical properties, and altered parameters for bone turnover, especially in bone formation. Moreover, the effects of GIP on bone mass were site-specific and compensatory mechanism developed over time and ameliorated the impact of the loss of GIP signaling on bone mass. Further, GIPR-/- mice had earlier age-related changes than wild-type mice in body composition, including bone mass, lean body mass, and fat percentage. In summary, our results indicate that GIP has an anabolic effect on bone mass and bone quality and suggests that GIP may be a hormonal link between nutrient ingestion and utilization.

  7. Important role of heparan sulfate in postnatal islet growth and insulin secretion

    SciTech Connect

    Takahashi, Iwao; Noguchi, Naoya; Nata, Koji; Yamada, Shuhei; Kaneiwa, Tomoyuki; Mizumoto, Shuji; Ikeda, Takayuki; Sugihara, Kazushi; Asano, Masahide; Yoshikawa, Takeo; Yamauchi, Akiyo; Shervani, Nausheen Jamal; Uruno, Akira; Kato, Ichiro; Unno, Michiaki; Sugahara, Kazuyuki; Takasawa, Shin; and others

    2009-05-22

    Heparan sulfate (HS) binds with several signaling molecules and regulates ligand-receptor interactions, playing an essential role in embryonic development. Here we showed that HS was intensively expressed in pancreatic islet {beta}-cells after 1 week of age in mice. The enzymatic removal of HS in isolated islets resulted in attenuated glucose-induced insulin secretion with a concomitant reduction in gene expression of several key components in the insulin secretion machinery. We further depleted islet HS by inactivating the exostosin tumor-like 3 gene specifically in {beta}-cells. These mice exhibited abnormal islet morphology with reduced {beta}-cell proliferation after 1 week of age and glucose intolerance due to defective insulin secretion. These results demonstrate that islet HS is involved in the regulation of postnatal islet maturation and required to ensure normal insulin secretion.

  8. MGO3 and GIP1 act synergistically for the maintenance of centromeric cohesion.

    PubMed

    Batzenschlager, Morgane; Schmit, Anne-Catherine; Herzog, Etienne; Fuchs, Joerg; Schubert, Veit; Houlné, Guy; Chabouté, Marie-Edith

    2017-01-02

    The control of genomic maintenance during S phase is crucial in eukaryotes. It involves the establishment of sister chromatid cohesion, ensuring faithful chromosome segregation, as well as proper DNA replication and repair to preserve genetic information. In animals, nuclear periphery proteins - including inner nuclear membrane proteins and nuclear pore-associated components - are key factors which regulate DNA integrity. Corresponding functional homologues are not so well known in plants which may have developed specific mechanisms due to their sessile life. We have already characterized the Gamma-tubulin Complex Protein 3-interacting proteins (GIPs) as essential regulators of centromeric cohesion at the nuclear periphery. GIPs were also shown to interact with TSA1, first described as a partner of the epigenetic regulator MGOUN3 (MGO3)/BRUSHY1 (BRU1)/TONSOKU (TSK) involved in genomic maintenance. Here, using genetic analyses, we show that the mgo3gip1 mutants display an impaired and pleiotropic development including fasciation. We also provide evidence for the contribution of both MGO3 and GIP1 to the regulation of centromeric cohesion in Arabidopsis.

  9. Beneficial effects of a N-terminally modified GIP agonist on tissue-level bone material properties.

    PubMed

    Mabilleau, Guillaume; Mieczkowska, Aleksandra; Irwin, Nigel; Simon, Yannick; Audran, Maurice; Flatt, Peter R; Chappard, Daniel

    2014-06-01

    Bone remodeling is under complex regulation from nervous, hormonal and local signals, including gut hormones. Among the gut hormones, a role for the glucose-dependent insulinotropic polypeptide (GIP) has been suggested. However, the rapid degradation of GIP in the bloodstream by the ubiquitous enzyme dipeptidyl peptidase-4 (DPP-4) precludes therapeutic use. To circumvent this problem, a series of N-terminally modified GIP agonists have been developed, with N-AcGIP being the most promising. The aims of the present study were to investigate the effects of N-AcGIP on bone at the micro-level using trabecular and cortical microstructural morphology, and at the tissue-level in rats. Copenhagen rats were randomly assigned into control or N-AcGIP-treated groups and received daily injection for 4 weeks. Bone microstructural morphology was assessed by microCT and dynamic histomorphometry and tissue-level properties by nanoindentation, qBEI and infra-red microscopy. Four week treatment with N-AcGIP did not alter trabecular or cortical microstructural morphology. In addition, no significant modifications of mechanical response and properties at the tissue-level were observed in trabecular bone. However, significant augmentations in maximum load (12%), hardness (14%), indentation modulus (13%) and dissipated energy (16%) were demonstrated in cortical bone. These beneficial modifications of mechanical properties at the tissue-level were associated with increased mineralization (22%) and collagen maturity (13%) of the bone matrix. Taken together, the results support a beneficial role of GIP, and particularly stable analogs such as N-AcGIP, on tissue material properties of bone.

  10. Fetal endocannabinoids orchestrate the organization of pancreatic islet microarchitecture.

    PubMed

    Malenczyk, Katarzyna; Keimpema, Erik; Piscitelli, Fabiana; Calvigioni, Daniela; Björklund, Peyman; Mackie, Kenneth; Di Marzo, Vincenzo; Hökfelt, Tomas G M; Dobrzyn, Agnieszka; Harkany, Tibor

    2015-11-10

    Endocannabinoids are implicated in the control of glucose utilization and energy homeostasis by orchestrating pancreatic hormone release. Moreover, in some cell niches, endocannabinoids regulate cell proliferation, fate determination, and migration. Nevertheless, endocannabinoid contributions to the development of the endocrine pancreas remain unknown. Here, we show that α cells produce the endocannabinoid 2-arachidonoylglycerol (2-AG) in mouse fetuses and human pancreatic islets, which primes the recruitment of β cells by CB1 cannabinoid receptor (CB1R) engagement. Using subtractive pharmacology, we extend these findings to anandamide, a promiscuous endocannabinoid/endovanilloid ligand, which impacts both the determination of islet size by cell proliferation and α/β cell sorting by differential activation of transient receptor potential cation channel subfamily V member 1 (TRPV1) and CB1Rs. Accordingly, genetic disruption of TRPV1 channels increases islet size whereas CB1R knockout augments cellular heterogeneity and favors insulin over glucagon release. Dietary enrichment in ω-3 fatty acids during pregnancy and lactation in mice, which permanently reduces endocannabinoid levels in the offspring, phenocopies CB1R(-/-) islet microstructure and improves coordinated hormone secretion. Overall, our data mechanistically link endocannabinoids to cell proliferation and sorting during pancreatic islet formation, as well as to life-long programming of hormonal determinants of glucose homeostasis.

  11. Fetal endocannabinoids orchestrate the organization of pancreatic islet microarchitecture

    PubMed Central

    Malenczyk, Katarzyna; Keimpema, Erik; Piscitelli, Fabiana; Calvigioni, Daniela; Björklund, Peyman; Mackie, Kenneth; Di Marzo, Vincenzo; Hökfelt, Tomas G. M.; Dobrzyn, Agnieszka; Harkany, Tibor

    2015-01-01

    Endocannabinoids are implicated in the control of glucose utilization and energy homeostasis by orchestrating pancreatic hormone release. Moreover, in some cell niches, endocannabinoids regulate cell proliferation, fate determination, and migration. Nevertheless, endocannabinoid contributions to the development of the endocrine pancreas remain unknown. Here, we show that α cells produce the endocannabinoid 2-arachidonoylglycerol (2-AG) in mouse fetuses and human pancreatic islets, which primes the recruitment of β cells by CB1 cannabinoid receptor (CB1R) engagement. Using subtractive pharmacology, we extend these findings to anandamide, a promiscuous endocannabinoid/endovanilloid ligand, which impacts both the determination of islet size by cell proliferation and α/β cell sorting by differential activation of transient receptor potential cation channel subfamily V member 1 (TRPV1) and CB1Rs. Accordingly, genetic disruption of TRPV1 channels increases islet size whereas CB1R knockout augments cellular heterogeneity and favors insulin over glucagon release. Dietary enrichment in ω-3 fatty acids during pregnancy and lactation in mice, which permanently reduces endocannabinoid levels in the offspring, phenocopies CB1R−/− islet microstructure and improves coordinated hormone secretion. Overall, our data mechanistically link endocannabinoids to cell proliferation and sorting during pancreatic islet formation, as well as to life-long programming of hormonal determinants of glucose homeostasis. PMID:26494286

  12. Structural and pharmacological characterization of novel potent and selective monoclonal antibody antagonists of glucose-dependent insulinotropic polypeptide receptor.

    PubMed

    Ravn, Peter; Madhurantakam, Chaithanya; Kunze, Susan; Matthews, Evelyn; Priest, Claire; O'Brien, Siobhan; Collinson, Andie; Papworth, Monika; Fritsch-Fredin, Maria; Jermutus, Lutz; Benthem, Lambertus; Gruetter, Markus; Jackson, Ronald H

    2013-07-05

    Glucose-dependent insulinotropic polypeptide (GIP) is an endogenous hormonal factor (incretin) that, upon binding to its receptor (GIPr; a class B G-protein-coupled receptor), stimulates insulin secretion by beta cells in the pancreas. There has been a lack of potent inhibitors of the GIPr with prolonged in vivo exposure to support studies on GIP biology. Here we describe the generation of an antagonizing antibody to the GIPr, using phage and ribosome display libraries. Gipg013 is a specific competitive antagonist with equally high potencies to mouse, rat, dog, and human GIP receptors with a Ki of 7 nm for the human GIPr. Gipg013 antagonizes the GIP receptor and inhibits GIP-induced insulin secretion in vitro and in vivo. A crystal structure of Gipg013 Fab in complex with the human GIPr extracellular domain (ECD) shows that the antibody binds through a series of hydrogen bonds from the complementarity-determining regions of Gipg013 Fab to the N-terminal α-helix of GIPr ECD as well as to residues around its highly conserved glucagon receptor subfamily recognition fold. The antibody epitope overlaps with the GIP binding site on the GIPr ECD, ensuring competitive antagonism of the receptor. This well characterized antagonizing antibody to the GIPr will be useful as a tool to further understand the biological roles of GIP.

  13. The Pancreatic Islet Regulome Browser

    PubMed Central

    Mularoni, Loris; Ramos-Rodríguez, Mireia; Pasquali, Lorenzo

    2017-01-01

    The pancreatic islet is a highly specialized tissue embedded in the exocrine pancreas whose primary function is that of controlling glucose homeostasis. Thus, understanding the transcriptional control of islet-cell may help to puzzle out the pathogenesis of glucose metabolism disorders. Integrative computational analyses of transcriptomic and epigenomic data allows predicting genomic coordinates of putative regulatory elements across the genome and, decipher tissue-specific functions of the non-coding genome. We herein present the Islet Regulome Browser, a tool that allows fast access and exploration of pancreatic islet epigenomic and transcriptomic data produced by different labs worldwide. The Islet Regulome Browser is now accessible on the internet or may be installed locally. It allows uploading custom tracks as well as providing interactive access to a wealth of information including Genome-Wide Association Studies (GWAS) variants, different classes of regulatory elements, together with enhancer clusters, stretch-enhancers and transcription factor binding sites in pancreatic progenitors and adult human pancreatic islets. Integration and visualization of such data may allow a deeper understanding of the regulatory networks driving tissue-specific transcription and guide the identification of regulatory variants. We believe that such tool will facilitate the access to pancreatic islet public genomic datasets providing a major boost to functional genomics studies in glucose metabolism related traits including diabetes. PMID:28261261

  14. An engineered macroencapsulation membrane releasing FTY720 to precondition pancreatic islet transplantation.

    PubMed

    Bowers, Daniel T; Olingy, Claire E; Chhabra, Preeti; Langman, Linda; Merrill, Parker H; Linhart, Ritu S; Tanes, Michael L; Lin, Dan; Brayman, Kenneth L; Botchwey, Edward A

    2017-02-27

    Macroencapsulation is a powerful approach to increase the efficiency of extrahepatic pancreatic islet transplant. FTY720, a small molecule that activates signaling through sphingosine-1-phosphate receptors, is immunomodulatory and pro-angiogenic upon sustained delivery from biomaterials. While FTY720 (fingolimod, Gilenya) has been explored for organ transplantation, in the present work the effect of locally released FTY720 from novel nanofiber-based macroencapsulation membranes is explored for islet transplantation. We screened islet viability during culture with FTY720 and various biodegradable polymers. Islet viability is significantly reduced by the addition of high doses (≥500 ng/mL) of soluble FTY720. Among the polymers screened, islets have the highest viability when cultured with poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). Therefore, PHBV was blended with polycaprolactone (PCL) for mechanical stability and electrospun into nanofibers. Islets had no detectable function ex vivo following 5 days or 12 h of subcutaneous implantation within our engineered device. Subsequently, we explored a preconditioning scheme in which islets are transplanted 2 weeks after FTY720-loaded nanofibers are implanted. This allows FTY720 to orchestrate a local regenerative milieu while preventing premature transplantation into avascular sites that contain high concentrations of FTY720. These results provide a foundation and motivation for further investigation into the use of FTY720 in preconditioning sites for efficacious islet transplantation. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2017.

  15. Angiopoetin-2 Signals Do Not Mediate the Hypervascularization of Islets in Type 2 Diabetes

    PubMed Central

    Shah, Payal; Lueschen, Navina; Ardestani, Amin; Oberholzer, Jose; Olerud, Johan; Carlsson, Per-Ola; Maedler, Kathrin

    2016-01-01

    Aims Changes in the islet vasculature have been implicated in the regulation of β-cell survival and function during the progression to type 2 diabetes (T2D). Failure of the β-cell to compensate for the increased insulin demand in obesity eventually leads to diabetes; as a result of the complex interplay of genetic and environmental factors (e.g. ongoing inflammation within the islets) and impaired vascular function. The Angiopoietin/Tie (Ang/Tie) angiogenic system maintains vasculature and is closely related to organ inflammation and angiogenesis. In this study we aimed to identify whether the vessel area within the islets changes in diabetes and whether such changes would be triggered by the Tie-antagonist Ang-2. Methods Immunohistochemical and qPCR analyses to follow islet vascularization and Ang/Tie levels were performed in human pancreatic autopsies and isolated human and mouse islets. The effect of Ang-2 was assessed in β-cell-specific Ang-2 overexpressing mice during high fat diet (HFD) feeding. Results Islet vessel area was increased in autopsy pancreases from patients with T2D. The vessel markers Tie-1, Tie-2 and CD31 were upregulated in mouse islets upon HFD feeding from 8 to 24 weeks. Ang-2 was transiently upregulated in mouse islets at 8 weeks of HFD and under glucolipotoxic conditions (22.2 mM glucose/ 0.5 mM palmitate) in vitro in human and mouse islets, in contrast to its downregulation by cytokines (IL-1β, IFN-ɣ and TNF-α). Ang-1 on the other hand was oppositely regulated, with a significant loss under glucolipotoxic condition, a trend to reduce in islets from patients with T2D and an upregulation by cytokines. Modulation of such changes in Ang-2 by its overexpression or the inhibition of its receptor Tie-2 impaired β-cell function at basal conditions but protected islets from cytokine induced apoptosis. In vivo, β-cell-specific Ang-2 overexpression in mice induced hypervascularization under normal diet but contrastingly led to

  16. Pancreatic islet blood flow and its measurement.

    PubMed

    Jansson, Leif; Barbu, Andreea; Bodin, Birgitta; Drott, Carl Johan; Espes, Daniel; Gao, Xiang; Grapensparr, Liza; Källskog, Örjan; Lau, Joey; Liljebäck, Hanna; Palm, Fredrik; Quach, My; Sandberg, Monica; Strömberg, Victoria; Ullsten, Sara; Carlsson, Per-Ola

    2016-05-01

    Pancreatic islets are richly vascularized, and islet blood vessels are uniquely adapted to maintain and support the internal milieu of the islets favoring normal endocrine function. Islet blood flow is normally very high compared with that to the exocrine pancreas and is autonomously regulated through complex interactions between the nervous system, metabolites from insulin secreting β-cells, endothelium-derived mediators, and hormones. The islet blood flow is normally coupled to the needs for insulin release and is usually disturbed during glucose intolerance and overt diabetes. The present review provides a brief background on islet vascular function and especially focuses on available techniques to measure islet blood perfusion. The gold standard for islet blood flow measurements in experimental animals is the microsphere technique, and its advantages and disadvantages will be discussed. In humans there are still no methods to measure islet blood flow selectively, but new developments in radiological techniques hold great hopes for the future.

  17. Pancreatic islet blood flow and its measurement

    PubMed Central

    Jansson, Leif; Barbu, Andreea; Bodin, Birgitta; Drott, Carl Johan; Espes, Daniel; Gao, Xiang; Grapensparr, Liza; Källskog, Örjan; Lau, Joey; Liljebäck, Hanna; Palm, Fredrik; Quach, My; Sandberg, Monica; Strömberg, Victoria; Ullsten, Sara; Carlsson, Per-Ola

    2016-01-01

    Pancreatic islets are richly vascularized, and islet blood vessels are uniquely adapted to maintain and support the internal milieu of the islets favoring normal endocrine function. Islet blood flow is normally very high compared with that to the exocrine pancreas and is autonomously regulated through complex interactions between the nervous system, metabolites from insulin secreting β-cells, endothelium-derived mediators, and hormones. The islet blood flow is normally coupled to the needs for insulin release and is usually disturbed during glucose intolerance and overt diabetes. The present review provides a brief background on islet vascular function and especially focuses on available techniques to measure islet blood perfusion. The gold standard for islet blood flow measurements in experimental animals is the microsphere technique, and its advantages and disadvantages will be discussed. In humans there are still no methods to measure islet blood flow selectively, but new developments in radiological techniques hold great hopes for the future. PMID:27124642

  18. Unraveling pancreatic islet biology by quantitative proteomics

    SciTech Connect

    Zhou, Jianying; Dann, Geoffrey P.; Liew, Chong W.; Smith, Richard D.; Kulkarni, Rohit N.; Qian, Weijun

    2011-08-01

    The pancreatic islets of Langerhans play a critical role in maintaining blood glucose homeostasis by secreting insulin and several other important peptide hormones. Impaired insulin secretion due to islet dysfunction is linked to the pathogenesis underlying both Type 1 and Type 2 diabetes. Over the past 5 years, emerging proteomic technologies have been applied to dissect the signaling pathways that regulate islet functions and gain an understanding of the mechanisms of islet dysfunction relevant to diabetes. Herein, we briefly review some of the recent quantitative proteomic studies involving pancreatic islets geared towards gaining a better understanding of islet biology relevant to metabolic diseases.

  19. OB-Rb gene transfer to leptin-resistant islets reverses diabetogenic phenotype

    PubMed Central

    Wang, May-Yun; Koyama, Kazunori; Shimabukuro, Michio; Newgard, Christopher B.; Unger, Roger H.

    1998-01-01

    In obese Zucker diabetic fatty (ZDF) rats with mutant leptin receptors, pancreatic islets have an ≈50-fold increase in fat (TG), overproduce nitric oxide (NO), and lack a normal proinsulin mRNA response to fatty acids. We overexpressed the wild-type full-length “b” isoform of the leptin receptor (OB-Rb) in ZDF islets by perfusing ZDF pancreata with recombinant adenovirus containing the cDNA encoding OB-Rb. In cultured islets isolated from these animals, leptin lowered islet TG by 87% and completely blocked TG formation from free fatty acids. Overproduction of NO was reduced, and the preproinsulin mRNA response to free fatty acids was restored. This establishes defective leptin action as the proximate cause of lipotoxic diabetes in ZDF rats. PMID:9435258

  20. Glucose-dependent insulinotropic polypeptide confers early phase insulin release to oral glucose in rats: demonstration by a receptor antagonist.

    PubMed

    Lewis, J T; Dayanandan, B; Habener, J F; Kieffer, T J

    2000-10-01

    A novel GIP receptor antagonist was developed to evaluate the acute role of glucose-dependent insulinotropic polypeptide (GIP) in the insulin response to oral glucose in rats. Antisera to an extracellular epitope of the GIP receptor (GIPR) detected immunoreactive GIPR on rat pancreatic beta-cells. Purified GIPR antibody (GIPR Ab) specifically displaced GIP binding to the receptor and blocked GIP-mediated increases in intracellular cAMP. When delivered to rats by ip injection, GIPR Ab had a half-life of approximately 4 days. Treatment with GIPR Ab (1 microg/g BW) blocked the potentiation of glucose-stimulated insulin secretion by GIP (60 pmol) but not glucagon-like peptide-1 (GLP-1, 60 pmol) in anesthetized rats. The insulin response to oral glucose was delayed in conscious unrestrained rats that were pretreated with GIPR Ab. Plasma insulin levels were approximately 35% lower at 10 min in GIPR Ab treated animals compared with controls. As a result, the glucose excursion was greater in the GIPR Ab treated group. Fasting plasma glucose levels were not altered by GIPR Ab. We conclude that release of GIP following oral glucose may act as an anticipatory signal to pancreatic beta-cells to promote rapid release of insulin for glucose disposal.

  1. Oxygenation of the Intraportally Transplanted Pancreatic Islet

    PubMed Central

    2016-01-01

    Intraportal islet transplantation (IT) is not widely utilized as a treatment for type 1 diabetes. Oxygenation of the intraportally transplanted islet has not been studied extensively. We present a diffusion-reaction model that predicts the presence of an anoxic core and a larger partly functional core within intraportally transplanted islets. Four variables were studied: islet diameter, islet fractional viability, external oxygen partial pressure (P) (in surrounding portal blood), and presence or absence of a thrombus on the islet surface. Results indicate that an islet with average size and fractional viability exhibits an anoxic volume fraction (AVF) of 14% and a function loss of 72% at a low external P. Thrombus formation increased AVF to 30% and function loss to 92%, suggesting that the effect of thrombosis may be substantial. External P and islet diameter accounted for the greatest overall impact on AVF and loss of function. At our institutions, large human alloislets (>200 μm diameter) account for ~20% of total islet number but ~70% of total islet volume; since most of the total transplanted islet volume is accounted for by large islets, most of the intraportal islet cells are likely to be anoxic and not fully functional. PMID:27872862

  2. Impact of islet size on pancreatic islet transplantation and potential interventions to improve outcome.

    PubMed

    Zorzi, Daria; Phan, Tammy; Sequi, Marco; Lin, Yong; Freeman, Daniel H; Cicalese, Luca; Rastellini, Cristiana

    2015-01-01

    Better results have been recently reported in clinical pancreatic islet transplantation (ITX) due mostly to improved isolation techniques and immunosuppression; however, some limitations still exist. It is known that following transplantation, 30% to 60% of the islets are lost. In our study, we have investigated 1) the role of size as a factor affecting islet engraftment and 2) potential procedural manipulations to increase the number of smaller functional islets that can be transplanted. C57/BL10 mice were used as donors and recipients in a syngeneic islet transplant model. Isolated islets were divided by size (large, >300 μm; medium 150-300 μm; small, <150 μm). Each size was transplanted in chemically induced diabetic mice as full (600 IEQ), suboptimal (400 IEQ), and marginal mass (200 IEQ). Control animals received all size islets. Engraftment was defined as reversal of diabetes by day 7 posttransplantation. When the superiority of smaller islets was observed, strategies of overdigestion and fragmentation were adopted during islet isolation in the attempt to reduce islet size and improve engraftment. Smaller islets were significantly superior in engraftment compared to medium, large, and control (all sizes) groups. This was more evident when marginal mass data were compared. In all masses, success decreased as islet size increased. Once islets were engrafted, functionality was not affected by size. When larger islets were fragmented, a significant decrease in islet functionality was observed. On the contrary, if pancreata were slightly overdigested, although not as successful as small naive islets, an increase in engraftment was observed when compared to the control group. In conclusion, smaller islets are superior in engraftment following islet transplantation. Fragmentation has a deleterious effect on islet engraftment. Islet isolations can be performed by reducing islet size with slight overdigestion, and it can be safely adopted to improve clinical

  3. Targeting pancreatic islets with phage display assisted by laser pressure catapult microdissection.

    PubMed

    Yao, Virginia J; Ozawa, Michael G; Trepel, Martin; Arap, Wadih; McDonald, Donald M; Pasqualini, Renata

    2005-02-01

    Heterogeneity of the microvasculature in different organs has been well documented by multiple methods including in vivo phage display. However, less is known about the diversity of blood vessels within functionally distinct regions of organs. Here, we combined in vivo phage display with laser pressure catapult microdissection to identify peptide ligands for vascular receptors in the islets of Langerhans in the murine pancreas. Protein database analyses of the peptides, CVSNPRWKC and CHVLWSTRC, showed sequence identity to two ephrin A-type ligand homologues, A2 and A4. Confocal microscopy confirmed that most immunoreactivity of CVSNPRWKC and CHVLWSTRC phage was associated with blood vessels in pancreatic islets. Antibodies recognizing EphA4, a receptor for ephrin-A ligands, were similarly associated with islet blood vessels. Importantly, binding of both islet-homing phage and anti-EphA4 antibody was strikingly increased in blood vessels of pancreatic islet tumors in RIP-Tag2 transgenic mice. These results indicate that endothelial cells of blood vessels in pancreatic islets preferentially express EphA4 receptors, and this expression is increased in tumors. Our findings show in vivo phage display and laser pressure catapult microdissection can be combined to reveal endothelial cell specialization within focal regions of the microvasculature.

  4. Fibroblast populated collagen matrix promotes islet survival and reduces the number of islets required for diabetes reversal.

    PubMed

    Jalili, Reza B; Moeen Rezakhanlou, Alireza; Hosseini-Tabatabaei, Azadeh; Ao, Ziliang; Warnock, Garth L; Ghahary, Aziz

    2011-07-01

    Islet transplantation represents a viable treatment for type 1 diabetes. However, due to loss of substantial mass of islets early after transplantation, islets from two or more donors are required to achieve insulin independence. Islet-extracellular matrix disengagement, which occurs during islet isolation process, leads to subsequent islet cell apoptosis and is an important contributing factor to early islet loss. In this study, we developed a fibroblast populated collagen matrix (FPCM) as a novel scaffold to improve islet cell viability and function post-transplantation. FPCM was developed by embedding fibroblasts within type-I collagen and used as scaffold for islet grafts. Viability and insulin secretory function of islets embedded within FPCM was evaluated in vitro and in a syngeneic murine islet transplantation model. Islets embedded within acellular matrix or naked islets were used as control. Islet cell survival and function was markedly improved particularly after embedding within FPCM. The composite scaffold significantly promoted islet isograft survival and reduced the critical islet mass required for diabetes reversal by half (from 200 to 100 islets per recipient). Fibroblast embedded within FPCM produced fibronectin and growth factors and induced islet cell proliferation. No evidence of fibroblast over-growth within composite grafts was noticed. These results confirm that FPCM significantly promotes islet viability and functionality, enhances engraftment of islet grafts and decreases the critical islet mass needed to reverse hyperglycemia. This promising finding offers a new approach to reducing the number of islet donors per recipient and improving islet transplant outcome.

  5. Chronic reduction of insulin receptors in the ventromedial hypothalamus produces glucose intolerance and islet dysfunction in the absence of weight gain.

    PubMed

    Paranjape, Sachin A; Chan, Owen; Zhu, Wanling; Horblitt, Adam M; Grillo, Claudia A; Wilson, Steven; Reagan, Lawrence; Sherwin, Robert S

    2011-11-01

    Insulin is believed to regulate glucose homeostasis mainly via direct effects on the liver, muscle, and adipose tissues. The contribution of insulin's central nervous system effects to disorders of glucose metabolism has received less attention. To evaluate whether postnatal reduction of insulin receptors (IRs) within the ventromedial hypothalamus (VMH), a brain region critical for glucose sensing, contributes to disorders of peripheral glucose metabolism, we microinjected a lentiviral vector expressing an antisense sequence to knockdown IRs or a control lentiviral vector into the VMH of nonobese nondiabetic rats. After 3-4 mo, we assessed 1) glucose tolerance, 2) hepatic insulin sensitivity, and 3) insulin and glucagon secretion, using the glucose clamp technique. Knockdown of IRs locally in the VMH caused glucose intolerance without altering body weight. Increments of plasma insulin during a euglycemic clamp study failed to suppress endogenous glucose production and produced a paradoxical rise in plasma glucagon in the VMH-IR knockdown rats. Unexpectedly, these animals also displayed a 40% reduction (P < 0.05) in insulin secretion in response to an identical hyperglycemic stimulus (∼220 mg/dl). Our data demonstrate that chronic suppression of VMH-IR gene expression is sufficient to impair glucose metabolism as well as α-cell and β-cell function in nondiabetic, nonobese rats. These data suggest that insulin resistance within the VMH may be a significant contributor to the development of type 2 diabetes.

  6. Anx7 is required for nutritional control of gene expression in mouse pancreatic islets of Langerhans.

    PubMed Central

    Srivastava, Meera; Eidelman, Ofer; Leighton, Ximena; Glasman, Mirta; Goping, Gertrude; Pollard, Harvey B.

    2002-01-01

    BACKGROUND: Gene expression in islets of Langerhans is profoundly sensitive to glucose and other nutrients. Islets of Langerhans in the Anx7(+/-) knockout mouse exhibit a profound reduction in ITPR3 protein expression, defective intracellular calcium signaling, and defective insulin secretion. Additional data presented here also show that mRNA for ITPR3 is virtually undetectable in isolated Anx7(+/-) islets. IP3Receptor type 3 (ITPR3) expression in islets of Langerhans is closely regulated by secretory stimuli, and it has been suggested that the level of the ITPR3 expression controls the ability of the islets to respond to nutritional signals. We report that although control islets respond to glucose in vitro by a transient increment in ITPR3 mRNA, the islets from the Anx7(+/-) mouse remain low. We therefore hypothesized that the Anx7/IP3 Receptor(3)/Ca(2+) signaling pathway plays a role in beta cell responses to glucose, and that in the absence of the Anx7/ITPR3 signaling system, the islets would be unable to discriminate between fed or fasted states in vivo. MATERIALS AND METHODS: To test this hypothesis, we subjected Anx7(+/-) and control mice to either food and water ad libidum or to an overnight fast with access to water only. We then isolated the respective islets and compared nutrient-dependent changes in global gene expression under the four conditions using genome-based microarray technology. RESULTS: Anx7 protein expression in these islets is only about 50% of control levels in normal littermate controls, and IPTR3 message and protein are virtually zero. cDNA microarray analyses show that in control animals gene expression is significantly affected by the fasting state. Many of the affected genes have historical relevance to development and differentiation of islets. These include preproglucagon, APOJ, cadherin2, phosphoglucoisomerase, oncostatin M, PAX6, HGF, and cytokeratin 18. However, there are also many other nutritionally sensitive genes in control

  7. In vitro reconstitution of pancreatic islets.

    PubMed

    Kojima, Nobuhiko

    2014-01-01

    The lack of transplantable pancreatic islets is a serious problem that affects the treatment of patients with type 1 diabetes mellitus. Beta cells can be induced from various sources of stem or progenitor cells, including induced pluripotent stem cells in the near future; however, the reconstitution of islets from β cells in culture dishes is challenging. The generation of highly functional islets may require three-dimensional spherical cultures that resemble intact islets. This review discusses recent advances in the reconstitution of islets. Several factors affect the reconstitution of pseudoislets with higher functions, such as architectural similarity, cell-to-cell contact, and the production method. The actual transplantation of naked or encapsulated pseudoislets and islet-like cell clusters from various stem cell sources is also discussed. Advancing our understanding of the methods used to reconstitute pseudoislets should expand the range of potential strategies available for developing de novo islets for therapeutic applications.

  8. Classification of microscopy images of Langerhans islets

    NASA Astrophysics Data System (ADS)

    Å vihlík, Jan; Kybic, Jan; Habart, David; Berková, Zuzana; Girman, Peter; Kříž, Jan; Zacharovová, Klára

    2014-03-01

    Evaluation of images of Langerhans islets is a crucial procedure for planning an islet transplantation, which is a promising diabetes treatment. This paper deals with segmentation of microscopy images of Langerhans islets and evaluation of islet parameters such as area, diameter, or volume (IE). For all the available images, the ground truth and the islet parameters were independently evaluated by four medical experts. We use a pixelwise linear classifier (perceptron algorithm) and SVM (support vector machine) for image segmentation. The volume is estimated based on circle or ellipse fitting to individual islets. The segmentations were compared with the corresponding ground truth. Quantitative islet parameters were also evaluated and compared with parameters given by medical experts. We can conclude that accuracy of the presented fully automatic algorithm is fully comparable with medical experts.

  9. Human pancreatic islets develop through fusion of distinct β and α/δ islets.

    PubMed

    Lee, Inchul

    2016-10-01

    Human pancreatic islets show unique architecture in which α and δ cells are mostly at the peripheral and perivascular areas. It has remained unknown how such prototype is realized in every islet. Here, I report that fetal islets develop first in two distinct types consisting of β or α/δ cells, respectively. The α/δ islets are variable in shape, composed of α and δ cells evenly intermixed. They are vascularized better but encapsulated poorer than β islets in general. During the development, the β and α/δ islets adjoin and fuse with each other in such a way that α and δ cells form a crescent on β cells and, then, progress to encompass and encroach into β cells. Most mature-form islets appear to develop through the fusion. Islets at various stages of fusion are present concurrently until late gestation, suggesting that the islet fusion is an ongoing developmental process. The α/δ islets appear to play a primary role for the process, approaching toward the fusion partner actively. Direct connection is present between the α/δ islets and neural ganglia undergoing active neurogenesis, suggesting an organ-wide neuroendocrine network development. The fusion of precursor islets appears to be a principle of human pancreatic development providing the prototype of mature islets. The complex development might be a reference for in vitro reproduction of biologically competent islets.

  10. Combinatorial human progenitor cell transplantation optimizes islet regeneration through secretion of paracrine factors.

    PubMed

    Bell, Gillian I; Meschino, Michael T; Hughes-Large, Jennifer M; Broughton, Heather C; Xenocostas, Anargyros; Hess, David A

    2012-07-20

    Transplanted human bone marrow (BM) and umbilical cord blood (UCB) progenitor cells activate islet-regenerative or revascularization programs depending on the progenitor subtypes administered. Using purification of multiple progenitor subtypes based on a conserved stem cell function, high aldehyde dehydrogenase (ALDH) activity (ALDH(hi)), we have recently shown that transplantation of BM-derived ALDH(hi) progenitors improved systemic hyperglycemia and augmented insulin secretion by increasing islet-associated proliferation and vascularization, without increasing islet number. Conversely, transplantation of culture-expanded multipotent-stromal cells (MSCs) derived from BM ALDH(hi) cells augmented total beta cell mass via formation of beta cell clusters associated with the ductal epithelium, without sustained islet vascularization. To identify paracrine effectors produced by islet-regenerative MSCs, culture-expanded BM ALDH(hi) MSCs were transplanted into streptozotocin-treated nonobese diabetic/severe combine immune deficient (SCID) mice and segregated into islet-regenerative versus nonregenerative cohorts based on hyperglycemia reduction, and subsequently compared for differential production of mRNA and secreted proteins. Regenerative MSCs showed increased expression of matrix metalloproteases, epidermal growth factor receptor (EGFR)-activating ligands, and downstream effectors of Wnt signaling. Regenerative MSC supernatant also contained increased levels of pro-angiogenic versus pro-inflammatory cytokines, and augmented the expansion of ductal epithelial but not beta cells in vitro. Conversely, co-culture with UCB ALDH(hi) cells induced beta cell but not ductal epithelial cell proliferation. Sequential transplantation of MSCs followed by UCB ALDH(hi) cells improved hyperglycemia and glucose tolerance by increasing beta cell mass associated with the ductal epithelium and by augmenting intra-islet capillary densities. Thus, combinatorial human progenitor cell

  11. Glucose-Dependent Insulinotropic Polypeptide Receptor Deficiency Leads to Impaired Bone Marrow Hematopoiesis.

    PubMed

    Mantelmacher, Fernanda Dana; Fishman, Sigal; Cohen, Keren; Pasmanik Chor, Metsada; Yamada, Yuichiro; Zvibel, Isabel; Varol, Chen

    2017-04-15

    The bone marrow (BM) contains controlled specialized microenvironments, or niches, that regulate the quiescence, proliferation, and differentiation of hematopoietic stem and progenitor cells (HSPC). The glucose-dependent insulinotropic polypeptide (GIP) is a gut-derived incretin hormone that mediates postprandial insulin secretion and has anabolic effects on adipose tissue. Previous studies demonstrated altered bone microarchitecture in mice deficient for GIP receptor (Gipr(-/-) ), as well as the expression of high-affinity GIP receptor by distinct cells constructing the BM HSPC niche. Nevertheless, the involvement of GIP in the process of BM hematopoiesis remains elusive. In this article, we show significantly reduced representation and proliferation of HSPC and myeloid progenitors in the BM of Gipr(-/-) mice. This was further manifested by reduced levels of BM and circulating differentiated immune cells in young and old adult mice. Moreover, GIP signaling was required for the establishment of supportive BM HSPC niches during HSPC repopulation in radioablated BM chimera mice. Finally, molecular profiling of various factors involved in retention, survival, and expansion of HSPC revealed significantly lower expression of the Notch-receptor ligands Jagged 1 and Jagged 2 in osteoblast-enriched bone extracts from Gipr(-/-) mice, which are important for HSPC expansion. In addition, there was increased expression of CXCL12, a factor important for HSPC retention and quiescence, in whole-BM extracts from Gipr(-/-) mice. Collectively, our data suggest that the metabolic hormone GIP plays an important role in BM hematopoiesis.

  12. Factors controlling pancreatic islet neogenesis.

    PubMed Central

    Vinik, A.; Pittenger, G.; Rafaeloff, R.; Rosenberg, L.

    1992-01-01

    We have established a model in which cellophane wrapping induces reiteration of the normal ontogeny of beta-cell differentiation from ductal tissue. The secretion of insulin is physiologic and coordinated to the needs of the animal. Streptozotocin-induced diabetes in hamsters can be "cured" at least half the time. There appears to be activation of growth factor(s) within the pancreas, acting in an autocrine, paracrine, or juxtacrine manner to induce ductal cell proliferation and differentiation into functioning beta cells. Given the results of our studies to date, it does not seem premature to envisage new approaches to the treatment of diabetes mellitus. Identification of the factor(s) regulating islet-cell proliferation and differentiation in our model may permit islets to be grown in culture. This concept could be extended to induce endocrine cell differentiation in vitro as well. Furthermore, islet-cell growth factors could be used to provide "trophic support" to islet transplants as a means of maintaining graft viability. There may also be greater scope for gene therapy when the growth factor(s) have been isolated, purified, sequenced, and cloned. Images FIG. 2 FIG. 5 FIG. 6 FIG. 9 PMID:1364089

  13. Feasibility of islet magnetic resonance imaging using ferumoxytol in intraportal islet transplantation.

    PubMed

    Jin, Sang-Man; Oh, Seung-Hoon; Oh, Bae Jun; Shim, Wooyoung; Choi, Jin Myung; Yoo, Dongkyeom; Hwang, Yong Hwa; Lee, Jung Hee; Lee, Dong Yun; Kim, Jae Hyeon

    2015-06-01

    There is a clinical need for an alternative labeling agent for magnetic resonance imaging (MRI) in islet transplantation. We aimed to evaluate the feasibility of islet MRI using ferumoxytol, which is the only clinically-available ultrasmall superparamagnetic iron oxide. We compared islet function and viability of control islets and islets labeled with ferumoxytol and/or a heparin-protamine complex (HPF). Efficacy of ferumoxytol labeling was assessed in both ex vivo and in vivo models. Labeling for 48 h with HPF, but not up to 800 μg/mL ferumoxytol, deranged ex vivo islet viability and function. The T2∗ relaxation time was optimal when islets were labeled with 800 μg/mL of ferumoxytol for 48 h. Prussian blue stain, iron content assay, transmission electron microscopy (TEM) supported internalization of ferumoxytol particles. However, the labeling intensity in the ex vivo MRI of islets labeled with ferumoxytol was much weaker than that of islets labeled with ferucarbotran. In syngeneic intraportal islet transplantation, there was a correlation between the total area of visualized islets and the transplanted islet mass. In conclusion, islet MRI using ferumoxytol was feasible in terms of in vitro and in vivo efficacy and safety. However, the weak labeling efficacy is still a hurdle for the clinical application.

  14. Melatonin and Pancreatic Islets: Interrelationships between Melatonin, Insulin and Glucagon

    PubMed Central

    Peschke, Elmar; Bähr, Ina; Mühlbauer, Eckhard

    2013-01-01

    The pineal hormone melatonin exerts its influence in the periphery through activation of two specific trans-membrane receptors: MT1 and MT2. Both isoforms are expressed in the islet of Langerhans and are involved in the modulation of insulin secretion from β-cells and in glucagon secretion from α-cells. De-synchrony of receptor signaling may lead to the development of type 2 diabetes. This notion has recently been supported by genome-wide association studies identifying particularly the MT2 as a risk factor for this rapidly spreading metabolic disturbance. Since melatonin is secreted in a clearly diurnal fashion, it is safe to assume that it also has a diurnal impact on the blood-glucose-regulating function of the islet. This factor has hitherto been underestimated; the disruption of diurnal signaling within the islet may be one of the most important mechanisms leading to metabolic disturbances. The study of melatonin–insulin interactions in diabetic rat models has revealed an inverse relationship: an increase in melatonin levels leads to a down-regulation of insulin secretion and vice versa. Elucidation of the possible inverse interrelationship in man may open new avenues in the therapy of diabetes. PMID:23535335

  15. Melatonin and pancreatic islets: interrelationships between melatonin, insulin and glucagon.

    PubMed

    Peschke, Elmar; Bähr, Ina; Mühlbauer, Eckhard

    2013-03-27

    The pineal hormone melatonin exerts its influence in the periphery through activation of two specific trans-membrane receptors: MT1 and MT2. Both isoforms are expressed in the islet of Langerhans and are involved in the modulation of insulin secretion from β-cells and in glucagon secretion from α-cells. De-synchrony of receptor signaling may lead to the development of type 2 diabetes. This notion has recently been supported by genome-wide association studies identifying particularly the MT2 as a risk factor for this rapidly spreading metabolic disturbance. Since melatonin is secreted in a clearly diurnal fashion, it is safe to assume that it also has a diurnal impact on the blood-glucose-regulating function of the islet. This factor has hitherto been underestimated; the disruption of diurnal signaling within the islet may be one of the most important mechanisms leading to metabolic disturbances. The study of melatonin-insulin interactions in diabetic rat models has revealed an inverse relationship: an increase in melatonin levels leads to a down-regulation of insulin secretion and vice versa. Elucidation of the possible inverse interrelationship in man may open new avenues in the therapy of diabetes.

  16. The role of endothelial cells on islet function and revascularization after islet transplantation

    PubMed Central

    Del Toro-Arreola, Alicia; Robles-Murillo, Ana Karina; Daneri-Navarro, Adrian; Rivas-Carrillo, Jorge David

    2016-01-01

    ABSTRACT Islet transplantation has become a widely accepted therapeutic option for selected patients with type 1 diabetes mellitus. However, in order to achieve insulin independence a great number of islets are often pooled from 2 to 4 pancreata donors. Mostly, it is due to the massive loss of islets immediately after transplant. The endothelium plays a key role in the function of native islets and during the revascularization process after islet transplantation. However, if a delayed revascularization occurs, even the remaining islets will also undergo to cell death and late graft dysfunction. Therefore, it is essential to understand how the signals are released from endothelial cells, which might regulate both differentiation of pancreatic progenitors and thereby maintenance of the graft function. New strategies to facilitate islet engraftment and a prompt revascularization could be designed to intervene and might lead to improve future results of islet transplantation. PMID:27002241

  17. Beneficial effects of the transgenic expression of human sTNF-αR-Fc and HO-1 on pig-to-mouse islet xenograft survival.

    PubMed

    Yan, Ji-Jing; Yeom, Hye-Jeong; Jeong, Jong Cheol; Lee, Jae-Ghi; Lee, Eun Won; Cho, Bumrae; Lee, Han Sin; Kim, Su Jin; Hwang, Jong-Ik; Kim, Sung Joo; Lee, Byeong-Chun; Ahn, Curie; Yang, Jaeseok

    2016-02-01

    Both human soluble tumor necrosis factor-α receptor-Fc (sTNF-αR-Fc) and heme oxygenase-1 (HO-1) transgenic pigs have been generated previously for xenotransplantation. Here, we investigated whether overexpression of sTNF-αR-Fc or HO-1 in pig islets prolongs islet xenograft survival. Adult porcine islets were isolated from human sTNF-αR-Fc or HO-1 transgenic and wild type pigs, and were transplanted into diabetic nude mice. Effects of the expression of both genes on islet apoptosis, chemokine expression, cellular infiltration, antibody production, and islet xenograft survival were analyzed. Human sTNF-αR-Fc transgenic pigs successfully expressed sTNF-αR-Fc in the islets; human HO-1 transgenic pigs expressed significant levels of HO-1 in the islets. Pig-to-mouse islet xenograft survival was significantly prolonged in both the sTNF-αR-Fc and HO-1 groups compared with that in the wild type group. Both the sTNF-αR-Fc and HO-1 groups exhibited suppressed intragraft expression of monocyte chemoattractant protein-1 (MCP-1) and decreased perigraft infiltration of immune cells. However, there was no difference in the anti-pig antibody levels between the groups. Apoptosis of islet cells during the early engraftment was suppressed only in the HO-1 group. Porcine islets from both sTNF-αR-Fc and HO-1 transgenic pigs prolonged xenograft survival by suppressing islet cell apoptosis or secondary inflammatory responses following islet death, indicating that these transgenic pigs might have applications in successful islet xenotransplantation.

  18. Taurine supplementation modulates glucose homeostasis and islet function.

    PubMed

    Carneiro, Everardo M; Latorraca, Marcia Q; Araujo, Eliana; Beltrá, Marta; Oliveras, Maria J; Navarro, Mónica; Berná, Genoveva; Bedoya, Francisco J; Velloso, Licio A; Soria, Bernat; Martín, Franz

    2009-07-01

    Taurine is a conditionally essential amino acid for human that is involved in the control of glucose homeostasis; however, the mechanisms by which the amino acid affects blood glucose levels are unknown. Using an animal model, we have studied these mechanisms. Mice were supplemented with taurine for 30 d. Blood glucose homeostasis was assessed by intraperitoneal glucose tolerance tests (IPGTT). Islet cell function was determined by insulin secretion, cytosolic Ca2+ measurements and glucose metabolism from isolated islets. Islet cell gene expression and translocation was examined via immunohistochemistry and quantitative real-time polymerase chain reaction. Insulin signaling was studied by Western blot. Islets from taurine-supplemented mice had: (i) significantly higher insulin content, (ii) increased insulin secretion at stimulatory glucose concentrations, (iii) significantly displaced the dose-response curve for glucose-induced insulin release to the left, (iv) increased glucose metabolism at 5.6 and 11.1-mmol/L concentrations; (v) slowed cytosolic Ca2+ concentration ([Ca2+]i) oscillations in response to stimulatory glucose concentrations; (vi) increased insulin, sulfonylurea receptor-1, glucokinase, Glut-2, proconvertase and pancreas duodenum homeobox-1 (PDX-1) gene expression and (vii) increased PDX-1 expression in the nucleus. Moreover, taurine supplementation significantly increased both basal and insulin stimulated tyrosine phosphorylation of the insulin receptor in skeletal muscle and liver tissues. Finally, taurine supplemented mice showed an improved IPGTT. These results indicate that taurine controls glucose homeostasis by regulating the expression of genes required for glucose-stimulated insulin secretion. In addition, taurine enhances peripheral insulin sensitivity.

  19. A Simple Method to Replace Islet Equivalents for Volume Quantification of Human Islets.

    PubMed

    Ramachandran, Karthik; Huang, Han-Hung; Stehno-Bittel, Lisa

    2015-01-01

    Human islets come in a variety of sizes and shapes, and the total volume of islets used for research or clinical transplants must be estimated in a manner that is simple and valid. Islet equivalent (IEQ) measurements are the standard estimate of islet volume. We published a new method (the Kansas method) for estimating rat islet volume using cell numbers that was reliable and valid. Here we modified the method for human islets. We measured the dimensions of isolated human islets showing that they are not spherical and became less so in larger islets, with an average smallest/largest diameter ratio of 0.73 in large islets and 0.85 in small islets. Human islets were individually loaded into 96-well plates, dissociated into single cells, and the total cell number per islet determined with computer-assisted cytometry. Based on the counted cell number per islet, a regression model was created to convert islet diameter to cell number with a high R(2) value (0.99). Separate regression equations for male and female donors or young and old donors were not significantly different than the pooled data and did not improve the regression values. There was an inverse correlation between the cell number per IEQ and islet size. The Kansas method was validated with ATP/cell and cell viability data. Compared to the actual cell count, conventional IEQ measurements overestimated tissue volume of large islets by nearly double. Examples of differences in results obtained from the same data sets normalized to IEQ or the Kansas method included viability and insulin secretion concentrations. The implications of the error associated with the current IEQ method of volume estimation are discussed.

  20. Pancreas preservation for pancreas and islet transplantation

    PubMed Central

    Iwanaga, Yasuhiro; Sutherland, David E.R.; Harmon, James V.; Papas, Klearchos K.

    2010-01-01

    Purpose of review To summarize advances and limitations in pancreas procurement and preservation for pancreas and islet transplantation, and review advances in islet protection and preservation. Recent findings Pancreases procured after cardiac death, with in-situ regional organ cooling, have been successfully used for islet transplantation. Colloid-free Celsior and histidine-tryptophan-ketoglutarate preservation solutions are comparable to University of Wisconsin solution when used for cold storage before pancreas transplantation. Colloid-free preservation solutions are inferior to University of Wisconsin solution for pancreas preservation prior to islet isolation and transplantation. Clinical reports on pancreas and islet transplants suggest that the two-layer method may not offer significant benefits over cold storage with the University of Wisconsin solution: improved oxygenation may depend on the graft size; benefits in experimental models may not translate to human organs. Improvements in islet yield and quality occurred from pancreases treated with inhibitors of stress-induced apoptosis during procurement, storage, isolation or culture. Pancreas perfusion may be desirable before islet isolation and transplantation and may improve islet yields and quality. Methods for real-time, noninvasive assessment of pancreas quality during preservation have been implemented and objective islet potency assays have been developed and validated. These innovations should contribute to objective evaluation and establishment of improved pancreas preservation and islet isolation strategies. Summary Cold storage may be adequate for preservation before pancreas transplants, but insufficient when pancreases are processed for islets or when expanded donors are used. Supplementation of cold storage solutions with cytoprotective agents and perfusion may improve pancreas and islet transplant outcomes. PMID:18685343

  1. Storage and microencapsulation of islets for transplantation.

    PubMed

    Charles, K; Harland, R C; Ching, D; Opara, E C

    2000-01-01

    Microencapsulation is an effective means of immunoisolation for pancreatic islet transplants. However, the process of isolating, purifying, encapsulating, and transplanting islets in a single day is labor intensive and difficult for routine use. There is an apparent need for reliable methods of islet storage, and cryopreservation has emerged as an attractive system of islet banking. While studies have shown that cryopreserved islets are viable when tested unencapsulated after thawing, it is not clear if the combination of freezing and encapsulation would affect islet function. The purpose of the present study was to determine the in vitro function of cryopreserved islets following thawing and microencapsulation. Islets were isolated from the pancreata of Sprague-Dawley rats and cryopreserved under liquid nitrogen for either 1 week or 1 month, following an overnight culture at 37 degrees C. Upon thawing, the islets were tested either unencapsulated or after encapsulation in polylysine-alginate membrane. In all experiments islets were preperifused for 1 h at 37 degrees C with a modified Krebs-Ringer bicarbonate buffer containing 3.3 mM (60 mg/dl) glucose and maintained at pH 7.4 by continuous gassing with 95% air/5% CO2. Following basal effluent sample collection on ice, the glucose concentration was raised to 16.7 mM (300 mg/dl). It was found that, within 10 min of high glucose stimulation, an average of twofold increase in insulin secretion (p < 0.01) was obtained in islets within or without microcapsules. We conclude that islets cryopreserved for 1 month prior to thawing and microencapsulation retained functional viability as determined in in vitro experiments.

  2. Ghrelin Attenuates cAMP-PKA Signaling to Evoke Insulinostatic Cascade in Islet β-Cells

    PubMed Central

    Dezaki, Katsuya; Damdindorj, Boldbaatar; Sone, Hideyuki; Dyachok, Oleg; Tengholm, Anders; Gylfe, Erik; Kurashina, Tomoyuki; Yoshida, Masashi; Kakei, Masafumi; Yada, Toshihiko

    2011-01-01

    OBJECTIVE Ghrelin reportedly restricts insulin release in islet β-cells via the Gαi2 subtype of G-proteins and thereby regulates glucose homeostasis. This study explored whether ghrelin regulates cAMP signaling and whether this regulation induces insulinostatic cascade in islet β-cells. RESEARCH DESIGN AND METHODS Insulin release was measured in rat perfused pancreas and isolated islets and cAMP production in isolated islets. Cytosolic cAMP concentrations ([cAMP]i) were monitored in mouse MIN6 cells using evanescent-wave fluorescence imaging. In rat single β-cells, cytosolic protein kinase-A activity ([PKA]i) and Ca2+ concentration ([Ca2+]i) were measured by DR-II and fura-2 microfluorometry, respectively, and whole cell currents by patch-clamp technique. RESULTS Ghrelin suppressed glucose (8.3 mmol/L)-induced insulin release in rat perfused pancreas and isolated islets, and these effects of ghrelin were blunted in the presence of cAMP analogs or adenylate cyclase inhibitor. Glucose-induced cAMP production in isolated islets was attenuated by ghrelin and enhanced by ghrelin receptor antagonist and anti-ghrelin antiserum, which counteract endogenous islet-derived ghrelin. Ghrelin inhibited the glucose-induced [cAMP]i elevation and [PKA]i activation in MIN6 and rat β-cells, respectively. Furthermore, ghrelin potentiated voltage-dependent K+ (Kv) channel currents without altering Ca2+ channel currents and attenuated glucose-induced [Ca2+]i increases in rat β-cells in a PKA-dependent manner. CONCLUSIONS Ghrelin directly interacts with islet β-cells to attenuate glucose-induced cAMP production and PKA activation, which lead to activation of Kv channels and suppression of glucose-induced [Ca2+]i increase and insulin release. PMID:21788571

  3. Quantification of β-Cell Mass in Intramuscular Islet Grafts Using Radiolabeled Exendin-4

    PubMed Central

    Espes, Daniel; Selvaraju, Ramkumar; Velikyan, Irina; Krajcovic, Martin; Carlsson, Per-Ola; Eriksson, Olof

    2016-01-01

    Background There is an increasing interest in alternative implantation sites to the liver for islet transplantation. Intramuscular implantation has even been tested clinically. Possibilities to monitor β-cell mass would be of huge importance not only for the understanding of islet engraftment but also for the decision of changing the immunosuppressive regime. We have therefore evaluated the feasibility of quantifying intramuscular β-cell mass using the radiolabeled glucagon like peptide-1 receptor agonist DO3A-VS-Cys40-Exendin-4. Methods One hundred to 400 islets were transplanted to the abdominal muscle of nondiabetic mice. After 3 to 4 weeks, 0.2 to 0.5 MBq [177Lu]DO3A-VS-Cys40-Exendin-4 was administered intravenously. Sixty minutes postinjection abdominal organs and graft bearing muscle were retrieved, and the radioactive uptake measured in a well counter within 10 minutes. The specific uptake in native and transplanted islets was assessed by autoradiography. The total insulin-positive area of the islet grafts was determined by immunohistochemistry. Results Intramuscular islet grafts could easily be visualized by this tracer, and the background uptake was very low. There was a linear correlation between the radioactivity uptake and the number of transplanted islets, both for standardized uptake values and the total radiotracer uptake in each graft (percentage of injected dose). The quantified total insulin area of surviving β cells showed an even stronger correlation to both standardized uptake values (R = 0.96, P = 0.0002) and percentage of injected dose (R = 0.88, P = 0.0095). There was no correlation to estimated α cell mass. Conclusions [177Lu]DO3A-VS-Cys40-Exendin-4 could be used to quantify β-cell mass after experimental intramuscular islet transplantation. This technique may well be transferred to the clinical setting by exchanging Lutetium-177 radionuclide to a positron emitting Gallium-68. PMID:27819034

  4. A protective effect of endomorphins on the oxidative injury of islet.

    PubMed

    Tian, L M; Liu, J; Sun, X L; Gao, C X; Fan, Y; Guo, Q

    2010-08-01

    The antioxidative capacity of endomorphins (EMs), endogenous μ-opioid receptor agonists, has been demonstrated by IN VIVO assays. In this study, we attempt to evaluate the effects of endomorphin 1 (EM1) and endomorphin 2 (EM2) on pancreatic islet injuries induced by streptozotocin (STZ), alloxan (ALX) and H(2)O(2), respectively. Wistar rats' islets were isolated and purified. The function of the islet cells, the insulin response to glucose stimulation was examined by insulin Radio Immuno Assay and the cell viability was measured by MTT assay. DNA fragments were performed to evaluate the apoptosis, while the cell cycle distribution was analyzed by PI staining flow cytometric analysis. Furthermore, the islet were treated with EM1, EM2 or ALX for 24 h, and the expression of p53 and p21 protein were determined by Western blot. The results showed that STZ, ALX, and H(2)O(2) displayed clear concentration-dependent inhibitory effects on the pancreatic islet cells. While EMs improved the viability of islet induced by STZ, ALX or H(2)O(2), and EMs enhanced insulin accumulation of the cell supernatant after ALX and STZ stimulation. Our data also showed both that EMs inhibited cell apoptosis and cell cycle G1 arrest induced by STZ and ALX through down-regulaing p53 and p21 expression. Taken together, these results demonstrate that EMs can protect islet cells from STZ, ALX and H(2)O(2) induced injuries. Our observations imply that the endomorphins may have protective effects on islet cells oxidative injury.

  5. Metabolomics applied to the pancreatic islet

    PubMed Central

    Gooding, Jessica R.; Jensen, Mette V.; Newgard, Christopher B.

    2016-01-01

    Metabolomics, the characterization of the set of small molecules in a biological system, is advancing research in multiple areas of islet biology. Measuring a breadth of metabolites simultaneously provides a broad perspective on metabolic changes as the islets respond dynamically to metabolic fuels, hormones, or environmental stressors. As a result, metabolomics has the potential to provide new mechanistic insights into islet physiology and pathophysiology. Here we summarize advances in our understanding of islet physiology and the etiologies of type-1 and type-2 diabetes gained from metabolomics studies. PMID:26116790

  6. Islet Transplantation in Type I Diabetes Mellitus

    PubMed Central

    Jamiolkowski, Ryan M.; Guo, Lucie Y.; Li, Yun Rose; Shaffer, Sydney M.; Naji, Ali

    2012-01-01

    For most patients with type I diabetes, insulin therapy and glucose monitoring are sufficient to maintain glycemic control. However, hypoglycemia is a potentially lethal side effect of insulin treatment in patients who are glycemically labile or have hypoglycemia-associated autonomic failure [1]. For those patients, an alternative therapy is beta cell replacement via pancreas or islet transplantation. Pancreas transplants using cadaveric donor organs reduce insulin dependence but carry risks involved in major surgery and chronic immunosuppression. Islet transplantation, in which islets are isolated from donor pancreases and intravenously infused, require no surgery and can utilize islets isolated from pancreases unsuitable for whole organ transplantation. However, islet transplantation also requires immunosuppression, and standard steroid regimens may be toxic to beta cells [2]. The 2000 Edmonton Trial demonstrated the first long-term successful islet transplantation by using a glucocorticoid-free immunosuppressive regimen (sirolimus and tacrolimus). The Clinical Islet Transplantation (CIT) Consortium seeks to improve upon the Edmonton Protocol by using anti-thymocyte globulin (ATG) and TNFα antagonist (etanercept). The trials currently in progress, in addition to research efforts to find new sources of islet cells, reflect enormous potential for islet transplantation in treatment of type I diabetes. PMID:22461742

  7. D-Ala2-GIP-glu-PAL is neuroprotective in a chronic Parkinson's disease mouse model and increases BNDF expression while reducing neuroinflammation and lipid peroxidation.

    PubMed

    Li, Yanwei; Liu, WeiZhen; Li, Lin; Hölscher, Christian

    2017-02-15

    Type 2 diabetes mellitus (T2DM) is a risk factor for Parkinson's disease (PD). Therefore, treatment to improve insulin resistance in T2DM may be useful for PD patients. Glucose dependent insulinotropic polypeptide (GIP) is a member of the incretin hormone family that can promote insulin release and improve insulin resistance. Several GIP analogues have been developed as potential treatments for T2DM. We had shown previously that D-Ala2-GIP-glu-PAL, a novel long-acting GIP analogue, can play a neuroprotective role in the PD mouse model induced by acute MPTP injection. The drug reduced damage to the dopaminergic neurons and increased CREB-mediated Bcl-2 expression to prevent apoptosis and reduced chronic inflammation in the brain. In the present study, we further tested the effects of chronic treatment by D-Ala2-GIP-glu-PAL in a chronic PD mouse model induced by MPTP (25mg/kg ip.) combination with probenecid (250mg/kg ip.) injection for 5 weeks. The results demonstrated that chronic treatment with D-Ala2-GIP-glu-PAL inhibits MPTP -induced Parkinsonism-like motor disorders in mice, and that the drug prevents dopaminergic neuronal loss in the substantia nigra pars compacta (SNpc). Moreover, D-Ala2-GIP-glu-PAL also inhibited the increased levels of expression of α-synuclein in the SNpc and striatum induced by MPTP. Furthermore, drug treatment reduced chronic neuroinflammation, oxidative stress and lipid peroxidation, and increased the expression of BDNF. These findings show that GIP signaling is neuroprotective and holds promise as a novel treatment of PD.

  8. Continuous Quadrupole Magnetic Separation of Islets during Digestion Improves Purified Porcine Islet Viability

    PubMed Central

    Kumar Sajja, Venkata Sunil; Rizzari, Michael D.; Scott III, William E.; Kitzmann, Jennifer P.; Kennedy, David J.; Todd, Paul W.; Balamurugan, Appakalai N.; Hering, Bernhard J.

    2016-01-01

    Islet transplantation (ITx) is an emerging and promising therapy for patients with uncontrolled type 1 diabetes. The islet isolation and purification processes require exposure to extended cold ischemia, warm-enzymatic digestion, mechanical agitation, and use of damaging chemicals for density gradient separation (DG), all of which reduce viable islet yield. In this paper, we describe initial proof-of-concept studies exploring quadrupole magnetic separation (QMS) of islets as an alternative to DG to reduce exposure to these harsh conditions. Three porcine pancreata were split into two parts, the splenic lobe (SPL) and the combined connecting/duodenal lobes (CDL), for paired digestions and purifications. Islets in the SPL were preferentially labeled using magnetic microparticles (MMPs) that lodge within the islet microvasculature when infused into the pancreas and were continuously separated from the exocrine tissue by QMS during the collection phase of the digestion process. Unlabeled islets from the CDL were purified by conventional DG. Islets purified by QMS exhibited significantly improved viability (measured by oxygen consumption rate per DNA, p < 0.03) and better morphology relative to control islets. Islet purification by QMS can reduce the detrimental effects of prolonged exposure to toxic enzymes and density gradient solutions and substantially improve islet viability after isolation. PMID:27843954

  9. Continuous Quadrupole Magnetic Separation of Islets during Digestion Improves Purified Porcine Islet Viability.

    PubMed

    Weegman, Bradley P; Kumar Sajja, Venkata Sunil; Suszynski, Thomas M; Rizzari, Michael D; Scott Iii, William E; Kitzmann, Jennifer P; Mueller, Kate R; Hanley, Thomas R; Kennedy, David J; Todd, Paul W; Balamurugan, Appakalai N; Hering, Bernhard J; Papas, Klearchos K

    2016-01-01

    Islet transplantation (ITx) is an emerging and promising therapy for patients with uncontrolled type 1 diabetes. The islet isolation and purification processes require exposure to extended cold ischemia, warm-enzymatic digestion, mechanical agitation, and use of damaging chemicals for density gradient separation (DG), all of which reduce viable islet yield. In this paper, we describe initial proof-of-concept studies exploring quadrupole magnetic separation (QMS) of islets as an alternative to DG to reduce exposure to these harsh conditions. Three porcine pancreata were split into two parts, the splenic lobe (SPL) and the combined connecting/duodenal lobes (CDL), for paired digestions and purifications. Islets in the SPL were preferentially labeled using magnetic microparticles (MMPs) that lodge within the islet microvasculature when infused into the pancreas and were continuously separated from the exocrine tissue by QMS during the collection phase of the digestion process. Unlabeled islets from the CDL were purified by conventional DG. Islets purified by QMS exhibited significantly improved viability (measured by oxygen consumption rate per DNA, p < 0.03) and better morphology relative to control islets. Islet purification by QMS can reduce the detrimental effects of prolonged exposure to toxic enzymes and density gradient solutions and substantially improve islet viability after isolation.

  10. Single-cell transcriptomes identify human islet cell signatures and reveal cell-type–specific expression changes in type 2 diabetes

    PubMed Central

    Bolisetty, Mohan; Kursawe, Romy; Sun, Lili; Sivakamasundari, V.; Kycia, Ina

    2017-01-01

    Blood glucose levels are tightly controlled by the coordinated action of at least four cell types constituting pancreatic islets. Changes in the proportion and/or function of these cells are associated with genetic and molecular pathophysiology of monogenic, type 1, and type 2 (T2D) diabetes. Cellular heterogeneity impedes precise understanding of the molecular components of each islet cell type that govern islet (dys)function, particularly the less abundant delta and gamma/pancreatic polypeptide (PP) cells. Here, we report single-cell transcriptomes for 638 cells from nondiabetic (ND) and T2D human islet samples. Analyses of ND single-cell transcriptomes identified distinct alpha, beta, delta, and PP/gamma cell-type signatures. Genes linked to rare and common forms of islet dysfunction and diabetes were expressed in the delta and PP/gamma cell types. Moreover, this study revealed that delta cells specifically express receptors that receive and coordinate systemic cues from the leptin, ghrelin, and dopamine signaling pathways implicating them as integrators of central and peripheral metabolic signals into the pancreatic islet. Finally, single-cell transcriptome profiling revealed genes differentially regulated between T2D and ND alpha, beta, and delta cells that were undetectable in paired whole islet analyses. This study thus identifies fundamental cell-type–specific features of pancreatic islet (dys)function and provides a critical resource for comprehensive understanding of islet biology and diabetes pathogenesis. PMID:27864352

  11. Expression of receptors for luteinizing hormone, gastric-inhibitory polypeptide, and vasopressin in normal adrenal glands and cortisol-secreting adrenocortical tumors in dogs.

    PubMed

    Galac, S; Kars, V J; Klarenbeek, S; Teerds, K J; Mol, J A; Kooistra, H S

    2010-07-01

    Hypercortisolism caused by an adrenocortical tumor (AT) results from adrenocorticotropic hormone (ACTH)-independent hypersecretion of glucocorticoids. Studies in humans demonstrate that steroidogenesis in ATs may be stimulated by ectopic or overexpressed eutopic G protein-coupled receptors. We report on a screening of 23 surgically removed, cortisol-secreting ATs for the expression of receptors for luteinizing hormone (LH), gastric-inhibitory polypeptide (GIP), and vasopressin (V(1a), V(1b), and V(2)). Normal adrenal glands served as control tissues. Abundance of mRNA for these receptors was quantified using quantitative polymerase chain reaction (QPCR), and the presence and localization of these receptors were determined by immunohistochemistry. In both normal adrenal glands and ATs, mRNA encoding for all receptors was present, although the expression abundance of the V(1b) receptor was very low. The mRNA expression abundance for GIP and V(2) receptors in ATs were significantly lower (0.03 and 0.01, respectively) than in normal adrenal glands. The zona fasciculata of normal adrenal glands stained immunonegative for the GIP receptor. In contrast, islands of GIP receptor-immunopositive cells were detected in about half of the ATs. The zona fasciculata of both normal adrenal glands and AT tissue were immunopositive for LH receptor; in ATs in a homogenous or heterogenous pattern. In normal adrenal glands, no immunolabeling for V(1b)R and V(2) receptor was present, but in ATs, V(2) receptor-immunopositive cells were detected. In conclusion, QPCR analysis did not reveal overexpression of LH, GIP, V(1a), V(1b), or V(2) receptors in the ATs. However, the ectopic expression of GIP and V(2) receptor proteins in tumorous zona fasciculata tissue may play a role in the pathogenesis of canine cortisol-secreting ATs.

  12. Immunosuppression With CD40 Costimulatory Blockade Plus Rapamycin for Simultaneous Islet-Kidney Transplantation in Nonhuman Primates.

    PubMed

    Oura, T; Hotta, K; Lei, J; Markmann, J; Rosales, I; Dehnadi, A; Kawai, K; Ndishabandi, D; Smith, R-N; Cosimi, A B; Kawai, T

    2017-03-01

    The lack of a reliable immunosuppressive regimen that effectively suppresses both renal and islet allograft rejection without islet toxicity hampers a wider clinical application of simultaneous islet-kidney transplantation (SIK). Seven MHC-mismatched SIKs were performed in diabetic cynomolgus monkeys. Two recipients received rabbit antithymocyte globulin (ATG) induction followed by daily tacrolimus and rapamycin (ATG/Tac/Rapa), and five recipients were treated with anti-CD40 monoclonal antibody (mAb) and rapamycin (aCD40/Rapa). Anti-inflammatory therapy, including anti-interleukin-6 receptor mAb and anti-tumor necrosis factor-α mAb, was given in both groups. The ATG/Tac/Rapa recipients failed to achieve long-term islet allograft survival (19 and 26 days) due to poor islet engraftment and cytomegalovirus pneumonia. In contrast, the aCD40/Rapa regimen provided long-term islet and kidney allograft survival (90, 94, >120, >120, and >120 days), with only one recipient developing evidence of allograft rejection. The aCD40/Rapa regimen was also tested in four kidney-alone transplant recipients. All four recipients achieved long-term renal allograft survival (100% at day 120), which was superior to renal allograft survival (62.9% at day 120) with triple immunosuppressive regimen (tacrolimus, mycophenolate mofetil, and steroids). The combination of anti-CD40 mAb and rapamycin is an effective and nontoxic immunosuppressive regimen that uses only clinically available agents for kidney and islet recipients.

  13. Internalization and desensitization of the human glucose-dependent-insulinotropic receptor is affected by N-terminal acetylation of the agonist.

    PubMed

    Ismail, Sadek; Dubois-Vedrenne, Ingrid; Laval, Marie; Tikhonova, Irina G; D'Angelo, Romina; Sanchez, Claire; Clerc, Pascal; Gherardi, Marie-Julie; Gigoux, Véronique; Magnan, Remi; Fourmy, Daniel

    2015-10-15

    How incretins regulate presence of their receptors at the cell surface and their activity is of paramount importance for the development of therapeutic strategies targeting these receptors. We have studied internalization of the human Glucose-Insulinotropic Polypeptide receptor (GIPR). GIP stimulated rapid robust internalization of the GIPR, the major part being directed to lysosomes. GIPR internalization involved mainly clathrin-coated pits, AP-2 and dynamin. However, neither GIPR C-terminal region nor β-arrestin1/2 was required. Finally, N-acetyl-GIP recognized as a dipeptidyl-IV resistant analogue, fully stimulated cAMP production with a ∼15-fold lower potency than GIP and weakly stimulated GIPR internalization and desensitization of cAMP response. Furthermore, docking N-acetyl-GIP in the binding site of modeled GIPR showed slighter interactions with residues of helices 6 and 7 of GIPR compared to GIP. Therefore, incomplete or partial activity of N-acetyl-GIP on signaling involved in GIPR desensitization and internalization contributes to the enhanced incretin activity of this peptide.

  14. Total Pancreatectomy With Islet Autotransplantation

    PubMed Central

    Bellin, Melena D.; Gelrud, Andres; Arreaza-Rubin, Guillermo; Dunn, Ty B.; Humar, Abhinav; Morgan, Katherine A.; Naziruddin, Bashoo; Rastellini, Cristiana; Rickels, Michael R.; Schwarzenberg, Sarah J.; Andersen, Dana K.

    2015-01-01

    A workshop sponsored by the National Institute of Diabetes and Digestive and Kidney Diseases focused on research gaps and opportunities in total pancreatectomy with islet autotransplantation (TPIAT) for the management of chronic pancreatitis. The session was held on July 23, 2014 and structured into 5 sessions: (1) patient selection, indications, and timing; (2) technical aspects of TPIAT; (3) improving success of islet autotransplantation; (4) improving outcomes after total pancreatectomy; and (5) registry considerations for TPIAT. The current state of knowledge was reviewed; knowledge gaps and research needs were specifically highlighted. Common themes included the need to identify which patients best benefit from and when to intervene with TPIAT, current limitations of the surgical procedure, diabetes remission and the potential for improvement, opportunities to better address pain remission, GI complications in this population, and unique features of children with chronic pancreatitis considered for TPIAT. The need for a multicenter patient registry that specifically addresses the complexities of chronic pancreatitis and total pancreatectomy outcomes and postsurgical diabetes outcomes was repeatedly emphasized. PMID:25599324

  15. Direct glucocorticoid inhibition of insulin secretion. An in vitro study of dexamethasone effects in mouse islets.

    PubMed Central

    Lambillotte, C; Gilon, P; Henquin, J C

    1997-01-01

    The direct effects of glucocorticoids on pancreatic beta cell function were studied with normal mouse islets. Dexamethasone inhibited insulin secretion from cultured islets in a concentration-dependent manner: maximum of approximately 75% at 250 nM and IC50 at approximately 20 nM dexamethasone. This inhibition was of slow onset (0, 20, and 40% after 1, 2, and 3 h) and only slowly reversible. It was prevented by a blocker of nuclear glucocorticoid receptors, by pertussis toxin, by a phorbol ester, and by dibutyryl cAMP, but was unaffected by an increase in the fuel content of the culture medium. Dexamethasone treatment did not affect islet cAMP levels but slightly reduced inositol phosphate formation. After 18 h of culture with or without 1 microM dexamethasone, the islets were perifused and stimulated by a rise in the glucose concentration from 3 to 15 mM. Both phases of insulin secretion were similarly decreased in dexamethasone-treated islets as compared with control islets. This inhibition could not be ascribed to a lowering of insulin stores (higher in dexamethasone-treated islets), to an alteration of glucose metabolism (glucose oxidation and NAD(P)H changes were unaffected), or to a lesser rise of cytoplasmic Ca2+ in beta cells (only the frequency of the oscillations was modified). Dexamethasone also inhibited insulin secretion induced by arginine, tolbutamide, or high K+. In this case also the inhibition was observed despite a normal rise of cytoplasmic Ca2+. In conclusion, dexamethasone inhibits insulin secretion through a genomic action in beta cells that leads to a decrease in the efficacy of cytoplasmic Ca2+ on the exocytotic process. PMID:9022074

  16. In Vivo Islet Protection by a Nuclear Import Inhibitor in a Mouse Model of Type 1 Diabetes

    PubMed Central

    Moore, Daniel J.; Zienkiewicz, Jozef; Kendall, Peggy L.; Liu, Danya; Liu, Xueyan; Veach, Ruth Ann; Collins, Robert D.; Hawiger, Jacek

    2010-01-01

    Background Insulin-dependent Type 1 diabetes (T1D) is a devastating autoimmune disease that destroys beta cells within the pancreatic islets and afflicts over 10 million people worldwide. These patients face life-long risks for blindness, cardiovascular and renal diseases, and complications of insulin treatment. New therapies that protect islets from autoimmune destruction and allow continuing insulin production are needed. Increasing evidence regarding the pathomechanism of T1D indicates that islets are destroyed by the relentless attack by autoreactive immune cells evolving from an aberrant action of the innate, in addition to adaptive, immune system that produces islet-toxic cytokines, chemokines, and other effectors of islet inflammation. We tested the hypothesis that targeting nuclear import of stress-responsive transcription factors evoked by agonist-stimulated innate and adaptive immunity receptors would protect islets from autoimmune destruction. Principal Findings Here we show that a first-in-class inhibitor of nuclear import, cSN50 peptide, affords in vivo islet protection following a 2-day course of intense treatment in NOD mice, which resulted in a diabetes-free state for one year without apparent toxicity. This nuclear import inhibitor precipitously reduces the accumulation of islet-destructive autoreactive lymphocytes while enhancing activation-induced cell death of T and B lymphocytes derived from autoimmune diabetes-prone, non-obese diabetic (NOD) mice that develop T1D. Moreover, in this widely used model of human T1D we noted attenuation of pro-inflammatory cytokine and chemokine production in immune cells. Conclusions These results indicate that a novel form of immunotherapy that targets nuclear import can arrest inflammation-driven destruction of insulin-producing beta cells at the site of autoimmune attack within pancreatic islets during the progression of T1D. PMID:20949090

  17. Glutathione Ethyl Ester Supplementation during Pancreatic Islet Isolation Improves Viability and Transplant Outcomes in a Murine Marginal Islet Mass Model

    PubMed Central

    Raposo do Amaral, Alexandre S.; Pawlick, Rena L.; Rodrigues, Erika; Costal, Flavia; Pepper, Andrew; Ferreira Galvão, Flávio H.; Correa-Giannella, Maria Lucia; Shapiro, A. M.James

    2013-01-01

    Background The success of pancreatic islet transplantation still faces many challenges, mainly related to cell damage during islet isolation and early post-transplant. The increased generation of reactive oxygen species (ROS) during islet isolation and the consumption of antioxidant defenses appear to be an important pathway related to islet damage. Methodology/Principal Findings In the present study we evaluated whether supplementation of glutathione-ethyl-ester (GEE) during islet isolation could improve islet viability and transplant outcomes in a murine marginal islet mass model. We also cultured human islets for 24 hours in standard CMRL media with or without GEE supplementation. Supplementation of GEE decreased the content of ROS in isolated islets, leading to a decrease in apoptosis and maintenance of islet viability. A higher percentage of mice transplanted with a marginal mass of GEE treated islets became euglycemic after transplant. The supplementation of 20 mM GEE in cultured human islets significantly reduced the apoptosis rate in comparison to untreated islets. Conclusions/Significance GEE supplementation was able to decrease the apoptosis rate and intracellular content of ROS in isolated islets and might be considered a potential intervention to improve islet viability during the isolation process and maintenance in culture before islet transplantation. PMID:23424628

  18. The heterogeneity of islet autoantibodies and the progression of islet failure in type 1 diabetic patients.

    PubMed

    Liu, Jin; Bian, Lingling; Ji, Li; Chen, Yang; Chen, Heng; Gu, Yong; Ma, Bingqin; Gu, Wei; Xu, Xinyu; Shi, Yun; Wang, Jian; Zhu, Dalong; Sun, Zilin; Ma, Jianhua; Jin, Hui; Shi, Xing; Miao, Heng; Xin, Bing; Zhu, Yan; Zhang, Zhenwen; Bu, Ruifang; Xu, Lan; Shi, Guangde; Tang, Wei; Li, Wei; Zhou, Dongmei; Liang, Jun; Cheng, Xingbo; Shi, Bimin; Dong, Jixiang; Hu, Ji; Fang, Chen; Zhong, Shao; Yu, Weinan; Lu, Weiping; Wu, Chenguang; Qian, Li; Yu, Jiancheng; Gao, Jialin; Fei, Xiaoqiang; Zhang, Qingqing; Wang, Xueqin; Cui, Shiwei; Cheng, Jinluo; Xu, Ning; Wang, Guofeng; Han, Guoqing; Xu, Chunrong; Xie, Yun; An, Minmin; Zhang, Wei; Wang, Zhixiao; Cai, Yun; Fu, Qi; Fu, Yu; Zheng, Shuai; Yang, Fan; Hu, Qingfang; Dai, Hao; Jin, Yu; Zhang, Zheng; Xu, Kuanfeng; Li, Yifan; Shen, Jie; Zhou, Hongwen; He, Wei; Zheng, Xuqin; Han, Xiao; Yu, Liping; She, Jinxiong; Zhang, Mei; Yang, Tao

    2016-09-01

    Type 1 diabetes mellitus is heterogeneous in many facets. The patients suffered from type 1 diabetes present several levels of islet function as well as variable number and type of islet-specific autoantibodies. This study was to investigate prevalence and heterogeneity of the islet autoantibodies and clinical phenotypes of type 1 diabetes mellitus; and also discussed the process of islet failure and its risk factors in Chinese type 1 diabetic patients. A total of 1,291 type 1 diabetic patients were enrolled in this study. Demographic information was collected. Laboratory tests including mixed-meal tolerance test, human leukocyte antigen alleles, hemoglobinA1c, lipids, thyroid function and islet autoantibodies were conducted. The frequency of islet-specific autoantibody in newly diagnosed T1DM patients (duration shorter than half year) was 73% in East China. According to binary logistic regressions, autoantibody positivity, longer duration and lower Body Mass Index were the risk factors of islet failure. As the disease developed, autoantibodies against glutamic acid decarboxylase declined as well as the other two autoantibodies against zinc transporter 8 and islet antigen 2. The decrease of autoantibodies was positively correlated with aggressive beta cell destruction. Autoantibodies can facilitate the identification of classic T1DM from other subtypes and predict the progression of islet failure. As there were obvious heterogeneity in autoantibodies and clinical manifestation in different phenotypes of the disease, we should take more factors into consideration when identifying type 1 diabetes mellitus.

  19. Differential responses of the incretin hormones GIP and GLP-1 to increasing doses of dietary carbohydrate but not dietary protein in lean rats

    PubMed Central

    Yang, Qing; Kindel, Tammy L.; Tso, Patrick

    2010-01-01

    Previous studies have shown that oral ingestion of nutrients stimulates secretion of the incretin hormones glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1); however, it is unclear whether there is a dose-dependent response between the amount of nutrient ingested and the secretion of the hormones in vivo. Using our lymph fistula rat model, we previously demonstrated that both GIP and GLP-1 responded dose dependently to increasing amounts of infused dietary lipid and that the GLP-1-secreting cells were more sensitive to changes in intestinal lipid content. In the present study, we investigated the dose-dependent relationships between incretin secretion and the two remaining macronutrients, carbohydrate and protein. To accomplish this objective, the major mesenteric lymphatic duct of male Sprague-Dawley rats was cannulated. Each animal received a single bolus (3 ml) of saline, dextrin, whey protein, or casein hydrolysate (0.275, 0.55, 1.1, 2.2, 4.4 kcal) via a surgically inserted duodenal or ileal feeding tube. Lymph was continuously collected for 3 h and analyzed for GIP and GLP-1 content. Both GIP and GLP-1 outputs responded dose dependently to increasing amounts of dietary carbohydrate but not protein. Additionally, we found that the GIP-secreting cells were more sensitive than the GLP-1-secreting cells to changes in intestinal carbohydrate content. PMID:20522638

  20. Noninvasive imaging of islet grafts using positron-emission tomography

    NASA Astrophysics Data System (ADS)

    Lu, Yuxin; Dang, Hoa; Middleton, Blake; Zhang, Zesong; Washburn, Lorraine; Stout, David B.; Campbell-Thompson, Martha; Atkinson, Mark A.; Phelps, Michael; Gambhir, Sanjiv Sam; Tian, Jide; Kaufman, Daniel L.

    2006-07-01

    Islet transplantation offers a potential therapy to restore glucose homeostasis in type 1 diabetes patients. However, islet transplantation is not routinely successful because most islet recipients gradually lose graft function. Furthermore, serological markers of islet function are insensitive to islet loss until the latter stages of islet graft rejection. A noninvasive method of monitoring islet grafts would aid in the assessment of islet graft survival and the evaluation of interventions designed to prolong graft survival. Here, we show that recombinant adenovirus can engineer isolated islets to express a positron-emission tomography (PET) reporter gene and that these islets can be repeatedly imaged by using microPET after transplantation into mice. The magnitude of signal from engineered islets implanted into the axillary cavity was directly related to the implanted islet mass. PET signals attenuated over the following weeks because of the transient nature of adenovirus-mediated gene expression. Because the liver is the preferred site for islet implantation in humans, we also tested whether islets could be imaged after transfusion into the mouse liver. Control studies revealed that both intrahepatic islet transplantation and hyperglycemia altered the biodistribution kinetics of the PET probe systemically. Although transplanted islets were dispersed throughout the liver, clear signals from the liver region of mice receiving PET reporter-expressing islets were detectable for several weeks. Viral transduction, PET reporter expression, and repeated microPET imaging had no apparent deleterious effects on islet function after implantation. These studies lay a foundation for noninvasive quantitative assessments of islet graft survival using PET. diabetes | transplantation

  1. GIP Contributions to the Regulation of Centromere at the Interface Between the Nuclear Envelope and the Nucleoplasm

    PubMed Central

    Chabouté, Marie-Edith; Berr, Alexandre

    2016-01-01

    Centromeres are known as specific chromatin domains without which eukaryotic cells cannot divide properly during mitosis. Despite the considerable efforts to understand the centromere/kinetochore assembly during mitosis, until recently, comparatively few studies have dealt with the regulation of centromere during interphase. Here, we briefly review and discuss past and recent advances about the architecture of centromeres and their regulation during the cell cycle. Furthermore, we highlight and discuss new findings and hypotheses regarding the specific regulation of centromeres in both plant and animal nuclei, especially with GIP proteins at the interface between the nuclear envelope and the nucleoplasm. PMID:26904080

  2. IDDM: an islet or an immune disease?

    PubMed

    Boitard, C; Larger, E; Timsit, J; Sempe, P; Bach, J F

    1994-09-01

    Insulin-dependent diabetes develops as a consequence of the selective destruction of insulin-producing cells by an autoimmune reaction. However, the precise series of events which trigger anti-islet autoreactive T cells is still being investigated. Major issues will need to be raised before a comprehensive view of the anti-islet autoimmune reaction can be delineated. These include defining the primary site of activation of autoreactive lymphocytes and exploring hypotheses to explain the chronicity of the diabetes process. These issues all relate with the more general dilemma of the actual role of the islets of Langerhans in breaking self tolerance to beta-cell antigens. By studying non-obese diabetic mice deprived of beta cells following a single injection of a high dose of alloxan at 3 weeks of age, we recently obtained evidence that the activation of autoreactive T cells requires the presence of target islet cells in order to develop.

  3. Trimeprazine increases IRS2 in human islets and promotes pancreatic β cell growth and function in mice

    PubMed Central

    Kuznetsova, Alexandra; Yu, Yue; Hollister-Lock, Jennifer; Opare-Addo, Lynn; Rozzo, Aldo; Sadagurski, Marianna; Norquay, Lisa; Reed, Jessica E.; El Khattabi, Ilham; Bonner-Weir, Susan; Weir, Gordon C.; Sharma, Arun

    2016-01-01

    The capacity of pancreatic β cells to maintain glucose homeostasis during chronic physiologic and immunologic stress is important for cellular and metabolic homeostasis. Insulin receptor substrate 2 (IRS2) is a regulated adapter protein that links the insulin and IGF1 receptors to downstream signaling cascades. Since strategies to maintain or increase IRS2 expression can promote β cell growth, function, and survival, we conducted a screen to find small molecules that can increase IRS2 mRNA in isolated human pancreatic islets. We identified 77 compounds, including 15 that contained a tricyclic core. To establish the efficacy of our approach, one of the tricyclic compounds, trimeprazine tartrate, was investigated in isolated human islets and in mouse models. Trimeprazine is a first-generation antihistamine that acts as a partial agonist against the histamine H1 receptor (H1R) and other GPCRs, some of which are expressed on human islets. Trimeprazine promoted CREB phosphorylation and increased the concentration of IRS2 in islets. IRS2 was required for trimeprazine to increase nuclear Pdx1, islet mass, β cell replication and function, and glucose tolerance in mice. Moreover, trimeprazine synergized with anti-CD3 Abs to reduce the progression of diabetes in NOD mice. Finally, it increased the function of human islet transplants in streptozotocin-induced (STZ-induced) diabetic mice. Thus, trimeprazine, its analogs, or possibly other compounds that increase IRS2 in islets and β cells without adverse systemic effects might provide mechanism-based strategies to prevent the progression of diabetes. PMID:27152363

  4. HLA class I sensitization in islet transplant recipients: report from the Collaborative Islet Transplant Registry.

    PubMed

    Naziruddin, Bashoo; Wease, Steve; Stablein, Donald; Barton, Franca B; Berney, Thierry; Rickels, Michael R; Alejandro, Rodolfo

    2012-01-01

    Pancreatic islet transplantation is a promising treatment option for patients severely affected with type 1 diabetes. This report from CITR presents pre- and posttransplant human leukocyte antigen (HLA) class I sensitization rates in islet-alone transplantation. Data came from 303 recipients transplanted with islet-alone between January 1999 and December 2008. HLA class I sensitization was determined by the presence of anti-HLA class I antibodies. Panel-reactive antibodies (PRA) from prior to islet infusion and at 6 months, and yearly posttransplant was correlated to measures of islet graft failure. The cumulative number of mismatched HLA alleles increased with each additional islet infusion from a median of 3 for one infusion to 9 for three infusions. Pretransplant PRA was not predictive of islet graft failure. However, development of PRA >20% posttransplant was associated with 3.6-fold (p < 0.001) increased hazard ratio for graft failure. Patients with complete graft loss who had discontinued immunosuppression had significantly higher rate of PRA ≥ 20% compared to those with functioning grafts who remained on immunosuppression. Exposure to repeat HLA class I mismatch at second or third islet infusions resulted in less frequent development of de novo HLA class I antibodies when compared to increased class I mismatch. The development of HLA class I antibodies while on immunosuppression is associated with subsequent islet graft failure. The risk of sensitization may be reduced by minimizing the number of islet donors used per recipient, and in the absence of donor-specific anti-HLA antibodies, repeating HLA class I mismatches with subsequent islet infusions.

  5. HLA Class I Sensitization in Islet Transplant Recipients – Report from the Collaborative Islet Transplant Registry

    PubMed Central

    Naziruddin, Bashoo; Wease, Steve; Stablein, Donald; Barton, Franca B.; Berney, Thierry; Rickels, Michael R.; Alejandro, Rodolfo

    2015-01-01

    Pancreatic islet transplantation is a promising treatment option for patients severely affected with type 1 diabetes. This report from CITR presents pre- and post-transplant human leukocyte antigen (HLA) class I sensitization rates in islet alone transplantation. Data came from 303 recipients transplanted with islet alone between January 1999 and December 2008. HLA class I sensitization was determined by the presence of anti-HLA class I antibodies. Panel-reactive antibodies (PRA) from prior to islet infusion and at 6 months, and yearly post-transplant was correlated to measures of islet graft failure. The cumulative number of mismatched HLA alleles increased with each additional islet infusion from a median of 3 for one infusion to 9 for three infusions. Pre-transplant PRA was not predictive of islet graft failure. However, development of PRA ≥20% post-transplant was associated with 3.6 fold (p=.001) increased hazard ratio for graft failure. Patients with complete graft loss who had discontinued immunosuppression had significantly higher rate of PRA ≥ 20% compared to those with functioning grafts who remained on immunosuppression. Exposure to repeat HLA class I mismatch at second or third islet infusions resulted in less frequent development of de novo HLA class I antibodies when compared to increased class I mismatch. The development of HLA class I antibodies while on immunosuppression is associated with subsequent islet graft failure. The risk of sensitization may be reduced by minimizing the number of islet donors used per recipient, and in the absence of donor-specific anti-HLA antibodies, repeating HLA class I mismatches with subsequent islet infusions. PMID:22080832

  6. Pancreatic Islet Cell Development and Regeneration

    PubMed Central

    Romer, Anthony I.; Sussel, Lori

    2015-01-01

    Purpose This review will discuss recent advances in understanding mouse and human pancreatic islet cell development, novel concepts related to β cell dysfunction and improved approaches for replenishing β cells to treat diabetes. Recent Findings Considerable knowledge about pancreatic islet development and function has been gained using model systems with subsequent validation in human tissues. Recently, several rodent studies have revealed that differentiated adult islet cells retain remarkable plasticity and can be converted to other islet cell types by perturbing their transcription factor profiles. Furthermore, significant advances have been made in the generation of β-like cells from stem cell populations. Therefore, the generation of functionally mature β cells by the in situ conversion of non-β cell populations or by the directed differentiation of human pluripotent stem cells could represent novel mechanisms for replenishing β cells in diabetic patients. Summary The overall conservation between mouse and human pancreatic development, islet physiology and etiology of diabetes encourages the translation of novel β cell replacement therapies to humans. Further deciphering the molecular mechanisms that direct islet cell regeneration, plasticity and function could improve and expand the β cell replacement strategies for treating diabetes. PMID:26087337

  7. Justifying clinical trials for porcine islet xenotransplantation.

    PubMed

    Ellis, Cara E; Korbutt, Gregory S

    2015-01-01

    The development of the Edmonton Protocol encouraged a great deal of optimism that a cell-based cure for type I diabetes could be achieved. However, donor organ shortages prevent islet transplantation from being a widespread solution as the supply cannot possibly equal the demand. Porcine islet xenotransplantation has the potential to address these shortages, and recent preclinical and clinical trials show promising scientific support. Consequently, it is important to consider whether the current science meets the ethical requirements for moving toward clinical trials. Despite the potential risks and the scientific unknowns that remain to be investigated, there is optimism regarding the xenotransplantation of some types of tissue, and enough evidence has been gathered to ethically justify clinical trials for the most safe and advanced area of research, porcine islet transplantation. Researchers must make a concerted effort to maintain a positive image for xenotransplantation, as a few well-publicized failed trials could irrevocably damage public perception of xenotransplantation. Because all of society carries the burden of risk, it is important that the public be involved in the decision to proceed. As new information from preclinical and clinical trials develops, policy decisions should be frequently updated. If at any point evidence shows that islet xenotransplantation is unsafe, then clinical trials will no longer be justified and they should be halted. However, as of now, the expected benefit of an unlimited supply of islets, combined with adequate informed consent, justifies clinical trials for islet xenotransplantation.

  8. How to Use Image Analysis for Islet Counting

    PubMed Central

    Girman, Peter; Berkova, Zuzana; Dobolilova, Eva; Saudek, Frantisek

    2008-01-01

    AIM: Assessment of islet mass before islet transplantation requires a reliable technique to enable exact analysis of islet volume. This study aimed to test the applicability of digital image analysis (DIA) for evaluation of samples of purified and non-purified islets. METHODS: Pancreatic islets were isolated from 10 Lewis rats. Samples of purified (n = 10) and non-purified islets (n = 30) were counted conventionally and by using a computerized method. The equipment for the computerized counting consisted of a digital camera installed on a stereomicroscope and connected to a personal computer. Images of 2272x1704 pixels were processed using a previously described non-commercial program originally developed for this purpose. Islets were converted to equivalents using globe and ellipsoid models. The insulin content of purified islets was assessed using radioimmunoassay and was correlated to the absolute and standardized islet number. RESULTS: Mean absolute numbers of purified islets ± SD were 908 ± 130 and 1049 ± 230 (manually and DIA respectively). Mean insulin content ± SD obtained from purified islets was 161 ± 45 mU. The mean equivalents of purified islets (1589 ± 555 for globe and 1219 ± 452 for ellipsoid) significantly correlated with insulin content. However, this correlation was not significant when absolute islet numbers were used, counted using either method. There was no significant difference in absolute non-purified islet numbers assessed by manual and computerized methods (average ± SD in 50 µl samples; 12.6 ± 4.1 and 13.3 ± 5.3 respectively; p = 0.22). The manual method showed a significantly higher yield of islet equivalents (IE; p < 0.001 for both globe and ellipsoid). CONCLUSION: The computer-based system for islet counting correlated better to insulin content than conventional islet estimation and prevented overestimation. Reproducibility and ease of assessment make it potentially applicable to clinical islet transplantation. PMID

  9. Dipeptidyl Peptidase IV Inhibition Activates CREB and Improves Islet Vascularization through VEGF-A/VEGFR-2 Signaling Pathway

    PubMed Central

    Samikannu, Balaji; Chen, Chunguang; Lingwal, Neelam; Padmasekar, Manju; Engel, Felix B.; Linn, Thomas

    2013-01-01

    Substitution of pancreatic islets is a potential therapy to treat diabetes and it depends on reconstitution of islet’s capillary network. In this study, we addressed the question whether stabilization of Glucagon-Like-Peptide-1 (GLP-1) by inhibiting Dipeptidyl Peptidase-IV (DPP-IV) increases β-cell mass by modulating vascularization. Mouse or porcine donor islets were implanted under kidney capsule of diabetic mice treated with DPP-IV inhibitor sitagliptin. Grafts were analyzed for insulin production, β-cell proliferation and vascularization. In addition, the effect of sitagliptin on sprouting and Vascular Endothelial Growth Factor (VEGF)-A expression was examined ex vivo. The cAMP response element-binding (CREB) and VEGF-A/ Vascular Endothelial Growth Factor Receptor (VEGFR)-2 signaling pathway leading to islet vascularization was explored. Sitagliptin increased mean insulin content of islet grafts and area of insulin-positive tissue as well as β-cell proliferation. Interestingly, sitagliptin treatment also markedly increased endothelial cell proliferation, microvessel density and blood flow. Finally, GLP-1 (7-36) stimulated sprouting and VEGF expression, which was significantly enhanced by sitagliptin- mediated inhibition of DPP-IV. Our in vivo data demonstrate that sitagliptin treatment phosphorylated CREB and induced islet vascularization through VEGF-A/VEGFR-2 signaling pathway. This study paves a new pathway for improvement of islet transplantation in treating diabetes mellitus. PMID:24349326

  10. Inhibitory effect of kisspeptins on insulin secretion from isolated mouse islets.

    PubMed

    Vikman, J; Ahrén, B

    2009-11-01

    Islet hormone secretion is regulated by a variety of factors, and many of these signal through G protein-coupled receptors (GPCRs). A novel islet GPCR is GPR54, which couples to the Gq isoform of G proteins, which in turn signal through the phospholipase C pathway. Ligands for GPR54 are kisspeptins, which are peptides encoded in the KISS1 gene and also expressed in islet beta-cells. The KISS1 gene encodes a hydrophobic 145-amino acid protein that is cleaved into a 54-amino acid protein, kisspeptin-54 or KP54. Shorter kisspeptins also exist, such as kisspeptin-10 (KP10) and kisspeptin-13 (KP13). The involvement of GPR54 and kisspeptins in the regulation of islet function is not known. To address this problem, we incubated isolated mouse islets in the presence of KP13 and KP54 for 60 min and measured insulin secretion. We found that both KP13 and KP54 at 10 nM, 100 nM and 1microM inhibited insulin secretion in the presence of 2.8 mM glucose. However, by increasing the glucose concentration, this inhibitory action of the kisspeptins vanished. Thus, at 11.1 mM glucose, KP13 and KP54 inhibited insulin secretion only at high doses, and at 16.7 mM they no longer inhibited insulin secretion in any of the doses. We conclude that kisspeptins inhibit insulin secretion at glucose concentrations below 11.1 mM. This suggests that kisspeptins are regulating insulin secretion at physiological concentrations of glucose. The mechanisms by which kisspeptins regulate islet function and insulin secretion are unknown and will be further investigated.

  11. Preservation of pancreatic islets in cold UW solution before transplantation.

    PubMed

    Ishii, Show; Saito, Takuro; Ise, Kazuya; Yamashita, Michitoshi; Sato, Yoshihiro; Saito, Takaharu; Tsukada, Manabu; Oshibe, Ikuro; Kenjo, Akira; Kimura, Takashi; Anazawa, Takayuki; Suzuki, Shigeya; Gotoh, Mitsukazu

    2012-01-01

    Culture of islets prior to transplantation needs to be revisited for maintaining functional islet capacity. This study was conducted to compare cold UW (University of Wisconsin) preservation with conventional culture based on insulin secretory capacity in vitro and in vivo. Islets isolated from Wistar rats were either cultured for 24 h at 37°C in RPMI1640 medium or DMEM containing various concentrations of glucose or preserved for the same period in UW solution or in DMEM solution at 4°C. The islet yield in UW group, but not in other groups, was maintained as comparable with that of fresh islets. Insulin secretory capacity in response to glucose was maintained only in the islets of UW group, but not in other groups. SCID mice given 300 IEQ islets of UW group showed gradual restoration of normoglycemia as found in the mice given freshly isolated islets. Meanwhile, those mice given cultured islets for 24 h at 37°C in RPMI1640 medium showed rapid decrease of blood glucose levels on day 1 followed by relatively elevated levels on day 2, suggesting unstable insulin secretory capacity of islets.   Morphological staining with anti-HMGB1 (high mobility group B1) antibody revealed central damage of islets in all culture groups regardless of glucose concentration and in islets of cold DMEM group, whereas those in the UW group were quite intact. These results suggest that cold preservation in UW solution is simple and beneficial in protecting islets morphologically and functionally before transplantation.

  12. Sensing and Sensibility: Single-Islet-based Quality Control Assay of Cryopreserved Pancreatic Islets with Functionalized Hydrogel Microcapsules.

    PubMed

    Chen, Wanyu; Shu, Zhiquan; Gao, Dayong; Shen, Amy Q

    2016-01-21

    Despite decades of research and clinical studies of islet transplantations, finding simple yet reliable islet quality assays that correlate accurately with in vivo potency is still a major challenge, especially for real-time and single-islet-based quality assessment. Herein, proof-of-concept studies of a cryopreserved microcapsule-based quality control assays are presented for single islets. Individual rat pancreatic islets and fluorescent oxygen-sensitive dye (FOSD) are encapsulated in alginate hydrogel microcapsules via a microfluidic device. To test the susceptibility of the microcapsules and the FOSD to cryopreservation, the islet microcapsules containing FOSD are cryopreserved and the islet functionalities (adenosine triphosphate, static insulin release measurement, and oxygen consumption rate) are assessed after freezing and thawing steps. The cryopreserved islet capsules with FOSD remain functional after encapsulation and freezing/thawing procedures, validating a simple yet reliable individual-islet-based quality control method for the entire islet processing procedure prior to transplantation. This work also demonstrates that the functionality of cryopreserved islets can be improved by introducing trehalose into the routinely used cryoprotectant dimethyl sulfoxide. The functionalized alginate hydrogel microcapsules with embedded FOSD and optimized cryopreservation protocol presented in this work serve as a versatile islet quality assay and offer tremendous promise for tackling existing challenges in islet transplantation procedures.

  13. Temporal and Regional Expression of Glucose-Dependent Insulinotropic Peptide and Its Receptor in Spinal Cord Injured Rats.

    PubMed

    Marcos, Ana Beatriz W; Forner, Stefania; Martini, Alessandra C; Patrício, Eliziane S; Clarke, Julia R; Costa, Robson; Felix-Alves, João; Vieira, Vilberto José; de Andrade, Edinéia Lemos; Mazzuco, Tânia Longo; Calixto, João Batista; Figueiredo, Claudia Pinto

    2016-02-01

    Spinal cord injury (SCI) results in loss of movement, sensibility, and autonomic control at the level of the lesion and at lower parts of the body. Several experimental strategies have been used in attempts to increase endogenous mechanisms of neuroprotection, neuroplasticity, and repair, but with limited success. It is known that glucose-dependent insulinotropic peptide (GIP) and its receptor (GIPR) can enhance synaptic plasticity, neurogenesis, and axonal outgrowth. However, their role in the injury has never been studied. The aim of this study was to evaluate the changes in expression levels of both GIP and GIPR in acute and chronic phases of SCI in rats. Following SCI (2 to 24 h after damage), the rat spinal cord showed a lesion in which the epicenter had a cavity with hemorrhage and necrosis. Furthermore, the lesion cavity also showed ballooned cells 14 and 28 days after injury. We found that SCI induced increases in GIPR expression in areas neighboring the site of injury at 6 h and 28 days after the injury. Moreover, higher GIP expression was observed in these regions on day 28. Neuronal projections from the injury epicenter showed an increase in GIP immunoreactivity 24 h and 14 and 28 days after SCI. Interestingly, GIP was also found in progenitor cells at the spinal cord canal 24 h after injury, whereas both GIP and GIPR were present in progenitor cells at the injury epicenter 14 days after in SCI animals. These results suggest that GIP and its receptor might be implicated with neurogenesis and the repair process after SCI.

  14. Macroporous Three Dimensional PDMS Scaffolds for Extrahepatic Islet Transplantation

    PubMed Central

    Pedraza, Eileen; Brady, Ann-Christina; Fraker, Christopher A.; Molano, R. Damaris; Sukert, Steven; Berman, Dora M.; Kenyon, Norma S.; Pileggi, Antonello; Ricordi, Camillo; Stabler, Cherie L.

    2015-01-01

    Clinical islet transplantation has demonstrated success in treating type 1 diabetes. A current limitation is the intrahepatic portal vein transplant site, which is prone to mechanical stress and inflammation. Transplantation of pancreatic islets into alternative sites is preferable, but challenging, as it may require a three-dimensional vehicle to confer mechanical protection and to confine islets to a well-defined, retrievable space where islet neovascularization can occur. We have fabricated biostable, macroporous scaffolds from poly(dimethylsiloxane) (PDMS) and investigated islet retention and distribution, metabolic function, and glucose-dependent insulin secretion within these materials. Islets from multiple sources, including rodents, non-human primates, and humans, were tested in vitro. We observed high islet retention and distribution within PDMS scaffolds, with retention of small islets (< 100 µm) improved through the post-loading addition of fibrin gel. Islets loaded within PDMS scaffolds exhibited viability and function comparable to standard culture conditions when incubated under normal oxygen tensions, but displayed improved viability compared to standard two-dimensional culture controls under low oxygen tensions. In vivo efficacy of scaffolds to support islet grafts was evaluated after transplantation in the omental pouch of chemically-induced diabetic syngeneic rats, which promptly achieved normoglycemia. Collectively, these results are promising in that they indicate the potential for transplanting islets into a clinically relevant, extrahepatic site that provides spatial distribution of islets, as well as intra-device vascularization. PMID:23031502

  15. Glucose cycling in islets from healthy and diabetic rats

    SciTech Connect

    Khan, A.; Chandramouli, V.; Ostenson, C.G.; Loew, H.L.; Landau, B.R.; Efendic, S. )

    1990-04-01

    Pancreatic islets from healthy (control) and neonatally streptozocin-induced diabetic (STZ-D) rats, a model for non-insulin-dependent diabetes mellitus, were incubated with {sup 3}H{sub 2}O and 5.5 or 16.7 mM glucose. At 5.5 mM glucose, no detectable ({sup 3}H)glucose was formed. At 16.7 mM, 2.2 patom.islet-1.h-1 of {sup 3}H was incorporated into glucose by the control islets and 5.4 patom.islet-1.h-1 by STZ-D islets. About 75% of the {sup 3}H was bound to carbon-2 of the glucose. Glucose utilization was 35.3 pmol.islet-1.h-1 by the control and 19.0 pmol.islet-1.h-1 by the STZ-D islets. Therefore, 4.5% of the glucose-6-phosphate formed by the control islets and 15.7% by the STZ-D islets was dephosphorylated. This presumably occurred in the beta-cells of the islets catalyzed by glucose-6-phosphatase. An increased glucose cycling, i.e., glucose----glucose-6-phosphate----glucose, in islets of STZ-D rats may contribute to the decreased insulin secretion found in these animals.

  16. Response of incretins (GIP and GLP-1) to an oral glucose load in female and male subjects with normal glucose tolerance.

    PubMed

    Matsuo, Toshihiro; Kusunoki, Yoshiki; Katsuno, Tomoyuki; Ikawa, Takashi; Akagami, Takafumi; Murai, Kazuki; Miuchi, Masayuki; Miyagawa, Jun-ichiro; Namba, Mitsuyoshi

    2014-11-01

    The aim of this study was to analyze the blood glucose profile and the response of incretins in healthy young subjects by the 75 g oral glucose tolerance test (OGTT). We first reported that plasma glucose and GIP levels were higher in males during the early phase of the OGTT.

  17. Inhibition of TLR4 protects rat islets against lipopolysaccharide-induced dysfunction.

    PubMed

    Wang, Xiao; Ge, Qin Min; Bian, Fan; Dong, Yan; Huang, Chun Mei

    2017-02-01

    Oxidative stress leads to dysfunction in pancreatic cells, causing a reduction in insulin secretion following exposure to glucose. Toll-like receptor 4 (TLR4) may be activated by exposure to lipopolysaccharide (LPS) stress. TLR4 may mediate the initiation of inflammatory and immune defense responses; however, the importance of the LPS/TLR4 interaction in apoptosis induced by oxidative stress in pancreatic β cells remains to be elucidated. The present study aimed to investigate the importance of TLR4 during LPS‑induced oxidative stress, apoptosis and dysfunction of insulin secretion in isolated islets of rats. LPS‑induced stimulation of TLR4 increased the production of reactive oxygen species and promoted apoptosis by upregulating the expression levels of caspase‑3, poly ADP ribose polymerase and altering the expression ratio of B‑cell lymphoma‑2 (Bcl‑2)/Bcl‑2 associated X protein. Additionally, the insulin secretion of islets cells was reduced. Anti‑TLR4 antibody and a knockdown of TLR4 by TLR4‑short hairpin RNA were used to inhibit TLR4 activity, which may reverse LPS‑induced events. The present study determined that in islets exposed to LPS oxidative stress, dysfunction may be partly mediated via the TLR4 pathway. Inhibition of TLR4 may prevent dysfunction of rat islets due to oxidative stress. The present study revealed that targeting the LPS/TLR4 signaling pathway and antioxidant therapy may be a novel treatment for the severely septic patients with hyperglycemia stress.

  18. Extensive Loss of Islet Mass Beyond the First Day After Intraportal Human Islet Transplantation in a Mouse Model.

    PubMed

    Liljebäck, Hanna; Grapensparr, Liza; Olerud, Johan; Carlsson, Per-Ola

    2016-01-01

    Clinical islet transplantation is characterized by a progressive deterioration of islet graft function, which renders many patients once again dependent on exogenous insulin administration within a couple of years. In this study, we aimed to investigate possible engraftment factors limiting the survival and viability of experimentally transplanted human islets beyond the first day after their transplantation to the liver. Human islets were transplanted into the liver of nude mice and characterized 1 or 30 days after transplantation by immunohistochemistry. The factors assessed were endocrine mass, cellular death, hypoxia, vascular density and amyloid formation in the transplanted islets. One day posttransplantation, necrotic cells, as well as apoptotic cells, were commonly observed. In contrast to necrotic death, apoptosis rates remained high 1 month posttransplantation, and the total islet mass was reduced by more than 50% between 1 and 30 days posttransplantation. Islet mass at 30 days posttransplantation correlated negatively to apoptotic death. Vascular density within the transplanted islets remained less than 30% of that in native human islets up to 30 days posttransplantation and was associated with prevailing hypoxia. Amyloid formation was rarely observed in the 1-day-old transplants, but was commonly observed in the 30-day-old islet transplants. We conclude that substantial islet cell death occurs beyond the immediate posttransplantation phase, particularly through apoptotic events. Concomitant low vascularization with prevailing hypoxia and progressive amyloid development was observed in the human islet grafts. Strategies to improve engraftment at the intraportal site or change of implantation site in the clinical setting are needed.

  19. Plasma glucose kinetics and response of insulin and GIP following a cereal breakfast in female subjects: effect of starch digestibility

    PubMed Central

    Péronnet, F; Meynier, A; Sauvinet, V; Normand, S; Bourdon, E; Mignault, D; St-Pierre, D H; Laville, M; Rabasa-Lhoret, R; Vinoy, S

    2015-01-01

    Background/Objectives: Foods with high contents of slowly digestible starch (SDS) elicit lower glycemic responses than foods with low contents of SDS but there has been debate on the underlying changes in plasma glucose kinetics, that is, respective contributions of the increase in the rates of appearance and disappearance of plasma glucose (RaT and RdT), and of the increase in the rate of appearance of exogenous glucose (RaE) and decrease in endogenous glucose production (EGP). Subjects/Methods: Sixteen young healthy females ingested in random order four types of breakfasts: an extruded cereal (0.3% SDS: Lo-SDS breakfast) or one of three biscuits (39–45% SDS: Hi-SDS breakfasts). The flour in the cereal products was labeled with 13C, and plasma glucose kinetics were measured using [6,6-2H2]glucose infusion, along with the response of plasma glucose, insulin and glucose-dependent insulinotropic peptide (GIP) concentrations. Results: When compared with the Lo-SDS breakfast, after the three Hi-SDS breakfasts, excursions in plasma glucose, the response of RaE, RaT and RdT, and the reduction in EGP were significantly lower (P<0.05). The amount of exogenous glucose absorbed over the 4.5-h postprandial period was also significantly lower by ~31% (P<0.001). These differences were associated with lower responses of GIP and insulin concentrations. Conclusions: Substituting extruded cereals with biscuits slows down the availability of glucose from the breakfast and its appearance in peripheral circulation, blunts the changes in plasma glucose kinetics and homeostasis, reduces excursions in plasma glucose, and possibly distributes the glucose ingested over a longer period following the meal. PMID:25852025

  20. Pancreatic Islets: Methods for Isolation and Purification of Juvenile and Adult Pig Islets.

    PubMed

    Brandhorst, Heide; Johnson, Paul R V; Brandhorst, Daniel

    2016-01-01

    The current situation of organ transplantation is mainly determined by the disbalance between the number of available organs and the number of patients on the waiting list. This obvious dilemma might be solved by the transplantation of porcine organs into human patients. The metabolic similarities which exist between both species made pancreatic islets of Langerhans to that donor tissue which will be most likely transplanted in human recipients. Nevertheless, the successful isolation of significant yields of viable porcine islets is extremely difficult and requires extensive experiences in the field. This review is focussing on the technical challenges, pitfalls and particularities that are associated with the isolation of islets from juvenile and adult pigs considering donor variables that can affect porcine islet isolation outcome.

  1. Microwell Scaffolds for the Extrahepatic Transplantation of Islets of Langerhans

    PubMed Central

    Buitinga, Mijke; Truckenmüller, Roman; Engelse, Marten A.; Moroni, Lorenzo; Ten Hoopen, Hetty W. M.; van Blitterswijk, Clemens A.; de Koning, Eelco JP.; van Apeldoorn, Aart A.; Karperien, Marcel

    2013-01-01

    Allogeneic islet transplantation into the liver has the potential to restore normoglycemia in patients with type 1 diabetes. However, the suboptimal microenvironment for islets in the liver is likely to be involved in the progressive islet dysfunction that is often observed post-transplantation. This study validates a novel microwell scaffold platform to be used for the extrahepatic transplantation of islet of Langerhans. Scaffolds were fabricated from either a thin polymer film or an electrospun mesh of poly(ethylene oxide terephthalate)-poly(butylene terephthalate) (PEOT/PBT) block copolymer (composition: 4000PEOT30PBT70) and were imprinted with microwells, ∼400 µm in diameter and ∼350 µm in depth. The water contact angle and water uptake were 39±2° and 52.1±4.0 wt%, respectively. The glucose flux through electrospun scaffolds was three times higher than for thin film scaffolds, indicating enhanced nutrient diffusion. Human islets cultured in microwell scaffolds for seven days showed insulin release and insulin content comparable to those of free-floating control islets. Islet morphology and insulin and glucagon expression were maintained during culture in the microwell scaffolds. Our results indicate that the microwell scaffold platform prevents islet aggregation by confinement of individual islets in separate microwells, preserves the islet’s native rounded morphology, and provides a protective environment without impairing islet functionality, making it a promising platform for use in extrahepatic islet transplantation. PMID:23737999

  2. Clinical Allogeneic and Autologous Islet Cell Transplantation: Update

    PubMed Central

    2011-01-01

    Islet cell transplantation is categorized as a β-cell replacement therapy for diabetic patients who lack the ability to secrete insulin. Allogeneic islet cell transplantation is for the treatment of type 1 diabetes, and autologous islet cell transplantation is for the prevention of surgical diabetes after a total pancreatectomy. The issues of allogeneic islet cell transplantation include poor efficacy of islet isolation, the need for multiple donor pancreata, difficulty maintaining insulin independence and undesirable side effects of immunosuppressive drugs. Those issues have been solved step by step and allogeneic islet cell transplantation is almost ready to be the standard therapy. The donor shortage will be the next issue and marginal and/or living donor islet cell transplantation might alleviate the issue. Xeno-islet cell transplantation, β-cell regeneration from human stem cells and gene induction of the naïve pancreas represent the next generation of β-cell replacement therapy. Autologous islet cell transplantation after total pancreatectomy for the treatment of chronic pancreatitis with severe abdominal pain is the standard therapy, even though only limited centers are able to perform this treatment. Remote center autologous islet cell transplantation is an attractive option for hospitals performing total pancreatectomies without the proper islet isolation facilities. PMID:21785738

  3. Pancreatic islet cell therapy for type I diabetes: understanding the effects of glucose stimulation on islets in order to produce better islets for transplantation.

    PubMed

    Ren, Jiaqiang; Jin, Ping; Wang, Ena; Liu, Eric; Harlan, David M; Li, Xin; Stroncek, David F

    2007-01-03

    While insulin replacement remains the cornerstone treatment for type I diabetes mellitus (T1DM), the transplantation of pancreatic islets of Langerhans has the potential to become an important alternative. And yet, islet transplant therapy is limited by several factors, including far too few donor pancreases. Attempts to expand mature islets or to produce islets from stem cells are far from clinical application. The production and expansion of the insulin-producing cells within the islet (so called beta cells), or even creating cells that secrete insulin under appropriate physiological control, has proven difficult. The difficulty is explained, in part, because insulin synthesis and release is complex, unique, and not entirely characterized. Understanding beta-cell function at the molecular level will likely facilitate the development of techniques to manufacture beta-cells from stem cells. We will review islet transplantation, as well as the mechanisms underlying insulin transcription, translation and glucose stimulated insulin release.

  4. Glucose Oscillations Can Activate an Endogenous Oscillator in Pancreatic Islets.

    PubMed

    McKenna, Joseph P; Dhumpa, Raghuram; Mukhitov, Nikita; Roper, Michael G; Bertram, Richard

    2016-10-01

    Pancreatic islets manage elevations in blood glucose level by secreting insulin into the bloodstream in a pulsatile manner. Pulsatile insulin secretion is governed by islet oscillations such as bursting electrical activity and periodic Ca2+ entry in β-cells. In this report, we demonstrate that although islet oscillations are lost by fixing a glucose stimulus at a high concentration, they may be recovered by subsequently converting the glucose stimulus to a sinusoidal wave. We predict with mathematical modeling that the sinusoidal glucose signal's ability to recover islet oscillations depends on its amplitude and period, and we confirm our predictions by conducting experiments with islets using a microfluidics platform. Our results suggest a mechanism whereby oscillatory blood glucose levels recruit non-oscillating islets to enhance pulsatile insulin output from the pancreas. Our results also provide support for the main hypothesis of the Dual Oscillator Model, that a glycolytic oscillator endogenous to islet β-cells drives pulsatile insulin secretion.

  5. Using selective withdrawal to encapsulate pancreatic islets for immunoisolation

    NASA Astrophysics Data System (ADS)

    Wyman, Jason; Murphy, William; Mrksich, Milan

    2005-11-01

    We apply selective-withdrawal for encapsulating insulin-producing pancreatic islets within thin poly(ethylene glycol) (PEG) coats. Islets placed in an aqueous PEG solution are drawn into the selective-withdrawal spout which then breaks up, leaving the islets surrounded by a thin, 20μm, polymer coat. These coats, whose thickness is independent of the size of the encapsulated islet, are photo-crosslinked to form hydrogel capsules. We can apply multiple coats of varying chemical composition. These coats provide a semi-permeable membrane which allows the islets to respond to changes in glucose concentration by producing insulin in a manner similar to that of unencapsulated islets. Furthermore, the hydrogel capsules exclude large molecules the size of the smallest antibodies. Our results suggest that this microencapsulation technique may be useful for the transplantation of islets for treatment of Type I diabetes.

  6. Pancreatic Ductal Perfusion at Organ Procurement Enhances Islet Yield in Human Islet Isolation

    PubMed Central

    Shimoda, Masayuki; Kanak, Mazhar A.; Shahbazov, Rauf; Kunnathodi, Faisal; Lawrence, Michael C.; Naziruddin, Bashoo; Levy, Marlon F.

    2015-01-01

    Objective Pancreas preservation is a major factor influencing the results of islet cell transplantation. This study evaluated the effects of two different solutions for pancreatic ductal perfusion (PDP) at organ procurement. Methods Eighteen human pancreases were assigned to three groups: non-PDP (control), PDP with ET-Kyoto solution, and PDP with cold storage/purification stock solution. Pancreatic islets were isolated according to the modified Ricordi method. Results No significant differences in donor characteristics, including cold ischemia time, were observed between the three groups. All islet isolations in the PDP groups had >400,000 IEQ in total islet yield post-purification, a significant increase when compared with the control (P = 0.04 and <0.01). The islet quality assessments—including an in vivo diabetic nude mice assay and the response of high-mobility group box protein 1 to cytokine stimulation—also showed no significant differences. The proportion of TUNEL-positive cells showing apoptosis in islets in the PDP groups was significantly lower than in the control group (P < 0.05). Conclusion Both ET-Kyoto solution and cold storage/purification stock solution are suitable for PDP and consistently resulted in isolation success. Further studies with a larger number of pancreas donors should be done to compare the effects of the PDP solutions. PMID:25058879

  7. The influence of porcine pancreas digestion parameters and islet histomorphology on islet isolation outcome.

    PubMed

    Kinasiewicz, J; Sabat, M; Antosiak-Iwańska, M; Godlewska, E; Sitarek, E; Orłowski, T

    2011-01-01

    Transplantation of the pig islets of Langerhans is considered as the future treatment for patients suffering from type I diabetes mellitus. Despite the adaptation of modified Ricordi method and highly purified collagenase, the results of pancreas digestions are precarious. Selection of proper donor and optimal digestion procedure are fundamental. The aim of this study was to assess the impact of pancreas procuring parameters on pig islets yield. The pancreata were harvested from 69 market sows weighting over 150 kg. After intraductal injection of cold collagenase solution pancreata were transported in UW solution or under conditions of two layer method (TLM). In laboratory pancreata were digested at 37 degrees C according to Ricordi isolation method or stationary in the bottle. The particular parameters of isolation procedure were considered as substantial. Pig weight, volume of infused collagenase solution, TLM application and pancreas dividing before digestion positively affected islet yield. Additionally, the influence of pancreatic islet tissue histomorphology on isolation outcome was studied. Proper donor selection as well as adequate digestion parameters could improve pig islet recovery during islet isolation.

  8. Islets of stability beyond period doubling

    SciTech Connect

    Mackay, R.S.

    1981-11-01

    Period doubling of a periodic orbit of an area preserving map appears to lead to the elimination of all closed invariant curves in the vicinity. We show, however, that there are always islets of stabilhty again beyond the accumulation point of the period doubling sequence.

  9. Suppression of ROS Production by Exendin-4 in PSC Attenuates the High Glucose-Induced Islet Fibrosis

    PubMed Central

    Kim, Ji-Won; Park, Shin-Young; You, Young-Hye; Ham, Dong-Sik; Lee, Seung-Hwan; Yang, Hae Kyung; Jeong, In-Kyung; Ko, Seung-Hyun

    2016-01-01

    Pancreatic stellate cells (PSCs) play a major role to fibrotic islet destruction observed in diabetic patients and animal model of diabetes. Exendin-4 (Ex-4) is a potent insulinotropic agent and has been approved for the treatment of type 2 diabetes. However, there have been no reports demonstrating the effects of Ex-4 on pancreatic islet fibrosis. In this study, Ex-4 treatment clearly attenuated fibrotic islet destruction and improved glucose tolerance and islet survival. GLP-1 receptor expression was upregulated during activation and proliferation of PSCs by hyperglycemia. The activation of PKA pathway by Ex-4 plays a role in ROS production and angiotensin II (Ang II) production. Exposure to high glucose stimulated ERK activation and Ang II-TGF- β1 production in PSCs. Interestingly, Ex-4 significantly reduced Ang II and TGF-β1 production by inhibition of ROS production but not ERK phosphorylation. Ex-4 may be useful not only as an anti-diabetic agent but also as an anti-fibrotic agent in type 2 diabetes due to its ability to inhibit PSC activation and proliferation and improve islet fibrosis in OLETF rats. PMID:27977690

  10. Kyoto islet isolation method: the optimized one for non-heart-beating donors with highly efficient islet retrieval.

    PubMed

    Okitsu, T; Matsumoto, S; Iwanaga, Y; Noguchi, H; Nagata, H; Yonekawa, Y; Maekawa, T; Tanaka, K

    2005-10-01

    The availability of pancreata for clinical cadaveric islet transplantation is restricted to non-heart-beating donors (NHBDs) in Japan. This forced us to modify the current standard islet isolation protocol that was made up for brain-dead donors and make it suitable for NHBDs. The Kyoto islet isolation method is the one with induction of several steps based on the ideas both already reported literally and invented originally by ourselves. Using this islet isolation method, we isolated islets from 13 human pancreata of NHBDs and transplanted 11 preparations to six type-1 diabetic patients. The rate to meet release criteria of Edmonton protocol was 84.6%. Establishment of this method allowed us to begin a clinical islet transplantation program in Japan and to continue to perform the preparation of islets from NHBDs with high rate to meet the release criteria of the Edmonton protocol.

  11. 3-D Imaging Reveals Participation of Donor Islet Schwann Cells and Pericytes in Islet Transplantation and Graft Neurovascular Regeneration.

    PubMed

    Juang, Jyuhn-Huarng; Kuo, Chien-Hung; Peng, Shih-Jung; Tang, Shiue-Cheng

    2015-02-01

    The primary cells that participate in islet transplantation are the endocrine cells. However, in the islet microenvironment, the endocrine cells are closely associated with the neurovascular tissues consisting of the Schwann cells and pericytes, which form sheaths/barriers at the islet exterior and interior borders. The two cell types have shown their plasticity in islet injury, but their roles in transplantation remain unclear. In this research, we applied 3-dimensional neurovascular histology with cell tracing to reveal the participation of Schwann cells and pericytes in mouse islet transplantation. Longitudinal studies of the grafts under the kidney capsule identify that the donor Schwann cells and pericytes re-associate with the engrafted islets at the peri-graft and perivascular domains, respectively, indicating their adaptability in transplantation. Based on the morphological proximity and cellular reactivity, we propose that the new islet microenvironment should include the peri-graft Schwann cell sheath and perivascular pericytes as an integral part of the new tissue.

  12. Third Exposure to a Reduced Carbohydrate Meal Lowers Evening Postprandial Insulin and GIP Responses and HOMA-IR Estimate of Insulin Resistance

    PubMed Central

    Lin, Po-Ju; Borer, Katarina T.

    2016-01-01

    Background Postprandial hyperinsulinemia, hyperglycemia, and insulin resistance increase the risk of type 2 diabetes (T2D) and cardiovascular disease mortality. Postprandial hyperinsulinemia and hyperglycemia also occur in metabolically healthy subjects consuming high-carbohydrate diets particularly after evening meals and when carbohydrate loads follow acute exercise. We hypothesized the involvement of dietary carbohydrate load, especially when timed after exercise, and mediation by the glucose-dependent insulinotropic peptide (GIP) in this phenomenon, as this incretin promotes insulin secretion after carbohydrate intake in insulin-sensitive, but not in insulin-resistant states. Methods Four groups of eight metabolically healthy weight-matched postmenopausal women were provided with three isocaloric meals (a pre-trial meal and two meals during the trial day) containing either 30% or 60% carbohydrate, with and without two-hours of moderate-intensity exercise before the last two meals. Plasma glucose, insulin, glucagon, GIP, glucagon-like peptide 1 (GLP-1), free fatty acids (FFAs), and D-3-hydroxybutyrate concentrations were measured during 4-h postprandial periods and 3-h exercise periods, and their areas under the curve (AUCs) were analyzed by mixed-model ANOVA, and insulin resistance during fasting and meal tolerance tests within each diet was estimated using homeostasis-model assessment (HOMA-IR). Results The third low-carbohydrate meal, but not the high-carbohydrate meal, reduced: (1) evening insulin AUC by 39% without exercise and by 31% after exercise; (2) GIP AUC by 48% without exercise and by 45% after exercise, and (3) evening insulin resistance by 37% without exercise and by 24% after exercise. Pre-meal exercise did not alter insulin-, GIP- and HOMA-IR- lowering effects of low-carbohydrate diet, but exacerbated evening hyperglycemia. Conclusions Evening postprandial insulin and GIP responses and insulin resistance declined by over 30% after three meals

  13. Microfluidic platform for assessing pancreatic islet functionality through dielectric spectroscopy

    PubMed Central

    Heileman, K.; Daoud, J.; Hasilo, C.; Gasparrini, M.; Paraskevas, S.; Tabrizian, M.

    2015-01-01

    Human pancreatic islets are seldom assessed for dynamic responses to external stimuli. Thus, the elucidation of human islet functionality would provide insights into the progression of diabetes mellitus, evaluation of preparations for clinical transplantation, as well as for the development of novel therapeutics. The objective of this study was to develop a microfluidic platform for in vitro islet culture, allowing the multi-parametric investigation of islet response to chemical and biochemical stimuli. This was accomplished through the fabrication and implementation of a microfluidic platform that allowed the perifusion of islet culture while integrating real-time monitoring using impedance spectroscopy, through microfabricated, interdigitated electrodes located along the microchamber arrays. Real-time impedance measurements provide important dielectric parameters, such as cell membrane capacitance and cytoplasmic conductivity, representing proliferation, differentiation, viability, and functionality. The perifusion of varying glucose concentrations and monitoring of the resulting impedance of pancreatic islets were performed as proof-of-concept validation of the lab-on-chip platform. This novel technique to elucidate the underlying mechanisms that dictate islet functionality is presented, providing new information regarding islet function that could improve the evaluation of islet preparations for transplantation. In addition, it will lead to a better understanding of fundamental diabetes-related islet dysfunction and the development of therapeutics through evaluation of potential drug effects. PMID:26339324

  14. A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

    PubMed Central

    Zmuda, Erik J.; Powell, Catherine A.; Hai, Tsonwin

    2011-01-01

    Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize their blood glucose levels, islet transplantation has been proposed to be a potential treatment for type 1 diabetes 1,2. More recently, advances in human islet transplantation have further strengthened this view 1,3. However, two major limitations prevent islet transplantation from being a widespread clinical reality: (a) the requirement for large numbers of islets per patient, which severely reduces the number of potential recipients, and (b) the need for heavy immunosuppression, which significantly affects the pediatric population of patients due to their vulnerability to long-term immunosuppression. Strategies that can overcome these limitations have the potential to enhance the therapeutic utility of islet transplantation. Islet transplantation under the mouse kidney capsule is a widely accepted model to investigate various strategies to improve islet transplantation. This experiment requires the isolation of high quality islets and implantation of islets to the diabetic recipients. Both procedures require surgical steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol. We also briefly discuss different transplantation models: syngeneic, allogeneic, syngeneic autoimmune, and allogeneic autoimmune. PMID:21525838

  15. Quadrupole Magnetic Sorting of Porcine Islets of Langerhans

    PubMed Central

    Shenkman, Rustin M.; Chalmers, Jeffrey J.; Hering, Bernhard J.; Kirchhof, Nicole

    2009-01-01

    Islet transplantation is emerging as a treatment option for selected patients with type 1 diabetes. Inconsistent isolation, purification, and recovery of large numbers of high-quality islets remain substantial impediments to progress in the field. Removing islets as soon as they are liberated from the pancreas during digestion and circumventing the need for density gradient purification is likely to result in substantially increased viable islet yields by minimizing exposure to proteolytic enzymes, reactive oxygen intermediates, and mechanical stress associated with centrifugation. This study capitalized on the hypervascularity of islets compared with acinar tissue to explore their preferential enrichment with magnetic beads to enable immediate separation in a magnetic field utilizing a quadrupole magnetic sorting. The results demonstrate that (1) preferential enrichment of porcine islets is achievable, but homogeneous bead distribution within the pancreas is difficult to achieve with current protocols; (2) greater than 70% of islets in the dissociated pancreatic tissue were recovered by quadrupole magnetic sorting, but their purity was low; and (3) infused islets purified by density gradients and subsequently passed through quadrupole magnetic sorting had similar potency as uninfused islets. These results demonstrate proof of concept and define the steps for implementation of this technology in pig and human islet isolation. PMID:19505179

  16. Experimental studies on islets isolation, purification and function in rats.

    PubMed

    Pang, Xinlu; Xue, Wujun; Feng, Xinshun; Tian, Xiaohui; Teng, Yan; Ding, Xiaoming; Pan, Xiaoming; Guo, Qi; He, Xiaoli

    2015-01-01

    To develop a simple and effective method of islet isolation and purification in rats. Collagenase P was injected into pancreatic duct followed by incubation in water bath to digest the pancreas and isolate islet, then discontinuous gravity gradient purification was used to purify the islet. The purified islets were identified by dithizone staining. The viability of islets was assessed by fluorescence staining of acridine orange (AO) and propidium iodide (PI). The function of purified islets was determined by glucose-stimulated insulin release test and transplantation of rat with streptozocin-induced diabetes. 738±193 islets were recovered after purification. The average purity was 77±13%, the viability of islets was more than 95%. When inspected by glucose stimulation, the secreted insulin concentration was 24.31±5.47 mIU/L when stimulated by low concentration glucose and 37.62±4.29 mIU/L by high concentration glucose. There was significant difference between the two phases (P<0.05). The blood sugar concentration recovered to normal level after two days in the animals with islet transplantation. In conclusion, islets can be procured with good function and shape by using the method of injecting collagenase into pancreatic duct followed by incubation in water bath and purification using discontinuous gravity gradient.

  17. Optimizing Porcine Islet Isolation to Markedly Reduce Enzyme Consumption Without Sacrificing Islet Yield or Function

    PubMed Central

    Holdcraft, Robert W.; Green, Michael L.; Breite, Andrew G.; Circle, Lisa; Meyer, Eric D.; Adkins, Hollie; Harbeck, Steven G.; Smith, Barry H.; Gazda, Lawrence S.

    2016-01-01

    Background Human allogeneic islet transplantation for treatment of type 1 diabetes provides numerous clinical benefits, such as fewer episodes of hypoglycemic unawareness and tighter control of blood glucose levels. Availability of human pancreas for clinical and research use, however, is severely limited. Porcine pancreas offers an abundant source of tissue for optimization of islet isolation methodology and future clinical transplantation, thereby increasing patient access to this potentially lifesaving procedure. Methods Porcine islet isolations were performed using varying amounts of collagenase (7.5, 3.75, or 2.5 Wunsch units per gram tissue) and neutral protease activity (12 000, 6000, or 4000 neutral protease units per gram tissue) and perfusion volumes (1.7 or 0.85 mL/g tissue) to assess their effects on isolation outcomes. Retention of dissociative enzymes within the pancreas during perfusion and digestion was evaluated, along with distribution of the perfusion solution within the tissue. Results Reducing enzyme usage by as much as 67% and perfusion volume by 50% led to equally successful islet isolation outcomes when compared with the control group (48 ± 7% of tissue digested and 1088 ± 299 islet equivalents per gram of pancreas vs 47 ± 11% and 1080 ± 512, respectively). Using margin-marking dye in the perfusion solution to visualize enzyme distribution demonstrated that increasing perfusion volume did not improve tissue infiltration. Conclusions Current protocols for porcine islet isolation consume excessive amounts of dissociative enzymes, elevating cost and limiting research and development. These data demonstrate that islet isolation protocols can be optimized to significantly reduce enzyme usage while maintaining yield and function and thus accelerating progress toward clinical application. PMID:27830180

  18. A Method for Performing Islet Transplantation Using Tissue-Engineered Sheets of Islets and Mesenchymal Stem Cells

    PubMed Central

    Hirabaru, Masataka; Kuroki, Tamotsu; Adachi, Tomohiko; Kitasato, Amane; Ono, Shinichiro; Tanaka, Takayuki; Matsushima, Hajime; Sakai, Yusuke; Soyama, Akihiko; Hidaka, Masaaki; Yamanouchi, Kosho; Takatsuki, Mitsuhisa; Okano, Teruo

    2015-01-01

    Mesenchymal stem cells (MSCs) are known to have a protective effect on islet cells. Cell sheets developed using tissue engineering help maintain the function of the cells themselves. This study describes a tissue engineering approach using islets with MSC sheets to improve the therapeutic effect of islet transplantation. MSCs were obtained from Fischer 344 rats and engineered into cell sheets using temperature-responsive culture dishes. The islets obtained from Fischer 344 rats were seeded onto MSC sheets, and the islets with MSC sheets were harvested by low-temperature treatment after coculture. The functional activity of the islets with MSC sheets was confirmed by a histological examination, insulin secretion assay, and quantification of the levels of cytokines. The therapeutic effects of the islets with MSC sheets were investigated by transplanting the sheets at subcutaneous sites in severe combined immunodeficiency (SCID) mice with streptozotocin-induced diabetes. Improvement of islet function and viability was shown in situ on the MSC sheet, and the histological examination showed that the MSC sheet maintained adhesion factor on the surface. In the recipient mice, normoglycemia was maintained for at least 84 days after transplantation, and neovascularization was observed. These results demonstrated that islet transplantation in a subcutaneous site would be possible by using the MSC sheet as a scaffold for islets. PMID:26066973

  19. The isolation and purification of rodent pancreatic islets of Langerhans.

    PubMed

    O'Dowd, Jacqueline F

    2009-01-01

    This chapter describes the detailed protocol for the isolation and purification of islets of Langerhans from rodent pancreas using collagenase digestion. The first step of the process is to separate and isolate the insulin-producing islets of Langerhans from the rest of the pancreas. The pancreas is excised from the animal, trimmed of non-pancreatic tissues before being inflated and chopped into small pieces. The connective tissue is then broken down with a collagenase enzyme solution to selectively digest the bulk of the exocrine tissue while leaving the endocrine islets intact and separated from their surrounding non-islet tissue. Once this process is completed, the islets of Langerhans are separated from the remaining mixture by centrifugation and purified by the means of hand picking. Once isolated, the subsequent islets can be used for a number of experimental processes.

  20. Heavy metals, islet function and diabetes development.

    PubMed

    Chen, Ya Wen; Yang, Ching Yao; Huang, Chun Fa; Hung, Dong Zong; Leung, Yuk Man; Liu, Shing Hwa

    2009-01-01

    It has long been believed that heavy metals possess many adverse health effects. Uncontrolled industrialization has released heavy metal pollution in the world. Heavy metal pollutants damage organ functions and disrupt physiological homeostasis. Diabetes mellitus is growing in prevalence worldwide. Several studies have indicated that the deficiency and efficiency of some essential trace metals may play a role in the islet function and development of diabetes mellitus. Some toxic metals have also been shown to be elevated in biological samples of diabetes mellitus patients. In the present work, we review the important roles of heavy metals in islet function and diabetes development in which the in vitro, in vivo or human evidences are associated with exposure to zinc, arsenic, cadmium, mercury and nickel. Through this work, we summarize the evidence which suggests that some heavy metals may play an important role in diabetes mellitus as environmental risk factors.

  1. Core-shell hydrogel microcapsules for improved islets encapsulation.

    PubMed

    Ma, Minglin; Chiu, Alan; Sahay, Gaurav; Doloff, Joshua C; Dholakia, Nimit; Thakrar, Raj; Cohen, Joshua; Vegas, Arturo; Chen, Delai; Bratlie, Kaitlin M; Dang, Tram; York, Roger L; Hollister-Lock, Jennifer; Weir, Gordon C; Anderson, Daniel G

    2013-05-01

    Islets microencapsulation holds great promise to treat type 1 diabetes. Currently used alginate microcapsules often have islets protruding outside capsules, leading to inadequate immuno-protection. A novel design of microcapsules with core-shell structures using a two-fluid co-axial electro-jetting is reported. Improved encapsulation and diabetes correction is achieved in a single step by simply confining the islets in the core region of the capsules.

  2. An immunocytochemical and morphometric study of the rat pancreatic islets.

    PubMed Central

    Elayat, A A; el-Naggar, M M; Tahir, M

    1995-01-01

    The rat pancreas has frequently been used as an animal model to study changes in islet cells in pathological conditions, such as diabetes mellitus and islet cell tumours, but detailed quantitative data on the islets are not available. This study was therefore undertaken to investigate (1) the volume density of pancreatic islets, (2) islet diameter, islet volume and islet cell number and (3) islet cell pattern, i.e. the distribution, volume and number of each cell type per islet. The study also investigated the possibility of differences in various pancreatic regions derived from the dorsal primordium. The rat pancreas was divided into 4 regions: lower duodenal (derived from the ventral primordium) and upper duodenal, gastric and splenic regions (derived from the dorsal primordium). Sections were stained immunocytochemically with anti-insulin (B cells), antiglucagon (A cells), antisomatostatin (D cells) and antipancreatic polypeptide (PP cells) antibodies, and were used for morphometric analysis. A total of 1292 islets was examined, 328 from the lower duodenal, 245 from the upper duodenal, 314 from the gastric and 405 from the splenic regions. The mean volume density of the islets per pancreatic tissue was found to be 2.6 +/- 0.1%, 2.3 +/- 0.1%, 2.9 +/- 0.2% and 3.3 +/- 0.2%, in the lower duodenal, upper duodenal, gastric and splenic regions, respectively. The size-frequency distribution of the profile diameters of the islets showed an overall shift of all the size classes towards smaller sizes in the upper duodenal region, and towards larger sizes in the splenic region, as compared with the corresponding classes of the other regions.(ABSTRACT TRUNCATED AT 250 WORDS) Images Fig. 4 Fig. 5 PMID:7559135

  3. 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

  4. CT features of nonfunctioning islet cell carcinoma

    SciTech Connect

    Eelkema, E.A.; Stephens, D.H.; Ward, E.M.; Sheedy, P.F. II

    1984-11-01

    To determine the computed tomographic (CT) characteristics of nonfunctioning islet cell carcinoma of the pancreas, the CT scans of 27 patients with that disease were reviewed. The pancreatic tumor was identified as a mass in 26 patients (96%) Of the 25 tumors evaluated with contrast enhancement, 20 became partially diffusely hyperdense relative to nearby normal pancreatic tissue. Hepatic metastases were identified in 15 patients (56%), regional lymphadenopathy in 10 (37%), atrophy of the gland proximal to the tumor in six (22%), dilatation of the biliary ducts in five (19%), and dilatation of the pancreatic duct in four (15%). The CT appearances of the nonfunctioning islet cell tumors were compared with those of 100 ordinary (ductal) pancreatic adenocarcinomas. Although the two types of tumors were sometimes indistinguishable, features found to be more characteristic of islet cell carcinoma included a pancreatic mass of unusually large size, calcification within the tumor, and contrast enhancement of either the primary tumor or hepatic metastases. Involvement of the celiac axis or proximal superior mesenteric artery was limited to ductal carcinoma.

  5. Islets of Langerhans from prohormone convertase-2 knockout mice show α-cell hyperplasia and tumorigenesis with elevated α-cell neogenesis.

    PubMed

    Jones, Huw B; Reens, Jaimini; Brocklehurst, Simon R; Betts, Catherine J; Bickerton, Sue; Bigley, Alison L; Jenkins, Richard P; Whalley, Nicky M; Morgan, Derrick; Smith, David M

    2014-02-01

    Antagonism of the effects of glucagon as an adjunct therapy with other glucose-lowering drugs in the chronic treatment of diabetes has been suggested to aggressively control blood glucose levels. Antagonism of glucagon effects, by targeting glucagon secretion or disabling the glucagon receptor, is associated with α-cell hyperplasia. We evaluated the influence of total glucagon withdrawal on islets of Langerhans using prohormone convertase-2 knockout mice (PC2-ko), in which α-cell hyperplasia is present from a young age and persists throughout life, in order to understand whether or not sustained glucagon deficit would lead to islet tumorigenesis. PC2-ko and wild-type (WT) mice were maintained drug-free, and cohorts of these groups sampled at 3, 12 and 18 months for plasma biochemical and morphological (histological, immunohistochemical, electron microscopical and image analytical) assessments. WT mice showed no islet tumours up to termination of the study, but PC2-ko animals displayed marked changes in islet morphology from α-cell hypertrophy/hyperplasia/atypical hyperplasia, to adenomas and carcinomas, these latter being first encountered at 6-8 months. Islet hyperplasias and tumours primarily consisted of α-cells associated to varying degrees with other islet endocrine cell types. In addition to substantial increases in islet neoplasia, increased α-cell neogenesis associated primarily with pancreatic duct(ule)s was present. We conclude that absolute blockade of the glucagon signal results in tumorigenesis and that the PC2-ko mouse represents a valuable model for investigation of islet tumours and pancreatic ductal neogenesis.

  6. 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.

  7. The effect of ghrelin on Kiss-1 and KissR gene transcription and insulin secretion in rat islets of Langerhans and CRI-D2 cell line

    PubMed Central

    Sagheb, Mandana Mahmoodzaeh; Azarpira, Negar; Mokhtary, Mokhtar

    2017-01-01

    Objective(s): Ghrelin is a peptide hormone that has been shown to have numerous central and peripheral effects. The central effects including GH secretion, food intake, and energy homeostasis are partly mediated by Kiss1- KissR signaling pathway. Ghrelin and its receptor are also expressed in the pancreatic islets. Ghrelin is one of the key metabolic factors controlling insulin secretion from the islets of Langerhans. We hypothesize that the inhibitory effect of ghrelin on KiSS-1 and KissR in the islet cells may be similar to the same inhibitory effect of ghrelin in the hypothalamus. Materials and Methods: To investigate the effect of ghrelin, we isolated the islets from adult male rats by collagenase and cultured CRI-D2 cell lines. Then, we incubated them with different concentrations of ghrelin for 24 hr. After RNA extraction and cDNA synthesis from both islets and CRI-D2 cells, the relative expression of KiSS-1 and KissR was evaluated by means of real-time PCR. Furthermore, we measured the amount of insulin secreted by the islets after incubation in different concentrations of ghrelin and glucose after 1 hr. Besides, we checked the viability of the cells after 24 hr cultivation. Results: Ghrelin significantly decreased the KiSS-1 and KissR mRNA transcription in rat islets and CRI-D2 cells. Besides, Ghrelin suppressed insulin secretion from pancreatic beta cells and CRI-D2 cells. Conclusion: These findings indicate the possibility that KiSS-1 and KissR mRNA expression is mediator of ghrelin function in the islets of Langerhans. PMID:28133522

  8. Imaging pancreatic islet cells by positron emission tomography

    PubMed Central

    Li, Junfeng; Karunananthan, Johann; Pelham, Bradley; Kandeel, Fouad

    2016-01-01

    It was estimated that every year more than 30000 persons in the United States - approximately 80 people per day - are diagnosed with type 1 diabetes (T1D). T1D is caused by autoimmune destruction of the pancreatic islet (β cells) cells. Islet transplantation has become a promising therapy option for T1D patients, while the lack of suitable tools is difficult to directly evaluate of the viability of the grafted islet over time. Positron emission tomography (PET) as an important non-invasive methodology providing high sensitivity and good resolution, is able to accurate detection of the disturbed biochemical processes and physiological abnormality in living organism. The successful PET imaging of islets would be able to localize the specific site where transplanted islets engraft in the liver, and to quantify the level of islets remain alive and functional over time. This information would be vital to establishing and evaluating the efficiency of pancreatic islet transplantation. Many novel imaging agents have been developed to improve the sensitivity and specificity of PET islet imaging. In this article, we summarize the latest developments in carbon-11, fluorine-18, copper-64, and gallium-68 labeled radioligands for the PET imaging of pancreatic islet cells. PMID:27721939

  9. Human Islets Exhibit Electrical Activity on Microelectrode Arrays (MEA).

    PubMed

    Schönecker, S; Kraushaar, U; Guenther, E; Gerst, F; Ullrich, S; Häring, H-U; Königsrainer, A; Barthlen, W; Drews, G; Krippeit-Drews, P

    2015-05-01

    This study demonstrates for the first time that the microelectrode array (MEA) technique allows analysis of electrical activity of islets isolated from human biopsies. We have shown before that this method, i.e., measuring beta cell electrical activity with extracellular electrodes, is a powerful tool to assess glucose responsiveness of isolated murine islets. In the present study, human islets were shown to exhibit glucose-dependent oscillatory electrical activity. The glucose responsiveness could be furthermore demonstrated by an increase of insulin secretion in response to glucose. Electrical activity was increased by tolbutamide and inhibited by diazoxide. In human islets bursts of electrical activity were markedly blunted by the Na(+) channel inhibitor tetrodotoxin which does not affect electrical activity in mouse islets. Thus, the MEA technique emerges as a powerful tool to decipher online the unique features of human islets.Additionally, this technique will enable research with human islets even if only a few islets are available and it will allow a fast and easy test of metabolic integrity of islets destined for transplantation.

  10. Comparison of volume estimation methods for pancreatic islet cells

    NASA Astrophysics Data System (ADS)

    Dvořák, JiřÃ.­; Å vihlík, Jan; Habart, David; Kybic, Jan

    2016-03-01

    In this contribution we study different methods of automatic volume estimation for pancreatic islets which can be used in the quality control step prior to the islet transplantation. The total islet volume is an important criterion in the quality control. Also, the individual islet volume distribution is interesting -- it has been indicated that smaller islets can be more effective. A 2D image of a microscopy slice containing the islets is acquired. The input of the volume estimation methods are segmented images of individual islets. The segmentation step is not discussed here. We consider simple methods of volume estimation assuming that the islets have spherical or ellipsoidal shape. We also consider a local stereological method, namely the nucleator. The nucleator does not rely on any shape assumptions and provides unbiased estimates if isotropic sections through the islets are observed. We present a simulation study comparing the performance of the volume estimation methods in different scenarios and an experimental study comparing the methods on a real dataset.

  11. Implication of Mitochondrial Cytoprotection in Human Islet Isolation and Transplantation

    PubMed Central

    Wang, Yong; Mendoza-Elias, Joshua E.; Qi, Meirigeng; Harvat, Tricia A.; Ahn, Sang Joon; Lee, Dongyoung; Gutierrez, Diana; Jeon, Hyojin; Paushter, Daniel; Oberholzer, José

    2012-01-01

    Islet transplantation is a promising therapy for type 1 diabetes mellitus; however, success rates in achieving both short- and long-term insulin independence are not consistent, due in part to inconsistent islet quality and quantity caused by the complex nature and multistep process of islet isolation and transplantation. Since the introduction of the Edmonton Protocol in 2000, more attention has been placed on preserving mitochondrial function as increasing evidences suggest that impaired mitochondrial integrity can adversely affect clinical outcomes. Some recent studies have demonstrated that it is possible to achieve islet cytoprotection by maintaining mitochondrial function and subsequently to improve islet transplantation outcomes. However, the benefits of mitoprotection in many cases are controversial and the underlying mechanisms are unclear. This article summarizes the recent progress associated with mitochondrial cytoprotection in each step of the islet isolation and transplantation process, as well as islet potency and viability assays based on the measurement of mitochondrial integrity. In addition, we briefly discuss immunosuppression side effects on islet graft function and how transplant site selection affects islet engraftment and clinical outcomes. PMID:22611495

  12. Polymeric Nanomaterials for Islet Targeting and Immunotherapeutic Delivery

    PubMed Central

    Ghosh, Kaustabh; Kanapathipillai, Mathumai; Korin, Netanel; McCarthy, Jason R.; Ingber, Donald E.

    2012-01-01

    Here we report a proof-of-concept for development of pancreatic islet-targeting nanoparticles for immunomodulatory therapy of autoimmune Type 1 Diabetes. Modified with a unique islet-homing peptide, these polymeric nanomaterials exhibit 3-fold greater binding to islet endothelial cells and a 200-fold greater anti-inflammatory effect through targeted islet endothelial cell delivery of an immunosuppressant drug. Our findings also underscore the need to carefully tailor drug loading and nanoparticle dosage to achieve maximal vascular targeting and immunosuppression. PMID:22196766

  13. Effect of polyethylene glycol grafted onto islet capsules on prevention of splenocyte and cytokine attacks.

    PubMed

    Lee, Dong Yun; Nam, Jong Hee; Byun, Youngro

    2004-01-01

    In the graft rejection of transplanted islets, the host's immune cells recognize the islets as antigens, which then stimulate the immune cells to begin the cytokine secretion and also the proliferation of immune cells. To prevent the recognition of islets by the immune cells, we grafted biocompatible polyethylene glycol (PEG) onto the collagen capsule of islets without incurring any changes in the morphology and function of islets. To evaluate the efficiency of PEG grafting, PEG-grafted islets were cultured with splenocytes consisting mainly of lymphocytes and macrophages. A splenocyte proliferation assessment using a BrdU incorporation assay showed that the PEG-grafted islets did not stimulate the splenocytes. In addition, the viability and microorganisms in islet cells of co-cultured PEG-grafted islets were not altered. However, in the co-culture of free islets (control) splenocytes were stimulated; they mainly secreted TNF-alpha and strongly affected the viability and structure of free islets. Furthermore, when islets were treated with the rat recombinant TNF-alpha for 7 days, the viabilities of PEG-grafted and free islets were significantly damaged, although the viability of PEG-grafted islets was higher than that of free islets by nearly three times. These results demonstrate that PEG grafted on the surface of islets could prevent the recognition of islets by splenocytes, but could not completely protect islets from cytokines.

  14. Different responses of mouse islets and MIN6 pseudo-islets to metabolic stimulation: a note of caution.

    PubMed

    Schulze, Torben; Morsi, Mai; Brüning, Dennis; Schumacher, Kirstin; Rustenbeck, Ingo

    2016-03-01

    MIN6 cells and MIN6 pseudo-islets are popular surrogates for the use of primary beta cells and islets. Even though it is generally agreed that the stimulus-secretion coupling may deviate from that of beta cells or islets, direct comparisons are rare. The present side-by-side comparison of insulin secretion, cytosolic Ca(2+) concentration ([Ca(2+)] i ) and oxygen consumption rate (OCR) points out where similarities and differences exist between MIN6 cells and normal mouse beta cells. In mouse islets and MIN6 pseudo-islets depolarization by 40 mM KCl was a more robust insulinotropic stimulus than 30 mM glucose. In MIN6 pseudo-islets, but not in mouse islets, the response to 30 mM glucose was much lower than to 40 mM KCl and could be suppressed by a preceding stimulation with 40 mM KCl. In MIN6 pseudo-islets, glucose was less effective to raise [Ca(2+)] i than in primary islets. In marked contrast to islets, the OCR response of MIN6 pseudo-islets to 30 mM glucose was smaller than to 40 mM KCl and was further diminished by a preceding stimulation with 40 mM KCl. The same pattern was observed when MIN6 pseudo-islets were cultured in 5 mM glucose. As with insulin secretion memory effects on the OCR remained after wash-out of a stimulus. The differences between MIN6 cells and primary beta cells were generally larger in the responses to glucose than to depolarization by KCl. Thus, the use of MIN6 cells in investigations on metabolic signalling requires particular caution.

  15. Staining Protocols for Human Pancreatic Islets

    PubMed Central

    Campbell-Thompson, Martha L.; Heiple, Tiffany; Montgomery, Emily; Zhang, Li; Schneider, Lynda

    2012-01-01

    Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and beta mass ranges from 500 to 1500 mg 1-3. With this known heterogeneity, a standard processing and staining procedure was developed so that pancreatic regions were clearly defined and islets characterized using rigorous histopathology and immunolocalization examinations. Standardized procedures for processing human pancreas recovered from organ donors are described in part 1 of this series. The pancreas is processed into 3 main regions (head, body, tail) followed by transverse sections. Transverse sections from the pancreas head are further divided, as indicated based on size, and numbered alphabetically to denote subsections. This standardization allows for a complete cross sectional analysis of the head region including the uncinate region which contains islets composed primarily of pancreatic polypeptide cells to the tail region. The current report comprises part 2 of this series and describes the procedures used for serial sectioning and histopathological characterization of the pancreatic paraffin sections with an emphasis on islet endocrine cells, replication, and T-cell infiltrates. Pathology of pancreatic sections is intended to characterize both exocrine, ductular, and endocrine components. The exocrine compartment is evaluated for the presence of pancreatitis (active or chronic), atrophy, fibrosis, and fat, as well as the duct system, particularly in relationship to the presence of pancreatic intraductal neoplasia4. Islets are evaluated for morphology, size, and density, endocrine cells, inflammation, fibrosis, amyloid, and the presence of replicating or apoptotic cells using H&E and IHC stains. The final component described in part 2 is the provision of the stained slides as digitized whole slide images. The digitized slides are organized by case and pancreas region in an online pathology database

  16. Staining protocols for human pancreatic islets.

    PubMed

    Campbell-Thompson, Martha L; Heiple, Tiffany; Montgomery, Emily; Zhang, Li; Schneider, Lynda

    2012-05-23

    Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and beta mass ranges from 500 to 1500 mg. With this known heterogeneity, a standard processing and staining procedure was developed so that pancreatic regions were clearly defined and islets characterized using rigorous histopathology and immunolocalization examinations. Standardized procedures for processing human pancreas recovered from organ donors are described in part 1 of this series. The pancreas is processed into 3 main regions (head, body, tail) followed by transverse sections. Transverse sections from the pancreas head are further divided, as indicated based on size, and numbered alphabetically to denote subsections. This standardization allows for a complete cross sectional analysis of the head region including the uncinate region which contains islets composed primarily of pancreatic polypeptide cells to the tail region. The current report comprises part 2 of this series and describes the procedures used for serial sectioning and histopathological characterization of the pancreatic paraffin sections with an emphasis on islet endocrine cells, replication, and T-cell infiltrates. Pathology of pancreatic sections is intended to characterize both exocrine, ductular, and endocrine components. The exocrine compartment is evaluated for the presence of pancreatitis (active or chronic), atrophy, fibrosis, and fat, as well as the duct system, particularly in relationship to the presence of pancreatic intraductal neoplasia. Islets are evaluated for morphology, size, and density, endocrine cells, inflammation, fibrosis, amyloid, and the presence of replicating or apoptotic cells using H&E and IHC stains. The final component described in part 2 is the provision of the stained slides as digitized whole slide images. The digitized slides are organized by case and pancreas region in an online pathology database creating a

  17. Stimulation of β-adrenergic receptors plays a protective role via increased expression of RAF-1 and PDX-1 in hyperglycemic rat pancreatic islet (RIN-m5F) cells

    PubMed Central

    Qvist, Rajes; Ong, Gracie; Karimian, Hamed; Imran, Muhammad; Shah, Ikram

    2016-01-01

    Introduction It is a widely held view that a progressive reduction of beta-cell mass occurs in the progression of diabetes. RAF-1 kinase and pancreas duodenal homeobox 1 (PDX-1) are major factors that promote survival of cells and maintain normal insulin functions. In this study we investigated the effect of a β-adrenergic receptor agonist and antagonist on RAF-1 and PDX-1, and their respective effects on apoptosis and insulin release in RIN-m5F cells. Material and methods RIN-m5F cells were cultured in normal (5 mM) and high (25 mM) glucose to mimic diabetic conditions, followed by treatment with 5 µM, 10 µM and 20 µM of isoproterenol and isoproterenol + propranolol for 6, 12 and 24 h. Western blotting and reverse transcription analysis were performed to examine the expression of RAF-1 and PDX-1. Annexin-V-FITC and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assays were used to investigate apoptosis. ELISA was used to measure insulin levels. Reverse transcription polymerase chain reaction was conducted to investigate the expression of genes. Results Stimulation of β-adrenergic receptors with isoproterenol significantly induced RAF-1 and PDX-1 genes in a concentration-dependent and time-independent manner. Changes were significant both at protein and mRNA levels. Up-regulation of RAF-1 and PDX-1 was accompanied by improved insulin levels and reduced apoptosis. Concentrations of 10 µM and 20 µM for 12 and 24 h were more effective in achieving significant differences in the experimental and control groups. Propranolol reversed the effect of isoproterenol mostly at maximum concentrations and time periods. Conclusions A positive effect of a β-adrenergic agonist on RAF-1 and PDX-1, reduction in β-cell apoptosis and improved insulin contents can help to understand the pathogenesis of diabetes and to develop novel approaches for the β-cell dysfunction in diabetes. PMID:28261303

  18. Alpha1-antitrypsin monotherapy prolongs islet allograft survival in mice.

    PubMed

    Lewis, Eli C; Shapiro, Leland; Bowers, Owen J; Dinarello, Charles A

    2005-08-23

    Islet transplantation for type 1 diabetic patients shows promising results with the use of nondiabetogenic immunosuppressive therapy. However, in addition to compromising the immune system of transplant recipients, long-term studies demonstrate that islet viability is impaired. Here, we demonstrate that, in the absence of immunosuppressive agents, monotherapy with clinical-grade human alpha1-antitrypsin (hAAT), the major serum serine-protease inhibitor, prolongs islet graft survival and normoglycemia in transplanted allogeneic diabetic mice, lasting until the development of anti-hAAT antibodies. Compared to untreated or albumin-control-treated graft recipients, which rejected islets at day 10, AAT-treated mice displayed diminished cellular infiltrates and intact intragraft insulin production throughout treatment. Using peritoneal infiltration models, we demonstrate that AAT decreases allogeneic fibroblast-elicited natural-killer-cell influx by 89%, CD3-positive cell influx by 44%, and thioglycolate-elicited neutrophil emigration by 66%. ATT also extended islet viability in mice after streptozotocin-induced beta cell toxicity. In vitro, several islet responses to IL-1beta/IFNgamma stimulation were examined. In the presence of AAT, islets displayed enhanced viability and inducible insulin secretion. Islets also released 36% less nitric oxide and 82% less macrophage inflammatory protein 1 alpha and expressed 63% fewer surface MHC class II molecules. TNFalpha release from IL-1beta/IFNgamma-stimulated islet cells was reduced by 99%, accompanied by an 8-fold increase in the accumulation of membrane TNFalpha on CD45-positive islet cells. In light of the established safety record and the nondiabetogenic potential of AAT, these data suggest that AAT may be beneficial as adjunctive therapy in patients undergoing islet transplantation.

  19. Enhancement of in vitro and in vivo function of agarose-encapsulated porcine islets by changes in the islet microenvironment.

    PubMed

    Holdcraft, Robert W; Gazda, Lawrence S; Circle, Lisa; Adkins, Hollie; Harbeck, Steven G; Meyer, Eric D; Bautista, Melissa A; Martis, Prithy C; Laramore, Melissa A; Vinerean, Horatiu V; Hall, Richard D; Smith, Barry H

    2014-01-01

    The transplantation of porcine islets of Langerhans to treat type 1 diabetes may provide a solution to the demand for insulin-producing cells. Porcine islets encapsulated in agarose-agarose macrobeads have been shown to function in nonimmunosuppressed xenogeneic models of both streptozotocin-induced and autoimmune type 1 diabetes. One advantage of agarose encapsulation is the ability to culture macrobeads for extended periods, permitting microbiological and functional assessment. Herein we describe optimization of the agarose matrix that results in improved islet function. Porcine islets (500 IEQs) from retired breeding sows were encapsulated in 1.5% SeaKem Gold (SG), 0.8% SG, or 0.8% Litex (Li) agarose, followed by an outer capsule of 5% SG agarose. Insulin production by the encapsulated islets exhibited an agarose-specific effect with 20% (0.8% SG) to 50% (0.8% Li) higher initial insulin production relative to 1.5% SG macrobeads. Insulin production was further increased by 40-50% from week 2 to week 12 in both agarose types at the 0.8% concentration, whereas islets encapsulated in 1.5% SG agarose increased insulin production by approximately 20%. Correspondingly, fewer macrobeads were required to restore normoglycemia in streptozotocin-induced diabetic female CD(SD) rats that received 0.8% Li (15 macrobeads) or 0.8% SG (17 macrobeads) as compared to 1.5% SG (19 macrobeads). Islet cell proliferation was also observed during the first 2 months postencapsulation, peaking at 4 weeks, where approximately 50% of islets contained proliferative cells, including β-cells, regardless of agarose type. These results illustrate the importance of optimizing the microenvironment of encapsulated islets to improve islet performance and advance the potential of islet xenotransplantation for the treatment of type 1 diabetes.

  20. Induction of Protective Genes Leads to Islet Survival and Function

    PubMed Central

    Wang, Hongjun; Ferran, Christiane; Attanasio, Chiara; Calise, Fulvio; Otterbein, Leo E.

    2011-01-01

    Islet transplantation is the most valid approach to the treatment of type 1 diabetes. However, the function of transplanted islets is often compromised since a large number of β cells undergo apoptosis induced by stress and the immune rejection response elicited by the recipient after transplantation. Conventional treatment for islet transplantation is to administer immunosuppressive drugs to the recipient to suppress the immune rejection response mounted against transplanted islets. Induction of protective genes in the recipient (e.g., heme oxygenase-1 (HO-1), A20/tumor necrosis factor alpha inducible protein3 (tnfaip3), biliverdin reductase (BVR), Bcl2, and others) or administration of one or more of the products of HO-1 to the donor, the islets themselves, and/or the recipient offers an alternative or synergistic approach to improve islet graft survival and function. In this perspective, we summarize studies describing the protective effects of these genes on islet survival and function in rodent allogeneic and xenogeneic transplantation models and the prevention of onset of diabetes, with emphasis on HO-1, A20, and BVR. Such approaches are also appealing to islet autotransplantation in patients with chronic pancreatitis after total pancreatectomy, a procedure that currently only leads to 1/3 of transplanted patients being diabetes-free. PMID:22220267

  1. Microencapsulation of pancreatic islets in a water insoluble polyacrylate.

    PubMed

    Sugamori, M E; Sefton, M V

    1989-01-01

    Rat pancreatic islets were encapsulated in a water insoluble polyacrylate (Eudragit RL), a model polymer, by coaxial extrusion and interfacial precipitation. Despite exposure to organic solvents and nonsolvents (diethyl phthalate, corn oil, and mineral oil) and to shear, the islets survived encapsulation. They continued to secrete insulin into the tissue culture medium and responded to glucose in both static glucose challenges and perifusion assays as well and as long as control islets which were not encapsulated, but were maintained in tissue culture alongside the encapsulated islets. Unfortunately, there was a great deal of variability in the performance of all islets studied, making unequivocal conclusions difficult. Some encapsulated islets survived more than 140 days in vitro and histologically appeared healthy. However, there appeared to be a general deterioration in insulin secretion capacity following prolonged culture in all islets, with corresponding changes (e.g., central necrosis) visible by microscopy. Although Eudragit RL is not practical as an encapsulation polymer, this study was useful in demonstrating that islets may be encapsulated in materials other than alginate-polylysine, and ultimately in materials that may have a more optimum blend of the desired properties: biocompatibility, permselectivity, and mechanical durability.

  2. Autologous Pancreatic Islet Transplantation in Human Bone Marrow

    PubMed Central

    Maffi, Paola; Balzano, Gianpaolo; Ponzoni, Maurilio; Nano, Rita; Sordi, Valeria; Melzi, Raffaella; Mercalli, Alessia; Scavini, Marina; Esposito, Antonio; Peccatori, Jacopo; Cantarelli, Elisa; Messina, Carlo; Bernardi, Massimo; Del Maschio, Alessandro; Staudacher, Carlo; Doglioni, Claudio; Ciceri, Fabio; Secchi, Antonio; Piemonti, Lorenzo

    2013-01-01

    The liver is the current site of choice for pancreatic islet transplantation, even though it is far from being ideal. We recently have shown in mice that the bone marrow (BM) may be a valid alternative to the liver, and here we report a pilot study to test feasibility and safety of BM as a site for islet transplantation in humans. Four patients who developed diabetes after total pancreatectomy were candidates for the autologous transplantation of pancreatic islet. Because the patients had contraindications for intraportal infusion, islets were infused in the BM. In all recipients, islets engrafted successfully as shown by measurable posttransplantation C-peptide levels and histopathological evidence of insulin-producing cells or molecular markers of endocrine tissue in BM biopsy samples analyzed during follow-up. Thus far, we have recorded no adverse events related to the infusion procedure or the presence of islets in the BM. Islet function was sustained for the maximum follow-up of 944 days. The encouraging results of this pilot study provide new perspectives in identifying alternative sites for islet infusion in patients with type 1 diabetes. Moreover, this is the first unequivocal example of successful engraftment of endocrine tissue in the BM in humans. PMID:23733196

  3. Islet and stem cell encapsulation for clinical transplantation.

    PubMed

    Krishnan, Rahul; Alexander, Michael; Robles, Lourdes; Foster, Clarence E; Lakey, Jonathan R T

    2014-01-01

    Over the last decade, improvements in islet isolation techniques have made islet transplantation an option for a certain subset of patients with long-standing diabetes. Although islet transplants have shown improved graft function, adequate function beyond the second year has not yet been demonstrated, and patients still require immunosuppression to prevent rejection. Since allogeneic islet transplants have experienced some success, the next step is to improve graft function while eliminating the need for systemic immunosuppressive therapy. Biomaterial encapsulation offers a strategy to avoid the need for toxic immunosuppression while increasing the chances of graft function and survival. Encapsulation entails coating cells or tissue in a semipermeable biocompatible material that allows for the passage of nutrients, oxygen, and hormones while blocking immune cells and regulatory substances from recognizing and destroying the cell, thus avoiding the need for systemic immunosuppressive therapy. Despite advances in encapsulation technology, these developments have not yet been meaningfully translated into clinical islet transplantation, for which several factors are to blame, including graft hypoxia, host inflammatory response, fibrosis, improper choice of biomaterial type, lack of standard guidelines, and post-transplantation device failure. Several new approaches, such as the use of porcine islets, stem cells, development of prevascularized implants, islet nanocoating, and multilayer encapsulation, continue to generate intense scientific interest in this rapidly expanding field. This review provides a comprehensive update on islet and stem cell encapsulation as a treatment modality in type 1 diabetes, including a historical outlook as well as current and future research avenues.

  4. Islet and Stem Cell Encapsulation for Clinical Transplantation

    PubMed Central

    Krishnan, Rahul; Alexander, Michael; Robles, Lourdes; Foster 3rd, Clarence E.; Lakey, Jonathan R.T.

    2014-01-01

    Over the last decade, improvements in islet isolation techniques have made islet transplantation an option for a certain subset of patients with long-standing diabetes. Although islet transplants have shown improved graft function, adequate function beyond the second year has not yet been demonstrated, and patients still require immunosuppression to prevent rejection. Since allogeneic islet transplants have experienced some success, the next step is to improve graft function while eliminating the need for systemic immunosuppressive therapy. Biomaterial encapsulation offers a strategy to avoid the need for toxic immunosuppression while increasing the chances of graft function and survival. Encapsulation entails coating cells or tissue in a semipermeable biocompatible material that allows for the passage of nutrients, oxygen, and hormones while blocking immune cells and regulatory substances from recognizing and destroying the cell, thus avoiding the need for systemic immunosuppressive therapy. Despite advances in encapsulation technology, these developments have not yet been meaningfully translated into clinical islet transplantation, for which several factors are to blame, including graft hypoxia, host inflammatory response, fibrosis, improper choice of biomaterial type, lack of standard guidelines, and post-transplantation device failure. Several new approaches, such as the use of porcine islets, stem cells, development of prevascularized implants, islet nanocoating, and multilayer encapsulation, continue to generate intense scientific interest in this rapidly expanding field. This review provides a comprehensive update on islet and stem cell encapsulation as a treatment modality in type 1 diabetes, including a historical outlook as well as current and future research avenues. PMID:25148368

  5. Assignment of the gastric inhibitory polypeptide receptor gene (GIPR) to chromosome bands 19q13.2-q13.3 by fluorescence in situ hybridization

    SciTech Connect

    Stoffel, M.; Fernald, A.A.; Bell, G.I.; Le Beau, M.M.

    1995-08-10

    The gastric inhibitory polypeptide receptor gene (GIPR) was localized, using fluorescence in situ hybridization (FISH), to human chromosome bands 19q13.2-q13.3. Gastric inhibitory polypeptide (GIP) is a potent stimulator of insulin secretion and mutations in the GIPR gene may be related to non-insulin-dependent diabetes mellitus (NIDDM). 13 refs., 1 fig.

  6. Morphological changes of post-isolation of caprine pancreatic islet.

    PubMed

    Hani, Homayoun; Allaudin, Zeenathul Nazariah; Tengku Ibrahim, Tengku Azmi; Mohd-Lila, Mohd-Azmi; Sarsaifi, Kazhal; Camalxaman, Siti Nazrina; Othman, Abas Mazni

    2015-02-01

    Pancreatic islet transplantation is commonly used to treat diabetes. Cell isolation and purification methods can affect the structure and function of the isolated islet cells. Thus, the development of cell isolation techniques that preserve the structure and function of pancreatic islet cells is essential for enabling successful transplantation procedures. The impact of purification procedures on cell function can be assessed by performing ultrastructure and in vivo studies. Thus, the aim of this study was to evaluate the effect of caprine islets purification procedure on islet cell ultrastructure and functional integrity prior to and post-isolation/purification. The islets were isolated from caprine pancreas by using an optimized collagenase XI-S concentration, and the cells were subsequently purified using Euro-Ficoll density gradient. In vitro viability of islets was determined by fluorescein diacetate and propidium iodide staining. Static incubation was used to assess functionality and insulin production by islet cells in culture media when exposed to various levels of glucose. Pancreatic tissues were examined by using light microscopy, fluorescence microscopy, scanning, and transmission electron microscopy. In vivo viability and functionality of caprine islets were assessed by evaluating the transplanted islets in diabetic mice. Insulin assay of glucose-stimulated insulin secretion test showed that the insulin levels increased with increasing concentration of glucose. Thus, purified islets stimulated with high glucose concentration (25 mM) secreted higher levels of insulin (0.542 ± 0.346 μg/L) than the insulin levels (0.361 ± 0.219, 0.303 ± 0.234 μg/L) secreted by exposure to low glucose concentrations (1.67 mM). Furthermore, insulin levels of recipient mice were significantly higher (p < 0.001) than those prior to xenotransplantation. In addition, following islets transplantation, there was significant enhancement in blood glucose

  7. Membranes to achieve immunoprotection of transplanted islets

    PubMed Central

    Schweicher, Julien; Nyitray, Crystal; Desai, Tejal A.

    2014-01-01

    Transplantation of islet or beta cells is seen as the cure for type 1 diabetes since it allows physiological regulation of blood glucose levels without requiring any compliance from the patients. In order to circumvent the use of immunosuppressive drugs (and their side effects), semipermeable membranes have been developed to encapsulate and immunoprotect transplanted cells. This review presents the historical developments of immunoisolation and provides an update on the current research in this field. A particular emphasis is laid on the fabrication, characterization and performance of membranes developed for immunoisolation applications. PMID:24389172

  8. Identification of prosomatostatin in pancreatic islets.

    PubMed Central

    Patzelt, C; Tager, H S; Carroll, R J; Steiner, D F

    1980-01-01

    A 12.5-kilodalton protein not related to insulin or glucagon was detected in pulse-chase-labeled rat islets of Langerhans. Although this protein reacted poorly with various somatostatin antisera, analysis of two-dimensional peptide maps showed that it contains all of the tryptic fragments of somatostatin, which is located at its COOH terminus. Proteolytic conversion of the putative prosomatostatin, which took place parallel to the processing of proinsulin and proglucagon in pulse-chase experiments, coincided with the appearance of newly synthesized somatostatin and proceeded without the apparent involvement of major intermediate forms. Images PMID:6771754

  9. Islet cell autoantigens in insulin-dependent diabetes.

    PubMed Central

    Atkinson, M A; Maclaren, N K

    1993-01-01

    A burgeoning number of antigenic targets of the islet cell autoimmunity in IDD have been identified, and more can be anticipated through improved methods for their identification. The challenge for those investigating the pathogenesis of IDD will be to assign the relative importance of these antigens to the development of the disease, and to resolve whether there is a dominant primary immunologic event that is followed by a series of secondary immunizations to a variety of normally sequestered islet cell antigens in the sequence of pathogenic events that culminate in IDD. One interesting observation that may have potential pathogenic implications is the observation that of all islet cell autoantigens described, only two (i.e., 64 kD/GAD, 38 kD) are reactive in their native configurations, implying that recognition of conformational epitopes is most important. This property argues for primary immunizing agents rather than secondary ones after release of denatured antigens and antigenic recognition through their epitopes. Given the complex and multiple physiological functions of islet cells and the continuous variation in their activity, it is reasonable to speculate that the speed of the progression to IDD could vary between individuals with respect to their insulin needs and the relative activities of their islets. Activated islets may express autoantigens that have only limited expression in quiescent islets. The often times striking variation in the severity of insulitis seen in different islets of a single pancreas may be explained by the level of activity of individual islets. Furthermore, disparity in HLA-DR/DQ associations with disease may involve differences in the immunological recognition of autoantigens. Whereas there is still much to learn, it is clear that disease predictability and disease intervention studies have been enhanced through the identification of the islet cell autoantigens in IDD. Images PMID:8408615

  10. Benefits of PEGylation in the early post-transplant period of intraportal islet transplantation as assessed by magnetic resonance imaging of labeled islets

    PubMed Central

    Jin, Sang-Man; Oh, Seung-Hoon; Oh, Bae Jun; Suh, Sunghwan; Bae, Ji Cheol; Lee, Jung Hee; Lee, Myung-Shik; Lee, Moon-Kyu; Kim, Kwang-Won; Kim, Jae Hyeon

    2014-01-01

    While a few studies have demonstrated the benefit of PEGylation in islet transplantation, most have employed renal subcapsular models and none have performed direct comparisons of islet mass in intraportal islet transplantation using islet magnetic resonance imaging (MRI). In this study, our aim was to demonstrate the benefit of PEGylation in the early post-transplant period of intraportal islet transplantation with a novel algorithm for islet MRI. Islets were PEGylated after ferucarbotran labeling in a rat syngeneic intraportal islet transplantation model followed by comparisons of post-transplant glycemic levels in recipient rats infused with PEGylated (n = 12) and non-PEGylated (n = 13) islets. The total area of hypointense spots and the number of hypointense spots larger than 1.758 mm2 of PEGylated and non-PEGylated islets were quantitatively compared. The total area of hypointense spots (P < 0.05) and the number of hypointense spots larger than 1.758 mm2 (P < 0.05) were higher in the PEGylated islet group 7 and 14 days post translation (DPT). These results translated into better post-transplant outcomes in the PEGylated islet group 28 DPT. In validation experiments, MRI parameters obtained 1, 7, and 14 DPT predicted normoglycemia 4 wk post-transplantation. We directly demonstrated the benefit of islet PEGylation in protection against nonspecific islet destruction in the early post-transplant period of intraportal islet transplantation using a novel algorithm for islet MRI. This novel algorithm could serve as a useful tool to demonstrate such benefit in future clinical trials of islet transplantation using PEGylated islets. PMID:25483878

  11. Benefits of PEGylation in the early post-transplant period of intraportal islet transplantation as assessed by magnetic resonance imaging of labeled islets.

    PubMed

    Jin, Sang-Man; Oh, Seung-Hoon; Oh, Bae Jun; Suh, Sunghwan; Bae, Ji Cheol; Lee, Jung Hee; Lee, Myung-Shik; Lee, Moon-Kyu; Kim, Kwang-Won; Kim, Jae Hyeon

    2014-01-01

    While a few studies have demonstrated the benefit of PEGylation in islet transplantation, most have employed renal subcapsular models and none have performed direct comparisons of islet mass in intraportal islet transplantation using islet magnetic resonance imaging (MRI). In this study, our aim was to demonstrate the benefit of PEGylation in the early post-transplant period of intraportal islet transplantation with a novel algorithm for islet MRI. Islets were PEGylated after ferucarbotran labeling in a rat syngeneic intraportal islet transplantation model followed by comparisons of post-transplant glycemic levels in recipient rats infused with PEGylated (n = 12) and non-PEGylated (n = 13) islets. The total area of hypointense spots and the number of hypointense spots larger than 1.758 mm(2) of PEGylated and non-PEGylated islets were quantitatively compared. The total area of hypointense spots (P < 0.05) and the number of hypointense spots larger than 1.758 mm(2) (P < 0.05) were higher in the PEGylated islet group 7 and 14 days post translation (DPT). These results translated into better post-transplant outcomes in the PEGylated islet group 28 DPT. In validation experiments, MRI parameters obtained 1, 7, and 14 DPT predicted normoglycemia 4 wk post-transplantation. We directly demonstrated the benefit of islet PEGylation in protection against nonspecific islet destruction in the early post-transplant period of intraportal islet transplantation using a novel algorithm for islet MRI. This novel algorithm could serve as a useful tool to demonstrate such benefit in future clinical trials of islet transplantation using PEGylated islets.

  12. Impact of Size in Pancreatic Islet Transplantation and Potential Interventions to Improve Outcome

    PubMed Central

    Zorzi, Daria; Phan, Tammy; Sequi, Marco; Lin, Yong; Freeman, Daniel H.; Cicalese, Luca; Rastellini, Cristiana

    2015-01-01

    Better results have been recently reported in clinical pancreatic islet transplantation (ITX) due mostly to improved isolation techniques and immunosuppression; however, some limitations still exist. It is known that following transplantation from 30 to 60% of the islets is lost. In our study, we have investigated: 1) the role of size as a factor affecting islet engraftment and, 2) potential procedural manipulations to increase the number of smaller functional islets that can be transplanted. C57/BL10 mice were used as donors and recipients in a syngeneic islet transplant model. Isolated islets were divided by size (large, >300 µm; medium 150–300 µm; small, ρ50 µm). Each size was transplanted in chemically induced diabetic mice as full (600 IEq), suboptimal (400 IEq), and marginal mass (200 IEq). Control animals received all size islets. Engraftment was defined as reversal of diabetes by day 7 post-transplantation. When the superiority of smaller islets was observed, strategies of over-digestion and fragmentation were adopted during islet isolation in the attempt to reduce islet size and improve engraftment. Smaller islets were significantly superior in engraftment as compared to medium, large, and control (all sizes) groups. This was more evident when marginal mass data were compared. In all masses, success decreased as islet sizes increased. Once islets were engrafted, functionality was not affected by size. When larger islets were fragmented, a significant decrease in islet functionality was observed. On the contrary, if pancreata were slightly over-digested, although not as successful as small naive islets, an increase in engraftment was observed when compared to the control group. In conclusion, smaller islets are superior in engraftment following islet transplantation. Fragmentation has a deleterious effect on islet engraftment. Islet isolations can be performed reducing islet size with slight over-digestion and it can be safely adopted to improve

  13. Umami Receptor Activation Increases Duodenal Bicarbonate Secretion via Glucagon-Like Peptide-2 Release in Rats

    PubMed Central

    Wang, Joon-Ho; Inoue, Takuya; Higashiyama, Masaaki; Guth, Paul H.; Engel, Eli; Kaunitz, Jonathan D.

    2011-01-01

    Luminal nutrient chemosensing during meal ingestion is mediated by intestinal endocrine cells, which regulate secretion and motility via the release of gut hormones. We have reported that luminal coperfusion of l-Glu and IMP, common condiments providing the umami or proteinaceous taste, synergistically increases duodenal bicarbonate secretion (DBS) possibly via taste receptor heterodimers, taste receptor type 1, member 1 (T1R1)/R3. We hypothesized that glucose-dependent insulinotropic peptide (GIP) or glucagon-like peptide (GLP) is released by duodenal perfusion with l-Glu/IMP. We measured DBS with pH and CO2 electrodes through a perfused rat duodenal loop in vivo. GIP, exendin (Ex)-4 (GLP-1 receptor agonist), or GLP-2 was intravenously infused (0.01–1 nmol/kg/h). l-Glu (10 mM) and IMP (0.1 mM) were luminally perfused with or without bolus intravenous injection (3 or 30 nmol/kg) of the receptor antagonists Pro3GIP, Ex-3(9-39), or GLP-2(3-33). GIP or GLP-2 infusion dose-dependently increased DBS, whereas Ex-4 infusion gradually decreased DBS. Luminal perfusion of l-Glu/IMP increased DBS, with no effect of Pro3GIP or Ex-3(9-39), whereas GLP-2(3-33) inhibited l-Glu/IMP-induced DBS. Vasoactive intestinal peptide (VIP)(6–28) intravenously or NG-nitro-l-arginine methyl ester coperfusion inhibited the effect of l-Glu/IMP. Perfusion of l-Glu/IMP increased portal venous concentrations of GLP-2, followed by a delayed increase of GLP-1, with no effect on GIP release. GLP-1/2 and T1R1/R3 were expressed in duodenal endocrine-like cells. These results suggest that luminal l-Glu/IMP-induced DBS is mediated via GLP-2 release and receptor activation followed by VIP and nitric oxide release. Because GLP-1 is insulinotropic and GLP-2 is intestinotrophic, umami receptor activation may have additional benefits in glucose metabolism and duodenal mucosal protection and regeneration. PMID:21846840

  14. Endoscopic biopsy of islet transplants in the gastric submucosal space provides evidence of islet graft rejection in diabetic pigs

    PubMed Central

    Tanaka, Takayuki; Fujita, Minoru; Bottino, Rita; Piganelli, Jon D.; McGrath, Kevin; Li, Jiang; Lee, Whayoung; Iwase, Hayato; Wijkstrom, Martin; Bertera, Suzanne; Long, Cassandra; Landsittel, Douglas; Haruma, Ken; Cooper, David K.C.; Hara, Hidetaka

    2016-01-01

    ABSTRACT Transplantation of islets into the gastric submucosal space (GSMS) has several advantages (e.g., avoidance of the instant blood-mediated inflammatory response [IBMIR], ability to biopsy). The aim of this study was to determine whether endoscopic biopsy of islet allografts transplanted into the GSMS in diabetic pigs can provide histopathological and immunohistochemical information that correlates with the clinical course (e.g.,, blood glucose level, insulin requirement). Islet allografts (Group1: 10,000 kIEq /kg [n = 4]; Group2: 15,000 kIEq /kg [n = 2]) were transplanted into the GSMS of diabetic pigs under immunosuppression. In Group2, the anti-oxidant, BMX-001 was applied during preservation, isolation, and culture of the islets, and at the time of transplantation. Endoscopic biopsies of the islet grafts were obtained one or 2 weeks after transplantation, and histopathological features were compared with the clinical course (e.g., blood glucose, insulin requirement). In Group1, in the absence of anti-oxidant therapy, most of the islets became fragmented, and there was no reduction in exogenous insulin requirement. In Group2, with an increased number of transplanted islets in the presence of BMX-001, more healthy insulin-positive islet masses were obtained at biopsy and necropsy (4 weeks), and these correlated with reductions in both blood glucose level and insulin requirement. In all cases, inflammatory cell infiltrates were present. After islet transplantation into the GSMS, endoscopic biopsy can provide information on graft rejection, which would be an immense advantage in clinical islet transplantation. PMID:26857703

  15. The release of GLP-1 and ghrelin, but not GIP and CCK, by glucose is dependent upon the length of small intestine exposed.

    PubMed

    Little, Tanya J; Doran, Selena; Meyer, James H; Smout, Andre J P M; O'Donovan, Deirdre G; Wu, Keng-Liang; Jones, Karen L; Wishart, Judith; Rayner, Christopher K; Horowitz, Michael; Feinle-Bisset, Christine

    2006-09-01

    Previous observations suggest that glucagon-like peptide-1 (GLP-1) is released into the bloodstream only when dietary carbohydrate enters the duodenum at rates that exceed the absorptive capacity of the proximal small intestine to contact GLP-1 bearing mucosa in more distal bowel. The aims of this study were to determine the effects of modifying the length of small intestine exposed to glucose on plasma concentrations of GLP-1 and also glucose-dependent insulinotropic peptide (GIP), insulin, cholecystokinin (CCK) and ghrelin, and antropyloric pressures. Glucose was infused at 3.5 kcal/min into the duodenum of eight healthy males (age 18-59 yr) over 60 min on the first day into an isolated 60-cm segment of the proximal small intestine ("short-segment infusion"); on the second day, the same amount of glucose was infused with access to the entire small intestine ("long-segment infusion"). Plasma GLP-1 increased and ghrelin decreased (P < 0.05 for both) during the long-, but not the short-, segment infusion. By contrast, increases in plasma CCK and GIP did not differ between days. The rises in blood glucose and plasma insulin were greater during the long- than during the short-segment infusion (P < 0.05). During the long- but not the short-segment infusion, antral pressure waves (PWs) were suppressed (P < 0.05). Isolated pyloric PWs and basal pyloric pressure were stimulated on both days. In conclusion, the release of GLP-1 and ghrelin, but not CCK and GIP, is dependent upon >60 cm of the intestine being exposed to glucose.

  16. Down-regulation of pancreatic transcription factors and incretin receptors in type 2 diabetes

    PubMed Central

    Kaneto, Hideaki; Matsuoka, Taka-aki

    2013-01-01

    Type 2 diabetes is one of the most prevalent and serious metabolic diseases. Under diabetic conditions, chronic hyperglycemia and subsequent induction of oxidative stress deteriorate pancreatic β-cell function, which leads to the aggravation of type 2 diabetes. Although such phenomena are well known as glucose toxicity, its molecular mechanism remains unclear. In this review article, we describe the possible molecular mechanism for β-cell dysfunction found in type 2 diabetes, focusing on (1) oxidative stress, (2) pancreatic transcription factors (PDX-1 and MafA) and (3) incretin receptors (GLP-1 and GIP receptors). Under such conditions, nuclear expression levels of PDX-1 and MafA are decreased, which leads to suppression of insulin biosynthesis and secretion. In addition, expression levels of GLP-1 and GIP receptors are decreased, which likely contributes to the impaired incretin effects found in diabetes. Taken together, it is likely that down-regulation of pancreatic transcription factors (PDX-1 and MafA) and down-regulation of incretin receptors (GLP-1 and GIP receptors) explain, at least in part, the molecular mechanism for β-cell dysfunction found in type 2 diabetes. PMID:24379916

  17. A map of open chromatin in human pancreatic islets

    PubMed Central

    Gaulton, Kyle J.; Nammo, Takao; Pasquali, Lorenzo; Simon, Jeremy M.; Giresi, Paul G.; Fogarty, Marie P.; Panhuis, Tami M.; Mieczkowski, Piotr; Secchi, Antonio; Bosco, Domenico; Berney, Thierry; Montanya, Eduard; Mohlke, Karen L.; Lieb, Jason D.; Ferrer, Jorge

    2010-01-01

    Tissue-specific transcriptional regulation is central to human disease1. To identify regulatory DNA active in human pancreatic islets, we profiled chromatin by FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements)2–4 coupled with high-throughput sequencing. We identified ~80,000 open chromatin sites. Comparison of islet FAIRE-seq to five non-islet cell lines revealed ~3,300 physically linked clusters of islet-selective open chromatin sites, which typically encompassed single genes exhibiting islet-specific expression. We mapped sequence variants to open chromatin sites and found that rs7903146, a TCF7L2 intronic variant strongly associated with type 2 diabetes (T2D)5, is located in islet-selective open chromatin. We show that rs7903146 heterozygotes exhibit allelic imbalance in islet FAIRE signal, and that the variant alters enhancer activity, indicating that genetic variation at this locus acts in cis with local chromatin and regulatory changes. These findings illuminate the tissue-specific organization of cis-regulatory elements, and show that FAIRE-seq can guide identification of regulatory variants important for disease. PMID:20118932

  18. Has the gap between pancreas and islet transplantation closed?

    PubMed

    Niclauss, Nadja; Morel, Philippe; Berney, Thierry

    2014-09-27

    Both pancreas and islet transplantations are therapeutic options for complicated type 1 diabetes. Until recent years, outcomes of islet transplantation have been significantly inferior to those of whole pancreas. Islet transplantation is primarily performed alone in patients with severe hypoglycemia, and recent registry reports have suggested that results of islet transplantation alone in this indication may be about to match those of pancreas transplant alone in insulin independence. Figures of 50% insulin independence at 5 years for either procedure have been cited. In this article, we address the question whether islet transplantation has indeed bridged the gap with whole pancreas. Looking at the evidence to answer this question, we propose that although pancreas may still be more efficient in taking recipients off insulin than islets, there are in fact numerous "gaps" separating both procedures that must be taken into the equation. These "gaps" relate to organ utilization, organ allocation, indication for transplantation, and morbidity. In-depth analysis reveals that islet transplantation, in fact, has an edge on whole pancreas in some of these aspects. Accordingly, attempts should be made to bridge these gaps from both sides to achieve the same level of success with either procedure. More realistically, it is likely that some of these gaps will remain and that both procedures will coexist and complement each other, to ensure that β cell replacement can be successfully implemented in the greatest possible number of patients with type 1 diabetes.

  19. Prediction of marginal mass required for successful islet transplantation.

    PubMed

    Papas, Klearchos K; Colton, Clark K; Qipo, Andi; Wu, Haiyan; Nelson, Rebecca A; Hering, Bernhard J; Weir, Gordon C; Koulmanda, Maria

    2010-02-01

    Islet quality assessment methods for predicting diabetes reversal (DR) following transplantation are needed. We investigated two islet parameters, oxygen consumption rate (OCR) and OCR per DNA content, to predict transplantation outcome and explored the impact of islet quality on marginal islet mass for DR. Outcomes in immunosuppressed diabetic mice were evaluated by transplanting mixtures of healthy and purposely damaged rat islets for systematic variation of OCR/DNA over a wide range. The probability of DR increased with increasing transplanted OCR and OCR/DNA. On coordinates of OCR versus OCR/DNA, data fell into regions in which DR occurred in all, some, or none of the animals with a sharp threshold of around 150-nmol/min mg DNA. A model incorporating both parameters predicted transplantation outcome with sensitivity and specificity of 93% and 94%, respectively. Marginal mass was not constant, depended on OCR/DNA, and increased from 2,800 to over 100,000 islet equivalents/kg body weight as OCR/DNA decreased. We conclude that measurements of OCR and OCR/DNA are useful for predicting transplantation outcome in this model system, and OCR/DNA can be used to estimate the marginal mass required for reversing diabetes. Because human clinical islet preparations in a previous study had OCR/DNA.

  20. Glucose Oscillations Can Activate an Endogenous Oscillator in Pancreatic Islets

    PubMed Central

    Mukhitov, Nikita; Roper, Michael G.; Bertram, Richard

    2016-01-01

    Pancreatic islets manage elevations in blood glucose level by secreting insulin into the bloodstream in a pulsatile manner. Pulsatile insulin secretion is governed by islet oscillations such as bursting electrical activity and periodic Ca2+ entry in β-cells. In this report, we demonstrate that although islet oscillations are lost by fixing a glucose stimulus at a high concentration, they may be recovered by subsequently converting the glucose stimulus to a sinusoidal wave. We predict with mathematical modeling that the sinusoidal glucose signal’s ability to recover islet oscillations depends on its amplitude and period, and we confirm our predictions by conducting experiments with islets using a microfluidics platform. Our results suggest a mechanism whereby oscillatory blood glucose levels recruit non-oscillating islets to enhance pulsatile insulin output from the pancreas. Our results also provide support for the main hypothesis of the Dual Oscillator Model, that a glycolytic oscillator endogenous to islet β-cells drives pulsatile insulin secretion. PMID:27788129

  1. Microfluidic device for multimodal characterization of pancreatic islets.

    PubMed

    Mohammed, Javeed Shaikh; Wang, Yong; Harvat, Tricia A; Oberholzer, Jose; Eddington, David T

    2009-01-07

    A microfluidic device to perfuse pancreatic islets while simultaneously characterizing their functionality through fluorescence imaging of the mitochondrial membrane potential and intracellular calcium ([Ca(2+)](i)) in addition to enzyme linked immunosorbent assay (ELISA) quantification of secreted insulin was developed and characterized. This multimodal characterization of islet function will facilitate rapid assessment of tissue quality immediately following isolation from donor pancreas and allow more informed transplantation decisions to be made which may improve transplantation outcomes. The microfluidic perfusion chamber allows flow rates of up to 1 mL min(-1), without any noticeable perturbation or shear of islets. This multimodal quantification was done on both mouse and human islets. The ability of this simple microfluidic device to detect subtle variations in islet responses in different functional assays performed in short time-periods demonstrates that the microfluidic perfusion chamber device can be used as a new gold standard to perform comprehensive islet analysis and obtain a more meaningful predictive value for islet functionality prior to transplantation into recipients, which is currently difficult to predict using a single functional assay.

  2. Biomolecular Surface Engineering of Pancreatic Islets with Thrombomodulin

    PubMed Central

    Wilson, John T.; Haller, Carolyn A.; Qu, Zheng; Cui, Wanxing; Urlam, Murali K.; Chaikof, Elliot L.

    2010-01-01

    Islet transplantation has emerged as a promising treatment for Type 1 diabetes, but its clinical impact remains limited by early islet destruction mediated by prothrombotic and innate inflammatory responses elicited upon transplantation. Thrombomodulin (TM) acts as an important regulator of thrombosis and inflammation through its capacity to channel the catalytic activity of thrombin towards generation of activated protein C (APC), a potent anti-coagulant and anti-inflammatory agent. We describe herein a novel biomolecular strategy for re-engineering the surface of pancreatic islets with TM. A biosynthetic approach was employed to generate recombinant human TM (rTM) bearing a C-terminal azide group, which facilitated site-specific biotinylation of rTM through Staudinger ligation. Murine pancreatic islets were covalently biotinylated through targeting of cell surface amines and aldehydes, and both islet viability and the surface density of streptavidin were maximized through optimization of biotinylation conditions. rTM was immobilized on islet surfaces through streptavidin-biotin interactions, resulting in a nearly three-fold increase in the catalytic capacity of islets to generate APC. PMID:20102751

  3. Photoacoustic imaging of angiogenesis in subdermal islet transplant sites

    NASA Astrophysics Data System (ADS)

    Shi, Wei; Pawlick, Rena; Bruni, Antonio; Rafiei, Yasmin; Pepper, Andrew R.; Gala-Lopez, Boris; Choi, Min; Malcolm, Andrew; Zemp, Roger J.; Shapiro, A. M. James

    2016-03-01

    Exogenous insulin administration is the mainstay treatment therapy for patients with Type-1 diabetes mellitus (T1DM). However, for select patients, clinical islet transplantation is an alternative therapeutic treatment. In this procedure, islets are transplanted into the hepatic portal vein, and despite improved success within the last decade, obstacles are still associated with this approach. It has been discovered that the subcutaneous space may be an effective alternative site for islet transplantation, and may provide advantages of easy access and potential for simple monitoring. The ability to monitor islet viability and the transplant microenvironment may be key to future success in islet transplantation. A subcutaneous device-less technique has been developed to facilitate angiogenesis in the islet transplant site, however, a method for monitoring the potential engraftment site have yet to be explored fully. Here we demonstrate the ability to track angiogenesis in mice with 1, 2, 3 and 4 weeks post-catheter implant on both sides of the abdomen using a FujiFilm VisualSonics Vevo-LAZR system. Quantitative analysis on vessel densities exhibited gradual vessel growth successfully induced by catheter implantation. Our study demonstrates the ability of employing photoacoustic and micro-ultrasound imaging to track angiogenesis around the catheter site prior to islet transplantation.

  4. Differential wiring of local excitatory and inhibitory synaptic inputs to islet cells in rat spinal lamina II demonstrated by laser scanning photostimulation

    PubMed Central

    Kato, Go; Kawasaki, Yasuhiko; Ji, Ru-Rong; Strassman, Andrew M

    2007-01-01

    The substantia gelatinosa (lamina II) of the spinal dorsal horn contains inhibitory and excitatory interneurons that are thought to play a critical role in the modulation of nociception. However, the organization of the intrinsic circuitry within lamina II remains poorly understood. We used glutamate uncaging by laser scanning photostimulation to map the location of neurons that give rise to local synaptic inputs to islet cells, a major class of inhibitory interneuron in lamina II. We also mapped the distribution of sites on the islet cells that exhibited direct (non-synaptic) responses to uncaging of excitatory and inhibitory transmitters. Local synaptic inputs to islet cells arose almost entirely from within lamina II, and these local inputs included both excitatory and inhibitory components. Furthermore, there was a striking segregation in the location of sites that evoked excitatory versus inhibitory synaptic inputs, such that inhibitory presynaptic neurons were distributed more proximal to the islet cell soma. This was paralleled in part by a differential distribution of transmitter receptor sites on the islet cell, in that inhibitory sites were confined to the peri-somatic region while excitatory sites were more widespread. This differential organization of excitatory and inhibitory inputs suggests a principle for the wiring of local circuitry within the substantia gelatinosa. PMID:17289782

  5. Serotonin- and Dopamine-Related Gene Expression in db/db Mice Islets and in MIN6 β-Cells Treated with Palmitate and Oleate

    PubMed Central

    Cataldo, L. R.; Olmos, P.; Galgani, J. E.; Valenzuela, R.; Aranda, E.; Cortés, V. A.; Santos, J. L.

    2016-01-01

    High circulating nonesterified fatty acids (NEFAs) concentration, often reported in diabetes, leads to impaired glucose-stimulated insulin secretion (GSIS) through not yet well-defined mechanisms. Serotonin and dopamine might contribute to NEFA-dependent β-cell dysfunction, since extracellular signal of these monoamines decreases GSIS. Moreover, palmitate-treated β-cells may enhance the expression of the serotonin receptor Htr2c, affecting insulin secretion. Additionally, the expression of monoamine-oxidase type B (Maob) seems to be lower in islets from humans and mice with diabetes compared to nondiabetic islets, which may lead to increased monoamine concentrations. We assessed the expression of serotonin- and dopamine-related genes in islets from db/db and wild-type (WT) mice. In addition, the effect of palmitate and oleate on the expression of such genes, 5HT content, and GSIS in MIN6 β-cell was determined. Lower Maob expression was found in islets from db/db versus WT mice and in MIN6 β-cells in response to palmitate and oleate treatment compared to vehicle. Reduced 5HT content and impaired GSIS in response to palmitate (−25%; p < 0.0001) and oleate (−43%; p < 0.0001) were detected in MIN6 β-cells. In conclusion, known defects of GSIS in islets from db/db mice and MIN6 β-cells treated with NEFAs are accompanied by reduced Maob expression and reduced 5HT content. PMID:27366756

  6. The role of rosiglitazone treatment in the modulation of islet hormones and hormone-like peptides: a combined in situ hybridization and immunohistochemical study.

    PubMed

    Yildirim, Sukriye; Bolkent, Sema; Sundler, Frank

    2008-12-01

    Rosiglitazone, peroxisome proliferator-activated receptor-gamma agonist, is an insulin sensitizing agent in peripheral tissues. This study investigated islet hormones and hormone-like peptides expression patterns in rosiglitazone treated streptozotocin (STZ)-diabetic rats by using immunohistochemistry and in situ hybridization methods. Animals were divided into four groups. I. Group: Intact control rats. II. Group: Rosiglitazone-treated controls. III. Group: STZ-diabetic rats. IV. Group: Rosiglitazone-treated diabetic animals. Rosiglitazone was given for 7 days at a dose of 20 mg/kg body weight. In the STZ-diabetic group, there were significant differences in islet hormones and hormone like peptides cell numbers compared to rosiglitazone control group and intact control group. There were significant differences in cocaine- and amphetamine-regulated transcript (CART) and pancreatic polypeptide (PP) cell numbers between rosiglitazone control group and rosiglitazone + STZ-diabetic group. We detected a significant decrease in glucagon mRNA signals in rosiglitazone-treated control group compared to intact controls. We found a statistically significant difference in islet amyloid polypeptide (IAPP) mRNA signals between the STZ-diabetic group and the rosiglitazone + STZ-diabetic group. Besides, we also demonstrated co-localization of peptides by using double and triple histochemistry. In conclusion, our results show that short-term rosiglitazone treatment had a preservative effect to some extent on the expression of islet hormones and hormone-like peptides to maintain the islet function.

  7. Extrahepatic islet transplantation with microporous polymer scaffolds in syngeneic mouse and allogeneic porcine models.

    PubMed

    Gibly, Romie F; Zhang, Xiaomin; Graham, Melanie L; Hering, Bernhard J; Kaufman, Dixon B; Lowe, William L; Shea, Lonnie D

    2011-12-01

    Intraportal transplantation of islets has successfully treated select patients with type 1 diabetes. However, intravascular infusion and the intrahepatic site contribute to significant early and late islet loss, yet a clinical alternative has remained elusive. We investigated non-encapsulating, porous, biodegradable polymer scaffolds as a vehicle for islet transplantation into extrahepatic sites, using syngeneic mouse and allogeneic porcine models. Scaffold architecture was modified to enhance cell infiltration leading to revascularization of the islets with minimal inflammatory response. In the diabetic mouse model, 125 islets seeded on scaffolds implanted into the epididymal fat pad restored normoglycemia within an average of 1.95 days and transplantation of only 75 islets required 12.1 days. Increasing the pore size to increase islet-islet interactions did not significantly impact islet function. The porcine model was used to investigate early islet engraftment. Increasing the islet seeding density led to a greater mass of engrafted islets, though the efficiency of islet survival decreased. Transplantation into the porcine omentum provided greater islet engraftment than the gastric submucosa. These results demonstrate scaffolds support murine islet transplantation with high efficiency, and feasibility studies in large animals support continued pre-clinical studies with scaffolds as a platform to control the transplant microenvironment.

  8. [Xenogeneic cell therapeutics: Treatment of type 1 diabetes using porcine pancreatic islets and islet cells].

    PubMed

    Godehardt, Antonia W; Schilling-Leiß, Dagmar; Sanzenbacher, Ralf; Tönjes, Ralf R

    2015-11-01

    In view of the existing shortage of human donor organs and tissues, xenogeneic cell therapeutics (xCT) offer an alternative for adequate treatment. In particular, porcine pancreatic islets and islet cells have already entered the field of experimental therapy for type-1 diabetes mellitus (T1DM) patients. Thereby, xCT depict challenging products with a glance on medical, ethical, and regulatory questions. With cross-species transplantation (xenotransplantation), the risk of immunological graft rejection as well as the risk of infectious transmission of microbial and viral pathogens must be considered. This includes the bidirectional transmission of microorganisms from graft to host as well as from host to graft. Crossing the border of species requires a critical risk-benefit evaluation as well as a thorough longtime surveillance of transplant recipients after treatment. The international legal and regulatory requirements for xCT are inter alia based on the World Health Organization criteria summarized in the Changsha Communiqué (2008). In the European Union, they were reflected by the European Medicines Agency (EMA) Guideline on Xenogeneic Cell-based Medicinal Products following the implementation of the Regulation on Advanced Therapies (ATMP). On the basis of this regulation, the first non-clinical and clinical experiences were obtained for porcine islets. The results suggest that supportive treatment of T1DM risk patients with xCT may be an alternative to established allogeneic organ transplantation in the future.

  9. Fibrillar dimer formation of islet amyloid polypeptides

    NASA Astrophysics Data System (ADS)

    Chiu, Chi-cheng; de Pablo, Juan J.

    2015-09-01

    Amyloid deposits of human islet amyloid polypeptide (hIAPP), a 37-residue hormone co-produced with insulin, have been implicated in the development of type 2 diabetes. Residues 20 - 29 of hIAPP have been proposed to constitute the amyloidogenic core for the aggregation process, yet the segment is mostly unstructured in the mature fibril, according to solid-state NMR data. Here we use molecular simulations combined with bias-exchange metadynamics to characterize the conformational free energies of hIAPP fibrillar dimer and its derivative, pramlintide. We show that residues 20 - 29 are involved in an intermediate that exhibits transient β-sheets, consistent with recent experimental and simulation results. By comparing the aggregation of hIAPP and pramlintide, we illustrate the effects of proline residues on inhibition of the dimerization of IAPP. The mechanistic insights presented here could be useful for development of therapeutic inhibitors of hIAPP amyloid formation.

  10. Fibrillar dimer formation of islet amyloid polypeptides

    SciTech Connect

    Chiu, Chi -cheng; de Pablo, Juan J.

    2015-05-08

    Amyloid deposits of human islet amyloid polypeptide (hIAPP), a 37-residue hormone co-produced with insulin, have been implicated in the development of type 2 diabetes. Residues 20 – 29 of hIAPP have been proposed to constitute the amyloidogenic core for the aggregation process, yet the segment is mostly unstructured in the mature fibril, according to solid-state NMR data. Here we use molecular simulations combined with bias-exchange metadynamics to characterize the conformational free energies of hIAPP fibrillar dimer and its derivative, pramlintide. We show that residues 20 – 29 are involved in an intermediate that exhibits transient β-sheets, consistent with recent experimental and simulation results. By comparing the aggregation of hIAPP and pramlintide, we illustrate the effects of proline residues on inhibition of the dimerization of IAPP. The mechanistic insights presented here could be useful for development of therapeutic inhibitors of hIAPP amyloid formation.

  11. Leptin rapidly suppresses insulin release from insulinoma cells, rat and human islets and, in vivo, in mice.

    PubMed Central

    Kulkarni, R N; Wang, Z L; Wang, R M; Hurley, J D; Smith, D M; Ghatei, M A; Withers, D J; Gardiner, J V; Bailey, C J; Bloom, S R

    1997-01-01

    Obesity is associated with diabetes, and leptin is known to be elevated in obesity. To investigate whether leptin has a direct effect on insulin secretion, isolated rat and human islets and cultured insulinoma cells were studied. In all cases, mouse leptin inhibited insulin secretion at concentrations within the plasma range reported in humans. Insulin mRNA expression was also suppressed in the cultured cells and rat islets. The long form of the leptin receptor (OB-Rb) mRNA was present in the islets and insulinoma cell lines. To determine the significance of these findings in vivo, normal fed mice were injected with two doses of leptin. A significant decrease in plasma insulin and associated rise in glucose concentration were observed. Fasted normal and leptin receptor-deficient db/db mice showed no response to leptin. A dose of leptin, which mimicked that found in normal mice, was administered to leptin-deficient, hyperinsulinemic ob/ob mice. This caused a marked lowering of plasma insulin concentration and a doubling of plasma glucose. Thus, leptin has a powerful acute inhibitory effect on insulin secretion. These results suggest that the action of leptin may be one mechanism by which excess adipose tissue could acutely impair carbohydrate metabolism. PMID:9389736

  12. In situ application of hydrogel-type fibrin-islet composite optimized for rapid glycemic control by subcutaneous xenogeneic porcine islet transplantation.

    PubMed

    Kim, Jung-Sik; Lim, Jong-Hyung; Nam, Hye-Young; Lim, Hyun-Ju; Shin, Jun-Seop; Shin, Jin-Young; Ryu, Ju-Hee; Kim, Kwangmeyung; Kwon, Ick-Chan; Jin, Sang-Man; Kim, Hang-Rae; Kim, Sang-Joon; Park, Chung-Gyu

    2012-09-10

    Maximum engraftment of transplanted islets is essential for the clinical application of a subcutaneous site. Significant barriers to the current approaches are associated with their low effectiveness, complexity and unproven biosafety. Here, we evaluated and optimized a fibrin-islet composite for effective glycemic control in a subcutaneous site whose environment is highly hypoxic due to low vascularization potential. In the setting of xenogeneic porcine islet transplantation into the subcutaneous space of a diabetic mouse, the in vivo islet functions were greatly affected by the concentrations of fibrinogen and thrombin. The optimized hydrogel-type fibrin remarkably reduced the marginal islet mass to approximately one tenth that of islets without fibrin. This marginal islet mass was comparable to that in the setting of the subcapsular space of the kidney, which is a highly vascularized organ. Highly vascularized structures were generated inside and on the outer surface of the grafts. A hydrogel-type fibrin-islet composite established early diabetic control within an average of 3.4days after the transplantation. In the mechanistic studies, fibrin promoted local angiogenesis, enhanced islet viability and prevented fragmentation of islets into single cells. In conclusion, in situ application of hydrogel-type fibrin-islet composite may be a promising modality in the clinical success of subcutaneous islet transplantation.

  13. Engineering a Local Microenvironment for Pancreatic Islet Replacement

    PubMed Central

    Coronel, Maria M.; Stabler, Cherie L.

    2013-01-01

    Intraportal islet transplantation has emerged as a promising treatment for type 1 diabetes mellitus (T1DM). Nevertheless, long-term efficacy has been limited to a marginal number of patients. Outcomes have been restricted, in part, by challenges associated with the transplant site, poor vascularization, and disruption of the native islet architecture during the isolation process. Engineering a biomaterial platform that recapitulates critical components of the pancreatic environment can serve to address these hurdles. This review highlights the challenges and opportunities in engineering 3-D niches for islets, specifically: the importance of site selection; the application of scaffold functionalization to present bioactive motifs; and the development of technologies for enhancing implant nutritional profiles. The potential of these novel approaches to improve islet engraftment and duration of function is discussed. PMID:23769320

  14. MULTIHORMONAL ISLET CELL CARCINOMAS IN THREE KOMODO DRAGONS (VARANUS KOMODOENSIS).

    PubMed

    Eustace, Ronan; Garner, Michael M; Cook, Kimberly; Miller, Christine; Kiupel, Matti

    2017-03-01

      Multihormonal pancreatic islet cell carcinomas were found in one female and two male captive geriatric Komodo dragons (Varanus komodoensis). Gross changes in the pancreas were visible in two of the cases. Clinical signs noted in the Komodo dragons were lethargy, weakness, and anorexia. Histologically, the tumors were comprised of nests and cords of well-differentiated neoplastic islet cells with scant amounts of eosinophilic cytoplasm and round, euchromatic nuclei, with rare mitoses. Infiltration by the islet cell tumor into the surrounding acinar tissue was observed in all cases, but no metastatic foci were seen. Multihormone expression was observed in all tumors, which labeled strongly positive for glucagon and somatostatin and focally positive for polypeptide. Pancreatic islet cell neoplasms should be considered in the differential diagnosis for geriatric Komodo dragons presenting with weakness, lethargy, and poor appetite.

  15. Islet cell xenotransplantation: a serious look toward the clinic.

    PubMed

    Samy, Kannan P; Martin, Benjamin M; Turgeon, Nicole A; Kirk, Allan D

    2014-01-01

    Type I diabetes remains a significant clinical problem in need of a reliable, generally applicable solution. Both whole organ pancreas and islet allotransplantation have been shown to grant patients insulin independence, but organ availability has restricted these procedures to an exceptionally small subset of the diabetic population. Porcine islet xenotransplantation has been pursued as a potential means of overcoming the limits of allotransplantation, and several preclinical studies have achieved near-physiologic function and year-long survival in clinically relevant pig-to-primate model systems. These proof-of-concept studies have suggested that xenogeneic islets may be poised for use in clinical trials. In this review, we examine recent progress in islet xenotransplantation, with a critical eye toward the gaps between the current state of the art and the state required for appropriate clinical investigation.

  16. Islet Cell Xenotransplantation: A Serious Look Towards the Clinic

    PubMed Central

    Samy, Kannan P.; Martin, Benjamin; Turgeon, Nicole A.; Kirk, Allan D.

    2014-01-01

    Type I diabetes remains a significant clinical problem in need of a reliable, generally applicable solution. Both whole organ pancreas and islet allotransplantation have been shown to grant patients insulin independence, but organ availability has restricted these procedures to an exceptionally small subset of the diabetic population. Porcine islet xenotransplantation has been pursued as a potential means of overcoming the limits of allotransplantation, and several preclinical studies have achieved near-physiologic function and yearlong survival in clinically relevant pig to primate model systems. These proof-of-concept studies have suggested that xenogeneic islets may be poised for use in clinical trials. In this commentary, we examine recent progress in islet xenotransplantation, with a critical eye towards the gaps between the current state of the art and the state required for appropriate clinical investigation. PMID:24806830

  17. Islet inflammation: A unifying target for diabetes treatment?

    PubMed Central

    Imai, Yumi; Dobrian, Anca D.; Morris, Margaret A.; Nadler, Jerry L.

    2013-01-01

    In the last decade, islet inflammation has emerged as a contributor to the loss of functional β cell mass in both type 1 (T1D) and type 2 diabetes (T2D). Evidence supports that over-nutrition and insulin resistance result in the production of proinflammatory mediators by β cells. In addition to compromising β cell function and survival, cytokines may recruit macrophages into islets, thus augmenting inflammation. Limited, but intriguing, data implies a role of adaptive immune response in islet dysfunction in T2D. Clinical trials validated anti-inflammatory therapies in T2D, while immune therapy for T1D remains challenging. Further research is required to improve our understanding of islet inflammatory pathways, and to identify more effective therapeutic targets for T1D and T2D. PMID:23484621

  18. A 3D map of the islet routes throughout the healthy human pancreas

    PubMed Central

    Ionescu-Tirgoviste, Constantin; Gagniuc, Paul A.; Gubceac, Elvira; Mardare, Liliana; Popescu, Irinel; Dima, Simona; Militaru, Manuella

    2015-01-01

    Islets of Langerhans are fundamental in understanding diabetes. A healthy human pancreas from a donor has been used to asses various islet parameters and their three-dimensional distribution. Here we show that islets are spread gradually from the head up to the tail section of the pancreas in the form of contracted or dilated islet routes. We also report a particular anatomical structure, namely the cluster of islets. Our observations revealed a total of 11 islet clusters which comprise of small islets that surround large blood vessels. Additional observations in the peripancreatic adipose tissue have shown lymphoid-like nodes and blood vessels captured in a local inflammatory process. Our observations are based on regional slice maps of the pancreas, comprising of 5,423 islets. We also devised an index of sphericity which briefly indicates various islet shapes that are dominant throughout the pancreas. PMID:26417671

  19. Pancreatic islet enhancer clusters enriched in type 2 diabetes risk-associated variants.

    PubMed

    Pasquali, Lorenzo; Gaulton, Kyle J; Rodríguez-Seguí, Santiago A; Mularoni, Loris; Miguel-Escalada, Irene; Akerman, Ildem; Tena, Juan J; Morán, Ignasi; Gómez-Marín, Carlos; van de Bunt, Martijn; Ponsa-Cobas, Joan; Castro, Natalia; Nammo, Takao; Cebola, Inês; García-Hurtado, Javier; Maestro, Miguel Angel; Pattou, François; Piemonti, Lorenzo; Berney, Thierry; Gloyn, Anna L; Ravassard, Philippe; Gómez-Skarmeta, José Luis; Müller, Ferenc; McCarthy, Mark I; Ferrer, Jorge

    2014-02-01

    Type 2 diabetes affects over 300 million people, causing severe complications and premature death, yet the underlying molecular mechanisms are largely unknown. Pancreatic islet dysfunction is central in type 2 diabetes pathogenesis, and understanding islet genome regulation could therefore provide valuable mechanistic insights. We have now mapped and examined the function of human islet cis-regulatory networks. We identify genomic sequences that are targeted by islet transcription factors to drive islet-specific gene activity and show that most such sequences reside in clusters of enhancers that form physical three-dimensional chromatin domains. We find that sequence variants associated with type 2 diabetes and fasting glycemia are enriched in these clustered islet enhancers and identify trait-associated variants that disrupt DNA binding and islet enhancer activity. Our studies illustrate how islet transcription factors interact functionally with the epigenome and provide systematic evidence that the dysregulation of islet enhancers is relevant to the mechanisms underlying type 2 diabetes.

  20. Connexin 36 mediates blood cell flow in mouse pancreatic islets.

    PubMed

    Short, Kurt W; Head, W Steve; Piston, David W

    2014-02-01

    The insulin-secreting β-cells are contained within islets of Langerhans, which are highly vascularized. Blood cell flow rates through islets are glucose-dependent, even though there are no changes in blood cell flow within in the surrounding exocrine pancreas. This suggests a specific mechanism of glucose-regulated blood flow in the islet. Pancreatic islets respond to elevated glucose with synchronous pulses of electrical activity and insulin secretion across all β-cells in the islet. Connexin 36 (Cx36) gap junctions between islet β-cells mediate this synchronization, which is lost in Cx36 knockout mice (Cx36(-/-)). This leads to glucose intolerance in these mice, despite normal plasma insulin levels and insulin sensitivity. Thus, we sought to investigate whether the glucose-dependent changes in intraislet blood cell flow are also dependent on coordinated pulsatile electrical activity. We visualized and quantified blood cell flow using high-speed in vivo fluorescence imaging of labeled red blood cells and plasma. With the use of a live animal glucose clamp, blood cell flow was measured during either hypoglycemia (∼50 mg/dl) or hyperglycemia (∼300 mg/dl). In contrast to the large glucose-dependent islet blood velocity changes observed in wild-type mice, only minimal differences are observed in both Cx36(+/-) and Cx36(-/-) mice. This observation supports a novel model where intraislet blood cell flow is regulated by the coordinated electrical activity in the islet β-cells. Because Cx36 expression and function is reduced in type 2 diabetes, the resulting defect in intraislet blood cell flow regulation may also play a significant role in diabetic pathology.

  1. Clinical islet transplant: current and future directions towards tolerance.

    PubMed

    Shapiro, A M James; Nanji, Sulaiman A; Lakey, Jonathan R T

    2003-12-01

    The ultimate goal of islet transplantation is to completely correct the diabetic state from an unlimited donor source, without the need for chronic immunosuppressive drug therapy. Although islet transplantation provides an opportunity to develop innovative strategies for tolerance in the clinic, both alloimmune and autoimmune barriers must be controlled, if stable graft function is to be maintained long-term. After islet extraction from the pancreas, the cellular graft may be stored in tissue culture or cryopreserved for banking, providing an opportunity not only to optimally condition the recipient but also to allow in vitro immunologic manipulation of the graft before transplantation, unlike solid organ grafts. As such, islets may be considered a "special case." Remarkable progress has occurred in the last three years, with dramatic improvements in outcomes after clinical islet transplantation. The introduction of a steroid-free, sirolimus-based, anti-rejection protocol and islets prepared from two (or rarely three) donors led to high rates of insulin independence. The "Edmonton Protocol" has been successfully replicated by other centers in an international multicenter trial. A number of key refinements in pancreas transportation, processing, purification on non-ficoll-based media, storage of islets in culture for two days and newer immunological conditioning and induction therapies have led to continued advancement through extensive collaboration between key centers. This review outlines the historical development of islet transplantation over the past 30 years, provides an update on current clinical outcomes, and summarizes a series of unique opportunities for development and early testing of tolerance protocols in patients.

  2. A preclinical evaluation of alternative site for islet allotransplantation

    PubMed Central

    He, Sirong; Yuan, Yujia; Han, Pengfei; Wang, Dan; Chen, Younan; Liu, Jingping; Tian, Bole; Yang, Guang; Yi, Shounan; Gao, Fabao; Zhong, Zhihui; Li, Hongxia; Cheng, Jingqiu; Lu, Yanrong

    2017-01-01

    The bone marrow cavity (BMC) has recently been identified as an alternative site to the liver for islet transplantation. This study aimed to compare the BMC with the liver as an islet allotransplantation site in diabetic monkeys. Diabetes was induced in Rhesus monkeys using streptozocin, and the monkeys were then divided into the following three groups: Group1 (islets transplanted in the liver with immunosuppressant), Group 2 (islets transplanted in the tibial BMC), and Group 3 (islets transplanted in the tibial BMC with immunosuppressant). The C-peptide and blood glucose levels were preoperatively measured. An intravenous glucose tolerance test (IVGTT) was conducted to assess graft function, and complete blood cell counts were performed to assess cell population changes. Cytokine expression was measured using an enzyme-linked immune sorbent assay (ELISA) and MILLIPLEX. Five monkeys in Group 3 exhibited a significantly increased insulin-independent time compared with the other groups (Group 1: 78.2 ± 19.0 days; Group 2: 58.8 ± 17.0 days; Group 3: 189.6 ± 26.2 days) and demonstrated increases in plasma C-peptide 4 months after transplantation. The infusion procedure was not associated with adverse effects. Functional islets in the BMC were observed 225 days after transplantation using the dithizone (DTZ) and insulin/glucagon stains. Our results showed that allogeneic islets transplanted in the BMC of diabetic Rhesus monkeys remained alive and functional for a longer time than those transplanted in the liver. This study was the first successful demonstration of allogeneic islet engraftment in the BMC of non-human primates (NHPs). PMID:28358858

  3. Compensatory Islet Response to Insulin Resistance Revealed by Quantitative Proteomics

    PubMed Central

    Gedeon, Nicholas; Kahraman, Sevim; De Jesus, Dario F.; Bhatt, Shweta; Kim, Jong-Seo; Clauss, Therese RW; Camp, David G.; Smith, Richard D.; Qian, Wei-Jun; Kulkarni, Rohit N.

    2015-01-01

    Compensatory islet response is a distinct feature of the pre-diabetic insulin resistant state in humans and rodents. To identify alterations in the islet proteome that characterize the adaptive response, we analyzed islets from five-month-old male control, high-fat diet fed (HFD) or obese ob/ob mice by LC-MS(/MS) and quantified ~1,100 islet proteins (at least two peptides) with a false discovery rate <1%. Significant alterations in abundance were observed for ~350 proteins between groups. A majority of alterations were common to both models, and the changes of a subset of ~40 proteins and 12 proteins were verified by targeted quantification using selected reaction monitoring and Western blots, respectively. The insulin resistant islets in both groups exhibited reduced expression of proteins controlling energy metabolism, oxidative phosphorylation, hormone processing, and secretory pathways. Conversely, an increased expression of molecules involved in protein synthesis and folding suggested effects in endoplasmic reticulum stress response, cell survival, and proliferation in both insulin resistant models. In summary, we report a unique comparison of the islet proteome that is focused on the compensatory response in two insulin resistant rodent models that are not overtly diabetic. These data provide a valuable resource of candidate proteins to the scientific community to undertake further studies aimed at enhancing β-cell mass in patients with diabetes. The data are available via the MassIVE repository, with accession MSV000079093. PMID:26151086

  4. Adaptation of pancreatic islet cyto-architecture during development

    NASA Astrophysics Data System (ADS)

    Striegel, Deborah A.; Hara, Manami; Periwal, Vipul

    2016-04-01

    Plasma glucose in mammals is regulated by hormones secreted by the islets of Langerhans embedded in the exocrine pancreas. Islets consist of endocrine cells, primarily α, β, and δ cells, which secrete glucagon, insulin, and somatostatin, respectively. β cells form irregular locally connected clusters within islets that act in concert to secrete insulin upon glucose stimulation. Varying demands and available nutrients during development produce changes in the local connectivity of β cells in an islet. We showed in earlier work that graph theory provides a framework for the quantification of the seemingly stochastic cyto-architecture of β cells in an islet. To quantify the dynamics of endocrine connectivity during development requires a framework for characterizing changes in the probability distribution on the space of possible graphs, essentially a Fokker-Planck formalism on graphs. With large-scale imaging data for hundreds of thousands of islets containing millions of cells from human specimens, we show that this dynamics can be determined quantitatively. Requiring that rearrangement and cell addition processes match the observed dynamic developmental changes in quantitative topological graph characteristics strongly constrained possible processes. Our results suggest that there is a transient shift in preferred connectivity for β cells between 1-35 weeks and 12-24 months.

  5. Compensatory islet response to insulin resistance revealed by quantitative proteomics

    SciTech Connect

    El Ouaamari, Abdelfattah; Zhou, Jian -Ying; Liew, Chong Wee; Shirakawa, Jun; Dirice, Ercument; Gedeon, Nicholas; Kahraman, Sevim; De Jesus, Dario F.; Bhatt, Shweta; Kim, Jong -Seo; Clauss, Therese R. W.; Camp, II, David G.; Smith, Richard D.; Qian, Wei -Jun; Kulkarni, Rohit N.

    2015-07-07

    Compensatory islet response is a distinct feature of the pre-diabetic insulin resistant state in humans and rodents. To identify alterations in the islet proteome that characterize the adaptive response, we analyzed islets from five-month-old male control, high-fat diet fed (HFD) or obese ob/ob mice by LC-MS(/MS) and quantified ~1,100 islet proteins (at least two peptides) with a false discovery rate <1%. Significant alterations in abundance were observed for ~350 proteins between groups. A majority of alterations were common to both models, and the changes of a subset of ~40 proteins and 12 proteins were verified by targeted quantification using selected reaction monitoring and Western blots, respectively. The insulin resistant islets in both groups exhibited reduced expression of proteins controlling energy metabolism, oxidative phosphorylation, hormone processing, and secretory pathways. Conversely, an increased expression of molecules involved in protein synthesis and folding suggested effects in endoplasmic reticulum stress response, cell survival, and proliferation in both insulin resistant models. In conclusion, we report a unique comparison of the islet proteome that is focused on the compensatory response in two insulin resistant rodent models that are not overtly diabetic. In conclusion, these data provide a valuable resource of candidate proteins to the scientific community to undertake further studies aimed at enhancing β-cell mass in patients with diabetes. The data are available via the MassIVE repository, with accession MSV000079093.

  6. Compensatory islet response to insulin resistance revealed by quantitative proteomics

    DOE PAGES

    El Ouaamari, Abdelfattah; Zhou, Jian -Ying; Liew, Chong Wee; ...

    2015-07-07

    Compensatory islet response is a distinct feature of the pre-diabetic insulin resistant state in humans and rodents. To identify alterations in the islet proteome that characterize the adaptive response, we analyzed islets from five-month-old male control, high-fat diet fed (HFD) or obese ob/ob mice by LC-MS(/MS) and quantified ~1,100 islet proteins (at least two peptides) with a false discovery rate <1%. Significant alterations in abundance were observed for ~350 proteins between groups. A majority of alterations were common to both models, and the changes of a subset of ~40 proteins and 12 proteins were verified by targeted quantification using selectedmore » reaction monitoring and Western blots, respectively. The insulin resistant islets in both groups exhibited reduced expression of proteins controlling energy metabolism, oxidative phosphorylation, hormone processing, and secretory pathways. Conversely, an increased expression of molecules involved in protein synthesis and folding suggested effects in endoplasmic reticulum stress response, cell survival, and proliferation in both insulin resistant models. In conclusion, we report a unique comparison of the islet proteome that is focused on the compensatory response in two insulin resistant rodent models that are not overtly diabetic. In conclusion, these data provide a valuable resource of candidate proteins to the scientific community to undertake further studies aimed at enhancing β-cell mass in patients with diabetes. The data are available via the MassIVE repository, with accession MSV000079093.« less

  7. Elevation of cytosolic calcium by imidazolines in mouse islets of Langerhans: implications for stimulus-response coupling of insulin release.

    PubMed Central

    Shepherd, R. M.; Hashmi, M. N.; Kane, C.; Squires, P. E.; Dunne, M. J.

    1996-01-01

    1. Microfluorimetry techniques with fura-2 were used to characterize the effects of efaroxan (200 microM), phenotolamine (200-500 microM) and idazoxan (200-500 microM) on the intracellular free Ca2+ concentration ([Ca2+]i) in mouse isolated islets of Langerhans. 2. The imidazoline receptor agonists efaroxan and phentolamine consistently elevated cytosolic Ca2+ by mechanisms that were dependent upon Ca2+ influx across the plasma membrane; there was no rise in [Ca2+]i when Ca2+ was removed from outside of the islets and diazoxide (100-250 microM) attenuated the responses. 3. Modulation of cytosolic [Ca2+]i by efaroxan and phentolamine was augmented by glucose (5-10 mM) which both potentiated the magnitude of the response and reduced the onset time of imidazoline-induced rises in [Ca2+]i. 4. Efaroxan- and phentolamine-evoked increases in [Ca2+]i were unaffected by overnight pretreatment of islets with the imidazolines. Idazoxan failed to increase [Ca2+]i under any experimental condition tested. 5. The putative endogenous ligand of imidazoline receptors, agmatine (1 microM-1 mM), blocked KATP channels in isolated patches of beta-cell membrane, but effects upon [Ca2+]i could not be further investigated since agmatine disrupts fura-2 fluorescence. 6. In conclusion, the present study shows that imidazolines will evoke rises in [Ca2+]i in intact islets, and this provides an explanation to account for the previously described effects of imidazolines on KATP channels, the cell membrane potential and insulin secretion in pancreatic beta-cells. PMID:8922740

  8. Biomembrane and receptor mechanisms

    SciTech Connect

    Chapman, D.; Bertoli, E.

    1987-01-01

    This book cover the reviews on biomembrane dynamics; recent spectroscopic studies. Topics covered are freeze fracture: Seeing and thinking biological membranes, membrane proteins and receptors: structure and organisation; techniques to determine the transbilayer distribution and mobility of phospholipids in biological membranes, transbilayer organisation of phospholipids in the plasma membranes of pro-erythroblasts and normal and abnormal red cells, aminophospholipid translocation in the erythroctye membrane is mediated by a specific AIP-dependent enzyme; membrane protein interactions, lipid-protein interactions: selectively and receptor binding, membrane fluidity in the regulation of membrane-linked enzymes, the lipid regulation of receptor functions, microheterogencity of biological membrane: structural and functional implications, fusion-fission reactions in biological membranes and in phospholpid bilayers, methods for studying the structure and function of the mitochondrial uncoupling protein, methods for studying metabolite transport in mitochondria, transport of metabolites in mitochondria, membrane gangliosides and allied glycosphingolipids: Biochemical features and physicochemical properties, the use of merocyanine 540 for monitoring aggregation properties of sialogangliosides in solution, hormone reception at the cell surface - an overview, double role for GIP in the stimulus secretion sequence of mast cells and neurophils, tumor promoters and hormone receptor coupling mechanisms in the anterior pituitary. The regulation of hormone-dependent adenylate cyclase in native membranes and systems reconstituted from purified components.- Immunological tools for the study of plasma membrane receptors.

  9. Chronic Glucose Exposure Systematically Shifts the Oscillatory Threshold of Mouse Islets: Experimental Evidence for an Early Intrinsic Mechanism of Compensation for Hyperglycemia

    PubMed Central

    Glynn, Eric; Thompson, Benjamin; Vadrevu, Suryakiran; Lu, Shusheng; Kennedy, Robert T.; Ha, Joon; Sherman, Arthur

    2016-01-01

    Mouse islets exhibit glucose-dependent oscillations in electrical activity, intracellular Ca2+ and insulin secretion. We developed a mathematical model in which a left shift in glucose threshold helps compensate for insulin resistance. To test this experimentally, we exposed isolated mouse islets to varying glucose concentrations overnight and monitored their glucose sensitivity the next day by measuring intracellular Ca2+, electrical activity, and insulin secretion. Glucose sensitivity of all oscillation modes was increased when overnight glucose was greater than 2.8mM. To determine whether threshold shifts were a direct effect of glucose or involved secreted insulin, the KATP opener diazoxide (Dz) was coapplied with glucose to inhibit insulin secretion. The addition of Dz or the insulin receptor antagonist s961 increased islet glucose sensitivity, whereas the KATP blocker tolbutamide tended to reduce it. This suggests insulin and glucose have opposing actions on the islet glucose threshold. To test the hypothesis that the threshold shifts were due to changes in plasma membrane KATP channels, we measured cell KATP conductance, which was confirmed to be reduced by high glucose pretreatment and further reduced by Dz. Finally, treatment of INS-1 cells with glucose and Dz overnight reduced high affinity sulfonylurea receptor (SUR1) trafficking to the plasma membrane vs glucose alone, consistent with insulin increasing KATP conductance by altering channel number. The results support a role for metabolically regulated KATP channels in the maintenance of glucose homeostasis. PMID:26697721

  10. Coxsackie–adenovirus receptor expression is enhanced in pancreas from patients with type 1 diabetes

    PubMed Central

    Hodik, M; Anagandula, M; Fuxe, J; Krogvold, L; Dahl-Jørgensen, K; Hyöty, H; Sarmiento, L; Frisk, G

    2016-01-01

    Objectives One of the theories connecting enterovirus (EV) infection of human islets with type 1 diabetes (T1D) is the development of a fertile field in the islets. This implies induction of appropriate proteins for the viral replication such as the coxsackie–adenovirus receptor (CAR). The aim of this study was to investigate to what extent CAR is expressed in human islets of Langerhans, and what conditions that would change the expression. Design Immunohistochemistry for CAR was performed on paraffin-embedded pancreatic tissue from patients with T1D (n=9 recent onset T1D, n=4 long-standing T1D), islet autoantibody-positive individuals (n=14) and non-diabetic controls (n=24) individuals. The expression of CAR was also examined by reverse transcription PCR on microdissected islets (n=5), exocrine tissue (n=5) and on explanted islets infected with EV or exposed to chemokines produced by EV-infected islet cells. Results An increased frequency of patients with T1D and autoantibody-positive individuals expressed CAR in the pancreas (p<0.039). CAR staining was detected more frequently in pancreatic islets from patients with T1D and autoantibody-positive subjects (15/27) compared with (6/24) non-diabetic controls (p<0.033). Also in explanted islets cultured in UV-treated culture medium from coxsackievirus B (CBV)-1-infected islets, the expression of the CAR gene was increased compared with controls. Laser microdissection of pancreatic tissue revealed that CAR expression was 10-fold higher in endocrine compared with exocrine cells of the pancreas. CAR was also expressed in explanted islets and the expression level decreased with time in culture. CBV-1 infection of explanted islets clearly decreased the expression of CAR (p<0.05). In contrast, infection with echovirus 6 did not affect the expression of CAR. Conclusions CAR is expressed in pancreatic islets of patients with T1D and the expression level of CAR is increased in explanted islets exposed to proinflammatory

  11. Maternal vitamin D-restricted diet has consequences in the formation of pancreatic islet/insulin-signaling in the adult offspring of mice.

    PubMed

    Maia-Ceciliano, Thais C; Barreto-Vianna, Andre R C; Barbosa-da-Silva, Sandra; Aguila, Marcia B; Faria, Tatiane S; Mandarim-de-Lacerda, Carlos A

    2016-10-01

    The maternal deficiency of vitamin D can act on organogenesis in mice offspring, being a risk factor for chronic diseases in adulthood. This study investigates the effects of maternal deficiency of vitamin D on structural islet remodeling and insulin-signaling pathway in the offspring. We studied male C57Bl/6 offspring at 3-month old (n = 10/group) from mother fed one of the two diets: control diet (C) or vitamin D-restricted diet (VitD(-)). After weaning, offspring only fed the control diet ad libitum. In the offspring, we studied insulin production, islet remodeling, and islet protein expression of the insulin-signaling pathway (Western blotting, isolated islet, n = 5/group). VitD(-) offspring showed greater glycemia (P = 0.012), smaller beta-cell mass (P = 0.014), and hypoinsulinemia (P = 0.024) than C offspring. Comparing VitD(-) offspring with C offspring, we observed lower protein levels in islet of insulin (P = 0.003), insulin receptor substrate-1 (P = 0.025), phosphatidylinositol-3-kinases (P = 0.045), 3-phosphoinositide-dependent protein kinase 1 (P = 0.017), protein kinase B (P = 0.028), with reduced expression of pancreas/duodenum homeobox-1 (PDX-1) (P = 0.016), glucose transporter-2 (P = 0.003), and glucokinase (P = 0.045). The maternal vitamin D-restricted diet modifies the development of the pancreas of the offspring, leading to islet remodeling and altered insulin-signaling pathway. The decrease of PDX-1 is probably significant to the changes in the beta-cell mass and insulin secretion in adulthood.

  12. Pancreatic Islet Survival and Engraftment Is Promoted by Culture on Functionalized Spider Silk Matrices.

    PubMed

    Johansson, Ulrika; Ria, Massimiliano; Åvall, Karin; Dekki Shalaly, Nancy; Zaitsev, Sergei V; Berggren, Per-Olof; Hedhammar, My

    2015-01-01

    Transplantation of pancreatic islets is one approach for treatment of diabetes, however, hampered by the low availability of viable islets. Islet isolation leads to disruption of the environment surrounding the endocrine cells, which contributes to eventual cell death. The reestablishment of this environment is vital, why we herein investigated the possibility of using recombinant spider silk to support islets in vitro after isolation. The spider silk protein 4RepCT was formulated into three different formats; 2D-film, fiber mesh and 3D-foam, in order to provide a matrix that can give the islets physical support in vitro. Moreover, cell-binding motifs from laminin were incorporated into the silk protein in order to create matrices that mimic the natural cell environment. Pancreatic mouse islets were thoroughly analyzed for adherence, necrosis and function after in vitro maintenance on the silk matrices. To investigate their suitability for transplantation, we utilized an eye model which allows in vivo imaging of engraftment. Interestingly, islets that had been maintained on silk foam during in vitro culture showed improved revascularization. This coincided with the observation of preserved islet architecture with endothelial cells present after in vitro culture on silk foam. Selected matrices were further evaluated for long-term preservation of human islets. Matrices with the cell-binding motif RGD improved human islet maintenance (from 36% to 79%) with preserved islets architecture and function for over 3 months in vitro. The islets established cell-matrix contacts and formed vessel-like structures along the silk. Moreover, RGD matrices promoted formation of new, insulin-positive islet-like clusters that were connected to the original islets via endothelial cells. On silk matrices with islets from younger donors (<35 year), the amount of newly formed islet-like clusters found after 1 month in culture were almost double compared to the initial number of islets

  13. Pancreatic Islet Survival and Engraftment Is Promoted by Culture on Functionalized Spider Silk Matrices

    PubMed Central

    Johansson, Ulrika; Dekki Shalaly, Nancy; Zaitsev, Sergei V.; Berggren, Per-Olof; Hedhammar, My

    2015-01-01

    Transplantation of pancreatic islets is one approach for treatment of diabetes, however, hampered by the low availability of viable islets. Islet isolation leads to disruption of the environment surrounding the endocrine cells, which contributes to eventual cell death. The reestablishment of this environment is vital, why we herein investigated the possibility of using recombinant spider silk to support islets in vitro after isolation. The spider silk protein 4RepCT was formulated into three different formats; 2D-film, fiber mesh and 3D-foam, in order to provide a matrix that can give the islets physical support in vitro. Moreover, cell-binding motifs from laminin were incorporated into the silk protein in order to create matrices that mimic the natural cell environment. Pancreatic mouse islets were thoroughly analyzed for adherence, necrosis and function after in vitro maintenance on the silk matrices. To investigate their suitability for transplantation, we utilized an eye model which allows in vivo imaging of engraftment. Interestingly, islets that had been maintained on silk foam during in vitro culture showed improved revascularization. This coincided with the observation of preserved islet architecture with endothelial cells present after in vitro culture on silk foam. Selected matrices were further evaluated for long-term preservation of human islets. Matrices with the cell-binding motif RGD improved human islet maintenance (from 36% to 79%) with preserved islets architecture and function for over 3 months in vitro. The islets established cell-matrix contacts and formed vessel-like structures along the silk. Moreover, RGD matrices promoted formation of new, insulin-positive islet-like clusters that were connected to the original islets via endothelial cells. On silk matrices with islets from younger donors (<35 year), the amount of newly formed islet-like clusters found after 1 month in culture were almost double compared to the initial number of islets

  14. Direct Stimulation of Islet Insulin Secretion by Glycolytic and Mitochondrial Metabolites in KCl-Depolarized Islets

    PubMed Central

    Deeney, Jude T.; Corkey, Barbara E.

    2016-01-01

    We have previously demonstrated that islet depolarization with 70 mM KCl opens Cx36 hemichannels and allows diffusion of small metabolites and cofactors through the β-cell plasma membrane. We have investigated in this islet “permeabilized” model whether glycolytic and citric acid cycle intermediates stimulate insulin secretion and how it correlates with ATP production (islet content plus extracellular nucleotide accumulation). Glycolytic intermediates (10 mM) stimulated insulin secretion and ATP production similarly. However, they showed differential sensitivities to respiratory chain or enzyme inhibitors. Pyruvate showed a lower secretory capacity and less ATP production than phosphoenolpyruvate, implicating an important role for glycolytic generation of ATP. ATP production by glucose-6-phosphate was not sensitive to a pyruvate kinase inhibitor that effectively suppressed the phosphoenolpyruvate-induced secretory response and islet ATP rise. Strong suppression of both insulin secretion and ATP production induced by glucose-6-phosphate was caused by 10 μM antimycin A, implicating an important role for the glycerophosphate shuttle in transferring reducing equivalents to the mitochondria. Five citric acid cycle intermediates were investigated for their secretory and ATP production capacity (succinate, fumarate, malate, isocitrate and α-ketoglutarate at 5 mM, together with ADP and/or NADP+ to feed the NADPH re-oxidation cycles). The magnitude of the secretory response was very similar among the different mitochondrial metabolites but α-ketoglutarate showed a more sustained second phase of secretion. Gabaculine (1 mM, a GABA-transaminase inhibitor) suppressed the second phase of secretion and the ATP-production stimulated by α-ketoglutarate, supporting a role for the GABA shuttle in the control of glucose-induced insulin secretion. None of the other citric acid intermediates essayed showed any suppression of both insulin secretion or ATP-production by the

  15. Quantitative Assessment of Islets of Langerhans Encapsulated in Alginate

    PubMed Central

    Johnson, Amy S.; O'Sullivan, Esther; D'Aoust, Laura N.; Omer, Abdulkadir; Bonner-Weir, Susan; Fisher, Robert J.; Weir, Gordon C.

    2011-01-01

    Improved methods have recently been developed for assessing islet viability and quantity in human islet preparations for transplantation, and these measurements have proven useful for predicting transplantation outcome. The objectives of this study were to adapt these methods for use with microencapsulated islets, to verify that they provide meaningful quantitative measurements, and to test them with two model systems: (1) barium alginate and (2) barium alginate containing a 70% (w/v) perfluorocarbon (PFC) emulsion, which presents challenges to use of these assays and is of interest in its own right as a means for reducing oxygen supply limitations to encapsulated tissue. Mitochondrial function was assessed by oxygen consumption rate measurements, and the analysis of data was modified to account for the increased solubility of oxygen in the PFC-alginate capsules. Capsules were dissolved and tissue recovered for nuclei counting to measure the number of cells. Capsule volume was determined from alginate or PFC content and used to normalize measurements. After low oxygen culture for 2 days, islets in normal alginate lost substantial viable tissue and displayed necrotic cores, whereas most of the original oxygen consumption rate was recovered with PFC alginate, and little necrosis was observed. All nuclei were recovered with normal alginate, but some nuclei from nonrespiring cells were lost with PFC alginate. Biocompatibility tests revealed toxicity at the islet periphery associated with the lipid emulsion used to provide surfactants during the emulsification process. We conclude that these new assay methods can be applied to islets encapsulated in materials as complex as PFC-alginate. Measurements made with these materials revealed that enhancement of oxygen permeability of the encapsulating material with a concentrated PFC emulsion improves survival of encapsulated islets under hypoxic conditions, but reformulation of the PFC emulsion is needed to reduce toxicity

  16. An 'alpha-beta' of pancreatic islet microribonucleotides.

    PubMed

    Dalgaard, Louise Torp; Eliasson, Lena

    2017-01-22

    MicroRNAs (miRNAs) are cellular, short, non-coding ribonucleotides acting as endogenous posttranscriptional repressors following incorporation in the RNA-induced silencing complex. Despite being chemically and mechanistically very similar, miRNAs exert a multitude of different cellular effects by acting on mRNA species, whose gene-products partake in a wide array of processes. Here, the aim was to review the knowledge of miRNA expression and action in the islet of Langerhans. We have focused on: 1) physiological consequences of islet or beta cell specific inhibition of miRNA processing, 2) mechanisms regulating processing of miRNAs in islet cells, 3) presence and function of miRNAs in alpha versus beta cells - the two main cell types of islets, and 4) miRNA mediators of beta cell decompensation. It is clear that miRNAs regulate pancreatic islet development, maturation, and function in vivo. Moreover, processing of miRNAs appears to be altered by obesity, diabetes, and aging. A number of miRNAs (such as miR-7, miR-21, miR-29, miR-34a, miR-212/miR-132, miR-184, miR-200 and miR-375) are involved in mediating beta cell dysfunction and/or compensation induced by hyperglycemia, oxidative stress, cytotoxic cytokines, and in rodent models of fetal metabolic programming prediabetes and overt diabetes. Studies of human type 2 diabetic islets underline that these miRNA families could have important roles also in human type 2 diabetes. Furthermore, there is a genuine gap of knowledge regarding miRNA expression and function in pancreatic alpha cells. Progress in this area would be enhanced by improved in vitro alpha cell models and better tools for islet cell sorting.

  17. Survival of encapsulated islets: More than a membrane story

    PubMed Central

    Barkai, Uriel; Rotem, Avi; de Vos, Paul

    2016-01-01

    At present, proven clinical treatments but no cures are available for diabetes, a global epidemic with a huge economic burden. Transplantation of islets of Langerhans by their infusion into vascularized organs is an experimental clinical protocol, the first approach to attain cure. However, it is associated with lifelong use of immunosuppressants. To overcome the need for immunosuppression, islets are encapsulated and separated from the host immune system by a permselective membrane. The lead material for this application is alginate which was tested in many animal models and a few clinical trials. This review discusses all aspects related to the function of transplanted encapsulated islets such as the basic requirements from a permselective membrane (e.g., allowable hydrodynamic radii, implications of the thickness of the membrane and relative electrical charge). Another aspect involves adequate oxygen supply, which is essential for survival/performance of transplanted islets, especially when using large retrievable macro-capsules implanted in poorly oxygenated sites like the subcutis. Notably, islets can survive under low oxygen tension and are physiologically active at > 40 Torr. Surprisingly, when densely crowded, islets are fully functional under hyperoxic pressure of up to 500 Torr (> 300% of atmospheric oxygen tension). The review also addresses an additional category of requirements for optimal performance of transplanted islets, named auxiliary technologies. These include control of inflammation, apoptosis, angiogenesis, and the intra-capsular environment. The review highlights that curing diabetes with a functional bio-artificial pancreas requires optimizing all of these aspects, and that significant advances have already been made in many of them. PMID:27011906

  18. Characterization of resident lymphocytes in human pancreatic islets.

    PubMed

    Radenkovic, M; Uvebrant, K; Skog, O; Sarmiento, L; Avartsson, J; Storm, P; Vickman, P; Bertilsson, P-A; Fex, M; Korgsgren, O; Cilio, C M

    2017-03-01

    The current view of type 1 diabetes (T1D) is that it is an immune-mediated disease where lymphocytes infiltrate the pancreatic islets, promote killing of beta cells and cause overt diabetes. Although tissue resident immune cells have been demonstrated in several organs, the composition of lymphocytes in human healthy pancreatic islets have been scarcely studied. Here we aimed to investigate the phenotype of immune cells associated with human islets of non-diabetic organ donors. A flow cytometry analysis of isolated islets from perfused pancreases (n = 38) was employed to identify alpha, beta, T, natural killer (NK) and B cells. Moreover, the expression of insulin and glucagon transcripts was evaluated by RNA sequencing. Up to 80% of the lymphocytes were CD3(+) T cells with a remarkable bias towards CD8(+) cells. Central memory and effector memory phenotypes dominated within the CD8(+) and CD4(+) T cells and most CD8(+) T cells were positive for CD69 and up to 50-70% for CD103, both markers of resident memory cells. The frequency of B and NK cells was low in most islet preparations (12 and 3% of CD45(+) cells, respectively), and the frequency of alpha and beta cells varied between donors and correlated clearly with insulin and glucagon mRNA expression. In conclusion, we demonstrated the predominance of canonical tissue resident memory CD8(+) T cells associated with human islets. We believe that these results are important to understand more clearly the immunobiology of human islets and the disease-related phenotypes observed in diabetes.

  19. The Effect of Leptin and Adiponectin on KiSS-1 and KissR mRNA Expression in Rat Islets of Langerhans and CRI-D2 Cell Line

    PubMed Central

    Mahmoodzadeh Sagheb, Mandana; Azarpira, Negar; Yaghobi, Ramin

    2014-01-01

    Background: Leptin and adiponectin are the two key metabolic hormones secreted from adipocytes to control food intake and energy expenditure. The action of both hormones in regulation of Gonadotropin Releasing Hormone (GnRH) secretion from the hypothalamus is mediated through Kisspeptins. Kisspeptins are products of KiSS-1 gene. Leptin and adiponectin are modulators of KiSS-1 expression in the hypothalamus. These peptides have also important roles in pancreatic β-cells to control insulin synthesis and secretion and their receptors are detected in Langerhans islets. We hypothesized that leptin and adiponectin might alter KiSS-1 and Kiss Receptor mRNA expression in the islets. Objectives: The aim of this study is to investigate any modulatory effect that leptin and adiponectin may have on the expression of Kiss-1 and KiSSR gene in Langerhans islets. Materials and Methods: We isolated the islets from adult male rats by collagenase and cultured CRI-D2 cell lines to investigate the effect of leptin and adiponectin. Then, we incubated them with different concentrations of leptin and adiponectin for 24 hours. After that, RNA was extracted from the islets and CRI-D2 cells and transcripted to cDNA. KiSS-1 and KissR expression levels were evaluated by real time PCR. Results: In islet and CRI-D2 cells, leptin increased the KiSS-1 mRNA expression significantly, but adiponectin decreased it was expected. Conclusions: These findings indicated the possibility that KiSS-1 mRNA expression is a mediator of leptin and adiponectin function in the islets. PMID:24910643

  20. Fibrillar dimer formation of islet amyloid polypeptides

    DOE PAGES

    Chiu, Chi -cheng; de Pablo, Juan J.

    2015-05-08

    Amyloid deposits of human islet amyloid polypeptide (hIAPP), a 37-residue hormone co-produced with insulin, have been implicated in the development of type 2 diabetes. Residues 20 – 29 of hIAPP have been proposed to constitute the amyloidogenic core for the aggregation process, yet the segment is mostly unstructured in the mature fibril, according to solid-state NMR data. Here we use molecular simulations combined with bias-exchange metadynamics to characterize the conformational free energies of hIAPP fibrillar dimer and its derivative, pramlintide. We show that residues 20 – 29 are involved in an intermediate that exhibits transient β-sheets, consistent with recent experimentalmore » and simulation results. By comparing the aggregation of hIAPP and pramlintide, we illustrate the effects of proline residues on inhibition of the dimerization of IAPP. The mechanistic insights presented here could be useful for development of therapeutic inhibitors of hIAPP amyloid formation.« less

  1. Optimal pig donor selection in islet xenotransplantation: current status and future perspectives.

    PubMed

    Zhu, Hai-tao; Yu, Liang; Lyu, Yi; Wang, Bo

    2014-08-01

    Islet transplantation is an attractive treatment of type 1 diabetes mellitus. Xenotransplantation, using the pig as a donor, offers the possibility of an unlimited supply of islet grafts. Published studies demonstrated that pig islets could function in diabetic primates for a long time (>6 months). However, pig-islet xenotransplantation must overcome the selection of an optimal pig donor to obtain an adequate supply of islets with high-quality, to reduce xeno-antigenicity of islet and prolong xenograft survival, and to translate experimental findings into clinical application. This review discusses the suitable pig donor for islet xenotransplantation in terms of pig age, strain, structure/function of islet, and genetically modified pig.

  2. Evaluation of the NanoCHIP® Gastrointestinal Panel (GIP) Test for Simultaneous Detection of Parasitic and Bacterial Enteric Pathogens in Fecal Specimens

    PubMed Central

    Ken Dror, Shifra; Pavlotzky, Elsa; Barak, Mira

    2016-01-01

    Infectious gastroenteritis is a global health problem associated with high morbidity and mortality rates. Rapid and accurate diagnosis is crucial to allow appropriate and timely treatment. Current laboratory stool testing has a long turnaround time (TAT) and demands highly qualified personnel and multiple techniques. The need for high throughput and the number of possible enteric pathogens compels the implementation of a molecular approach which uses multiplex technology, without compromising performance requirements. In this work we evaluated the feasibility of the NanoCHIP® Gastrointestinal Panel (GIP) (Savyon Diagnostics, Ashdod, IL), a molecular microarray-based screening test, to be used in the routine workflow of our laboratory, a big outpatient microbiology laboratory. The NanoCHIP® GIP test provides simultaneous detection of nine major enteric bacteria and parasites: Campylobacter spp., Salmonella spp., Shigella spp., Giardia sp., Cryptosporidium spp., Entamoeba histolytica, Entamoeba dispar, Dientamoeba fragilis, and Blastocystis spp. The required high-throughput was obtained by the NanoCHIP® detection system together with the MagNA Pure 96 DNA purification system (Roche Diagnostics Ltd., Switzerland). This combined system has demonstrated a higher sensitivity and detection yield compared to the conventional methods in both, retrospective and prospective samples. The identification of multiple parasites and bacteria in a single test also enabled increased efficiency of detecting mixed infections, as well as reduced hands-on time and work load. In conclusion, the combination of these two automated systems is a proper response to the laboratory needs in terms of improving laboratory workflow, turn-around-time, minimizing human errors and can be efficiently integrated in the routine work of the laboratory. PMID:27447173

  3. Transcriptome of pancreas-specific Bmpr1a-deleted islets links to TPH1–5-HT axis

    PubMed Central

    Jiang, Fang-Xu; Mishina, Yuji; Baten, Akma; Morahan, Grant; Harrison, Leonard C.

    2015-01-01

    ABSTRACT Bone morphogenetic protein (BMP) signaling is crucial for the development and function of numerous organs, but its role on the function of pancreatic islets is not completely clear. To explore this question, we applied the high throughput transcriptomic analyses on the islets isolated from mice with a pancreas-specific deletion of the gene, Bmpr1a, encoding the type 1a BMP receptor. Consistently, these pBmpr1aKO mice had impaired glucose homeostasis at 3 months, and were more severely affected at 12 months of age. These had lower fasting blood insulin concentrations, with reduced expression of several key regulators of β-cell function. Importantly, transcriptomic profiling of 3-month pBmpr1aKO islets and bioinformatic analyses revealed abnormal expression of 203 metabolic genes. Critically among these, the tryptophan hydroxylase 1 gene (Tph1), encoding the rate-limiting enzyme for the production of 5-hydroxytryptamine (5-HT) was the highest over-expressed one. 5-HT is an important regulator of insulin secretion from β cells. Treatment with excess 5-HT inhibited this secretion. Thus our transcriptomic analysis links two highly conserved molecular pathways the BMP signaling and the TPH1–5-HT axis on glucose homeostasis. PMID:26187948

  4. Human islet amyloid polypeptide expression in COS-1 cells. A model of intracellular amyloidogenesis.

    PubMed Central

    O'Brien, T. D.; Butler, P. C.; Kreutter, D. K.; Kane, L. A.; Eberhardt, N. L.

    1995-01-01

    Non-insulin-dependent diabetes mellitus is characterized by concurrent loss of beta-cells and deposition of islet amyloid derived from islet amyloid polypeptide (IAPP). We have previously demonstrated that IAPP-derived amyloid forms intracellularly in humans with chronic excess insulin expression (eg, insulinoma and insulin receptor antibody-induced insulin resistance). To determine whether overexpression of IAPP results in intracellular amyloid in mammalian cells, we transfected COS cells with vectors expressing amyloidogenic human IAPP or non-amyloidogenic rat IAPP. Transfected COS-1 cells secreted comparable amounts of human IAPP and rat IAPP (2.1 to 2.8 nmol/L/48 hours). After 96 hours, 90% of cells expressing human IAPP contained amyloid fibrils and were degenerating or dead, whereas cells transfected with rat IAPP lacked amyloid and were viable. Thus, overexpression of human IAPP can result in intracellular amyloid formation that is associated with cell death, suggesting that intracellular amyloid may play a role in beta-cell loss in non-insulin-dependent diabetes mellitus. Images Figure 1 Figure 2 PMID:7677175

  5. Regulation of pancreatic islet beta-cell mass by growth factor and hormone signaling.

    PubMed

    Huang, Yao; Chang, Yongchang

    2014-01-01

    Dysfunction and destruction of pancreatic islet beta cells is a hallmark of diabetes. Better understanding of cellular signals in beta cells will allow development of therapeutic strategies for diabetes, such as preservation and expansion of beta-cell mass and improvement of beta-cell function. During the past several decades, the number of studies analyzing the molecular mechanisms, including growth factor/hormone signaling pathways that impact islet beta-cell mass and function, has increased exponentially. Notably, somatolactogenic hormones including growth hormone (GH), prolactin (PRL), and insulin-like growth factor-1 (IGF-1) and their receptors (GHR, PRLR, and IGF-1R) are critically involved in beta-cell growth, survival, differentiation, and insulin secretion. In this chapter, we focus more narrowly on GH, PRL, and IGF-1 signaling, and GH-IGF-1 cross talk. We also discuss how these signaling aspects contribute to the regulation of beta-cell proliferation and apoptosis. In particular, our novel findings of GH-induced formation of GHR-JAK2-IGF-1R protein complex and synergistic effects of GH and IGF-1 on beta-cell signaling, proliferation, and antiapoptosis lead to a new concept that IGF-1R may serve as a proximal component of GH/GHR signaling.

  6. Protein-Mediated Interactions of Pancreatic Islet Cells

    PubMed Central

    Meda, Paolo

    2013-01-01

    The islets of Langerhans collectively form the endocrine pancreas, the organ that is soley responsible for insulin secretion in mammals, and which plays a prominent role in the control of circulating glucose and metabolism. Normal function of these islets implies the coordination of different types of endocrine cells, noticeably of the beta cells which produce insulin. Given that an appropriate secretion of this hormone is vital to the organism, a number of mechanisms have been selected during evolution, which now converge to coordinate beta cell functions. Among these, several mechanisms depend on different families of integral membrane proteins, which ensure direct (cadherins, N-CAM, occludin, and claudins) and paracrine communications (pannexins) between beta cells, and between these cells and the other islet cell types. Also, other proteins (integrins) provide communication of the different islet cell types with the materials that form the islet basal laminae and extracellular matrix. Here, we review what is known about these proteins and their signaling in pancreatic β-cells, with particular emphasis on the signaling provided by Cx36, given that this is the integral membrane protein involved in cell-to-cell communication, which has so far been mostly investigated for effects on beta cell functions. PMID:24278783

  7. Multicenter Australian trial of islet transplantation: improving accessibility and outcomes.

    PubMed

    O'Connell, P J; Holmes-Walker, D J; Goodman, D; Hawthorne, W J; Loudovaris, T; Gunton, J E; Thomas, H E; Grey, S T; Drogemuller, C J; Ward, G M; Torpy, D J; Coates, P T; Kay, T W

    2013-07-01

    Whilst initial rates of insulin independence following islet transplantation are encouraging, long-term function using the Edmonton Protocol remains a concern. The aim of this single-arm, multicenter study was to evaluate an immunosuppressive protocol of initial antithymocyte globulin (ATG), tacrolimus and mycophenolate mofetil (MMF) followed by switching to sirolimus and MMF. Islets were cultured for 24 h prior to transplantation. The primary end-point was an HbA1c of <7% and cessation of severe hypoglycemia. Seventeen recipients were followed for ≥ 12 months. Nine islet preparations were transported interstate for transplantation. Similar outcomes were achieved at all three centers. Fourteen of the 17 (82%) recipients achieved the primary end-point. Nine (53%) recipients achieved insulin independence for a median of 26 months (range 7-39 months) and 6 (35%) remain insulin independent. All recipients were C-peptide positive for at least 3 months. All subjects with unstimulated C-peptide >0.2 nmol/L had cessation of severe hypoglycemia. Nine of the 17 recipients tolerated switching from tacrolimus to sirolimus with similar graft outcomes. There was a small but significant reduction in renal function in the first 12 months. The combination of islet culture, ATG, tacrolimus and MMF is a viable alternative for islet transplantation.

  8. Groundwater dynamics of Fongafale Islet, Funafuti Atoll, Tuvalu.

    PubMed

    Nakada, Satoshi; Umezawa, Yu; Taniguchi, Makoto; Yamano, Hiroya

    2012-01-01

    Geoelectric and hydrologic surveys during spring tides revealed the spatiotemporal distribution of groundwater quality produced by tidal forcing in Fongafale Islet, Funafuti Atoll, Tuvalu. The observed low resistivity showed that saline water largely immersed the surficial Holocene aquifer, indicating that there is no thick freshwater lens in Fongafale Islet, unlike in other atoll islands of comparable size. Half of the islet was constructed by reclaiming the original swamp with porous, highly permeable coral blocks; this reclaimed area should not be considered as part of the islet width for calculation of the expected thickness of the freshwater lens. The degree of aquifer salinization depends on the topographic characteristics and the hydrologic controls on the inland propagation of the tidal forcing. Large changes in bulk resistivity and the electrical conductivity of groundwater from wells indicate that periodic salinization in phase with the semidiurnal tides was occurring widely, especially in areas at lower elevation than the high-tide level and in reclaimed areas with high permeability. Thin sheets of nearly fresh and brackish water were observed in the surficial aquifer in areas above the high-tide level and in taro swamps, respectively. The thinness of the brackish and freshwater sheets suggests that the taro swamps and the fresh groundwater resources of the islet are highly vulnerable to salinization from anticipated sea-level rise. An understanding of the inherent geologic and topographic features of an atoll is necessary to evaluate the groundwater resources of the atoll and assess the vulnerability of its water resources to climate change.

  9. Aldosterone decreases glucose-stimulated insulin secretion in vivo in mice and in murine islets

    PubMed Central

    Luo, P.; Kreger, M. T.; Brissova, M.; Dai, C.; Whitfield, T. T.; Kim, H. S.; Wasserman, D. H.; Powers, A. C.; Brown, N. J.

    2011-01-01

    Aims/hypothesis Aldosterone concentrations increase in obesity and predict the onset of diabetes. We investigated the effects of aldosterone on glucose homeostasis and insulin secretion in vivo and in vitro. Methods We assessed insulin sensitivity and insulin secretion in aldosterone synthase-deficient (As [also known as Cyp11b2]−/−)and wild-type mice using euglycaemic-hyperinsulinaemic and hyperglycaemic clamps, respectively. We also conducted studies during high sodium intake to normalise renin activity and potassium concentration in As−/− mice. We subsequently assessed the effect of aldosterone on insulin secretion in vitro in the presence or absence of mineralocorticoid receptor antagonists in isolated C57BL/6J islets and in the MIN6 beta cell line. Results Fasting glucose concentrations were reduced in As−/−mice compared with wild-type. During hyperglycaemic clamps, insulin and C-peptide concentrations increased to a greater extent in As−/− than in wild-type mice. This was not attributable to differences in potassium or angiotensin II, as glucose-stimulated insulin secretion was enhanced in As−/− mice even during high sodium intake. There was no difference in insulin sensitivity between As−/− and wild-type mice in euglycaemic-hyperinsulinaemic clamp studies. In islet and MIN6 beta cell studies, aldosterone inhibited glucose and isobutylmethylxanthine-stimulated insulin secretion, an effect that was not blocked by mineralocorticoid receptor antagonism, but was prevented by the superoxide dismutase mimetic tempol. Conclusions/interpretation We demonstrated that aldosterone deficiency or excess modulates insulin secretion in vivo and in vitro via reactive oxygen species and in a manner that is independent of mineralocorticoid receptors. These findings provide insight into the mechanism of glucose intolerance in conditions of relative aldosterone excess. PMID:21519965

  10. Pigment epithelium-derived factor (PEDF) regulates metabolism and insulin secretion from a clonal rat pancreatic beta cell line BRIN-BD11 and mouse islets.

    PubMed

    Chen, Younan; Carlessi, Rodrigo; Walz, Nikita; Cruzat, Vinicius Fernandes; Keane, Kevin; John, Abraham N; Jiang, Fang-Xu; Carnagarin, Revathy; Dass, Crispin R; Newsholme, Philip

    2016-05-05

    Pigment epithelium-derived factor (PEDF) is a multifunctional glycoprotein, associated with lipid catabolism and insulin resistance. In the present study, PEDF increased chronic and acute insulin secretion in a clonal rat β-cell line BRIN-BD11, without alteration of glucose consumption. PEDF also stimulated insulin secretion from primary mouse islets. Seahorse flux analysis demonstrated that PEDF did not change mitochondrial respiration and glycolytic function. The cytosolic presence of the putative PEDF receptor - adipose triglyceride lipase (ATGL) - was identified, and ATGL associated stimulation of glycerol release was robustly enhanced by PEDF, while intracellular ATP levels increased. Addition of palmitate or ex vivo stimulation with inflammatory mediators induced β-cell dysfunction, effects not altered by the addition of PEDF. In conclusion, PEDF increased insulin secretion in BRIN-BD11 and islet cells, but had no impact on glucose metabolism. Thus elevated lipolysis and enhanced fatty acid availability may impact insulin secretion following PEDF receptor (ATGL) stimulation.

  11. Identification of T cell subsets and class I and class II antigen expression in islet grafts and pancreatic islets of diabetic BioBreeding/Worcester rats.

    PubMed Central

    Weringer, E. J.; Like, A. A.

    1988-01-01

    The BioBreeding/Worcester (BB/Wor) rat develops a spontaneous disorder that closely resembles human insulin-dependent (Type I) diabetes mellitus. The syndrome is preceded by lymphocytic insulitis that destroys pancreatic beta cells. The morphologic features of the spontaneous insulitis lesions are also observed within islets transplanted beneath the renal capsule of diabetes-prone and diabetic animals. This study reports the results of experiments in which immunohistochemical techniques were used to characterize the phenotype of the infiltrating mononuclear cells and detect the expression of class I and class II MHC antigens in native islets and islet transplants in diabetic and diabetes-prone BB/Wor rats. The infiltrates within native pancreatic islets and islet grafts were comprised predominantly of Ia+ cells (dendritic cells and macrophages) CD4+ cells (helper/inducer lymphocytes and macrophages), CD5+ (pan-T) cells and smaller numbers of CD8+ (cytotoxic/suppressor and NK) cells. Pancreatic and graft insulitis were accompanied by markedly enhanced class I antigen expression on islet and exocrine cells. Class II (Ia) antigens were not detected on normal islet cells, islets undergoing insulitis or on islet transplants subjected to immune attack. In islet grafts stained with polymorphic MAbs that distinguish Ia antigens of donor and host origin, Ia antigen expression was limited to infiltrating dendritic cells and macrophages of host origin. It is concluded that the phenotypes of infiltrating mononuclear cells that comprise the insulitis lesion in spontaneous BB/Wor diabetes, and the inflammatory attack on islets transplanted into diabetic BB/Wor rats are the same, that pancreatic islet and graft insulitis occur in the presence of enhanced class I antigen expression but in the absence of class II antigen expression, and that infiltrating Ia+ cells within islet grafts are exclusively of recipient (BB/Wor) origin and may explain the initiation of immune insulitis

  12. Glucose-induced β cell production of IL-1β contributes to glucotoxicity in human pancreatic islets

    PubMed Central

    Maedler, Kathrin; Sergeev, Pavel; Ris, Frédéric; Oberholzer, José; Joller-Jemelka, Helen I.; Spinas, Giatgen A.; Kaiser, Nurit; Halban, Philippe A.; Donath, Marc Y.

    2002-01-01

    In type 2 diabetes, chronic hyperglycemia is suggested to be detrimental to pancreatic β cells, causing impaired insulin secretion. IL-1β is a proinflammatory cytokine acting during the autoimmune process of type 1 diabetes. IL-1β inhibits β cell function and promotes Fas-triggered apoptosis in part by activating the transcription factor NF-κB. Recently, we have shown that increased glucose concentrations also induce Fas expression and β cell apoptosis in human islets. The aim of the present study was to test the hypothesis that IL-1β may mediate the deleterious effects of high glucose on human β cells. In vitro exposure of islets from nondiabetic organ donors to high glucose levels resulted in increased production and release of IL-1β, followed by NF-κB activation, Fas upregulation, DNA fragmentation, and impaired β cell function. The IL-1 receptor antagonist protected cultured human islets from these deleterious effects. β cells themselves were identified as the islet cellular source of glucose-induced IL-1β. In vivo, IL-1β–producing β cells were observed in pancreatic sections of type 2 diabetic patients but not in nondiabetic control subjects. Similarly, IL-1β was induced in β cells of the gerbil Psammomys obesus during development of diabetes. Treatment of the animals with phlorizin normalized plasma glucose and prevented β cell expression of IL-1β. These findings implicate an inflammatory process in the pathogenesis of glucotoxicity in type 2 diabetes and identify the IL-1β/NF-κB pathway as a target to preserve β cell mass and function in this condition. PMID:12235117

  13. Monomethylated-adenines potentiate glucose-induced insulin production and secretion via inhibition of phosphodiesterase activity in rat pancreatic islets.

    PubMed

    Boland, Brandon B; Alarcón, Cristina; Ali, Almas; Rhodes, Christopher J

    2015-01-01

    Monomethyladenines have effects on DNA repair, G-protein-coupled receptor antagonism and autophagy. In islet ß-cells, 3-methyladenine (3-MA) has been implicated in DNA-repair and autophagy, but its mechanism of action is unclear. Here, the effect of monomethylated adenines was examined in rat islets. 3-MA, N6-methyladenine (N6-MA) and 9-methyladenine (9-MA), but not 1- or 7-monomethylated adenines, specifically potentiated glucose-induced insulin secretion (3-4 fold; p ≤ 0.05) and proinsulin biosynthesis (∼2-fold; p ≤ 0.05). Using 3-MA as a 'model' monomethyladenine, it was found that 3-MA augmented [cAMP]i accumulation (2-3 fold; p ≤ 0.05) in islets within 5 minutes. The 3-, N6- and 9-MA also enhanced glucose-induced phosphorylation of the cAMP/protein kinase-A (PKA) substrate cAMP-response element binding protein (CREB). Treatment of islets with pertussis or cholera toxin indicated 3-MA mediated elevation of [cAMP]i was not mediated via G-protein-coupled receptors. Also, 3-MA did not compete with 9-cyclopentyladenine (9-CPA) for adenylate cyclase inhibition, but did for the pan-inhibitor of phosphodiesterase (PDE), 3-isobutyl-1-methylxanthine (IBMX). Competitive inhibition experiments with PDE-isoform specific inhibitors suggested 3-MA to have a preference for PDE4 in islet ß-cells, but this was likely reflective of PDE4 being the most abundant PDE isoform in ß-cells. In vitro enzyme assays indicated that 3-, N6- and 9-MA were capable of inhibiting most PDE isoforms found in ß-cells. Thus, in addition to known inhibition of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3'K)/m Target of Rapamycin (mTOR) signaling, 3-MA also acts as a pan-phosphodiesterase inhibitor in pancreatic ß-cells to elevate [cAMP]i and then potentiate glucose-induced insulin secretion and production in parallel.

  14. Genetically modified mesenchymal stem cells for improved islet transplantation.

    PubMed

    Wu, Hao; Ye, Zhaoyang; Mahato, Ram I

    2011-10-03

    The use of adult stem cells for therapeutic purposes has met with great success in recent years. Among several types of adult stem cells, mesenchymal stem cells (MSCs) derived from bone marrow (BM) and other sources have gained popularity for basic research and clinical applications because of their therapeutic potential in treating a variety of diseases. Because of their tissue regeneration potential and immune modulation effect, MSCs were recently used as cell-based therapy to promote revascularization, increase pancreatic β-cell proliferation, and avoid allograft rejection in islet transplantation. Taking advantage of the recent progress in gene therapy, genetically modified MSCs can further enhance and expand the therapeutic benefit of primary MSCs while retaining their stem-cell-like properties. This review aims to gain a thorough understanding of the current obstacles to successful islet transplantation and discusses the potential role of primary MSCs before or after genetic modification in islet transplantation.

  15. Functional tissue engineering of the liver and islets.

    PubMed

    Ohashi, Kazuo; Okano, Teruo

    2014-01-01

    Cell-based therapies by using hepatocytes and islets have recently been evaluated as a new therapeutic modality for patients with many forms of liver diseases and insulin-deficient diabetes mellitus. In most of the recently conducted clinical trials, cells have been delivered into liver vasculatures by infusing them through the portal circulation. More recently, tissue engineering-based approaches have spurred significant interests, using hepatocytes and islets in which small but functional new tissues would be created in vivo. Under circumstances in which a higher level of cell engraftment could be obtained, these approaches could provide therapeutic effects. Considerable efforts have been given to sustaining engineered tissues and maintaining their therapeutic effects. This review highlights several strategies that can achieve a higher level of cell survival for creating new functional liver and islet tissues.

  16. 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.

  17. Islet-intrinsic effects of CFTR mutation.

    PubMed

    Koivula, Fiona N Manderson; McClenaghan, Neville H; Harper, Alan G S; Kelly, Catriona

    2016-07-01

    Cystic fibrosis-related diabetes (CFRD) is the most significant extra-pulmonary comorbidity in cystic fibrosis (CF) patients, and accelerates lung decline. In addition to the traditional view that CFRD is a consequence of fibrotic destruction of the pancreas as a whole, emerging evidence may implicate a role for cystic fibrosis transmembrane-conductance regulator (CFTR) in the regulation of insulin secretion from the pancreatic islet. Impaired first-phase insulin responses and glucose homeostasis have also been reported in CF patients. CFTR expression in both human and mouse beta cells has been confirmed, and recent studies have shown differences in endocrine pancreatic morphology from birth in CF. Recent experimental evidence suggests that functional CFTR channels are required for insulin exocytosis and the regulation of membrane potential in the pancreatic beta cell, which may account for the impairments in insulin secretion observed in many CF patients. These novel insights suggest that the pathogenesis of CFRD is more complicated than originally thought, with implications for diabetes treatment and screening in the CF population. This review summarises recent emerging evidence in support of a primary role for endocrine pancreatic dysfunction in the development of CFRD. Summary • CF is an autosomal recessive disorder caused by mutations in the CFTR gene • The vast majority of morbidity and mortality in CF results from lung disease. However CFRD is the largest extra-pulmonary co-morbidity and rapidly accelerates lung decline • Recent experimental evidence shows that functional CFTR channels are required for normal patterns of first phase insulin secretion from the pancreatic beta cell • Current clinical recommendations suggest that insulin is more effective than oral glucose-lowering drugs for the treatment of CFRD. However, the emergence of CFTR corrector and potentiator drugs may offer a personalised approach to treating diabetes in the CF population.

  18. Phosphatidylinositol hydrolysis in isolated guinea-pig islets of Langerhans.

    PubMed Central

    Schrey, M P; Montague, W

    1983-01-01

    Previous studies have reported an increased turnover of phospholipid in isolated islets of Langerhans in response to raised glucose concentrations. The present investigation was thus undertaken to determine the nature of any phospholipases that may be implicated in this phenomenon by employing various radiolabelled exogenous phospholipids. Hydrolysis of 1-acyl-2-[14C]arachidonoylglycerophosphoinositol by a sonicated preparation of islets optimally released radiolabelled lysophosphatidylinositol, arachidonic acid and 1,2-diacylglycerol at pH 5,7 and 9 respectively. This indicates the presence of a phospholipase A1 and a phospholipase C. However, the lack of any labelled lysophosphatidylinositol production when 2-acyl-1-[14C]stearoylglycerophosphoinositol was hydrolysed argues against a role for phospholipase A2 in the release of arachidonic acid. Phospholipase C activity as measured by phosphatidyl-myo-[3H]inositol hydrolysis was optimal around pH8, required Ca2+ for activity and was predominantly cytosolic in origin. The time course of phosphatidylinositol hydrolysis at pH 6 indicated a precursor-product relationship for 1,2-diacylglycerol and arachidonic acid respectively. The release of these two products when phosphatidylinositol was hydrolysed by either islet or acinar tissue was similar. However, phospholipase A1 activity was 20-fold higher in acinar tissue. Substrate specificity studies with islet tissue revealed that arachidonic acid release from phosphatidylethanolamine and phosphatidylcholine was only 8% and 2.5% respectively of that from phosphatidylinositol. Diacylglycerol lipase was also demonstrated in islet tissue being predominantly membrane bound and stimulated by Ca2+. The availability of non-esterified arachidonic acid in islet cells could be regulated by changes in the activity of a phosphatidylinositol-specific phospholipase C acting in concert with a diacylglycerol lipase. PMID:6362663

  19. DAMP production by human islets under low oxygen and nutrients in the presence or absence of an immunoisolating-capsule and necrostatin-1

    PubMed Central

    Paredes-Juarez, Genaro A.; Sahasrabudhe, Neha M.; Tjoelker, Reina S.; de Haan, Bart J.; Engelse, Marten A.; de Koning, Eelco J. P.; Faas, Marijke M.; de Vos, Paul

    2015-01-01

    In between the period of transplantation and revascularization, pancreatic islets are exposed to low-oxygen and low-nutrient conditions. In the present study we mimicked those conditions in vitro to study the involvement of different cell death processes, release of danger-associated molecular patterns (DAMP), and associated in vitro immune activation. Under low-oxygen and low-nutrient conditions, apoptosis, autophagy and necroptosis occur in human islets. Necroptosis is responsible for DAMP-release such as dsDNA, uric acid, and HMGB1. The sensors of the innate immune system able to recognize these DAMPs are mainly TLR, NOD receptors, and C-type lectins. By using cell-lines with a non-functional adaptor molecule MyD88, we were able to show that the islet-derived DAMPs signal mainly via TLR. Immunoisolation in immunoprotective membranes reduced DAMP release and immune activation via retention of the relative large DAMPs in the capsules. Another effective strategy was suppressing necroptosis using the inhibitor nec-1. Although the effect on cell-survival was minor, nec-1 was able to reduce the release of HMGB1 and its associated immune activation. Our data demonstrate that in the immediate post-transplant period islets release DAMPs that in vitro enhance responses of innate immune cells. DAMP release can be reduced in vitro by immunoisolation or intervention with nec-1. PMID:26419792

  20. Improved hypothermic short-term storage of isolated mouse islets by adding serum to preservation solutions.

    PubMed

    Kimura, Yasuko; Okitsu, Teru; Xibao, Liu; Teramae, Hiroki; Okonogi, Atsuhito; Toyoda, Kentaro; Uemoto, Shinji; Fukushima, Masanori

    2013-01-01

    Preserving isolated islets at low temperature appears attractive because it can keep islet quantity comparable to freshly isolated islets. In this study, we evaluated the effect of serum as an additive to preservation solutions on islet quality after short-term hypothermic storage. Isolated mouse islets were preserved at 4°C in University of Wisconsin solution (UW) alone, UW with serum, M-Kyoto solution (MK) alone or MK with serum. We then assessed islet quantity, morphology, viability and function in vitro as well as in vivo. Islet quantity after storage in all four solutions was well maintained for up to 120 h. However, islets functioned for different duration; glucose-stimulated insulin release assay revealed that the duration was 72 h when islets were stored in UW with serum and MK with serum, but only 24 h in UW alone, and the islet function disappeared immediately in MK alone. Viability assay confirmed that more than 70% islet cells survived for up to 48 h when islets are preserved in UW with serum and MK with serum, but the viability decreased rapidly in UW alone and MK alone. In in vivo bioassays using 48-h preserved isogeneic islets, all recipient mice restored normal blood glucose concentrations by transplants preserved in UW with serum or MK with serum, whereas 33.3% recipients and no recipient restored diabetes by transplants preserved in UW alone and in MK alone respectively. Adding serum to both UW and MK improves their capability to store isolated islets by maintaining islet functional viability.

  1. A central role for GRB10 in regulation of islet function in man.

    PubMed

    Prokopenko, Inga; Poon, Wenny; Mägi, Reedik; Prasad B, Rashmi; Salehi, S Albert; Almgren, Peter; Osmark, Peter; Bouatia-Naji, Nabila; Wierup, Nils; Fall, Tove; Stančáková, Alena; Barker, Adam; Lagou, Vasiliki; Osmond, Clive; Xie, Weijia; Lahti, Jari; Jackson, Anne U; Cheng, Yu-Ching; Liu, Jie; O'Connell, Jeffrey R; Blomstedt, Paul A; Fadista, Joao; Alkayyali, Sami; Dayeh, Tasnim; Ahlqvist, Emma; Taneera, Jalal; Lecoeur, Cecile; Kumar, Ashish; Hansson, Ola; Hansson, Karin; Voight, Benjamin F; Kang, Hyun Min; Levy-Marchal, Claire; Vatin, Vincent; Palotie, Aarno; Syvänen, Ann-Christine; Mari, Andrea; Weedon, Michael N; Loos, Ruth J F; Ong, Ken K; Nilsson, Peter; Isomaa, Bo; Tuomi, Tiinamaija; Wareham, Nicholas J; Stumvoll, Michael; Widen, Elisabeth; Lakka, Timo A; Langenberg, Claudia; Tönjes, Anke; Rauramaa, Rainer; Kuusisto, Johanna; Frayling, Timothy M; Froguel, Philippe; Walker, Mark; Eriksson, Johan G; Ling, Charlotte; Kovacs, Peter; Ingelsson, Erik; McCarthy, Mark I; Shuldiner, Alan R; Silver, Kristi D; Laakso, Markku; Groop, Leif; Lyssenko, Valeriya

    2014-04-01

    Variants in the growth factor receptor-bound protein 10 (GRB10) gene were in a GWAS meta-analysis associated with reduced glucose-stimulated insulin secretion and increased risk of type 2 diabetes (T2D) if inherited from the father, but inexplicably reduced fasting glucose when inherited from the mother. GRB10 is a negative regulator of insulin signaling and imprinted in a parent-of-origin fashion in different tissues. GRB10 knock-down in human pancreatic islets showed reduced insulin and glucagon secretion, which together with changes in insulin sensitivity may explain the paradoxical reduction of glucose despite a decrease in insulin secretion. Together, these findings suggest that tissue-specific methylation and possibly imprinting of GRB10 can influence glucose metabolism and contribute to T2D pathogenesis. The data also emphasize the need in genetic studies to consider whether risk alleles are inherited from the mother or the father.

  2. XIAP inhibition of β-cell apoptosis reduces the number of islets required to restore euglycemia in a syngeneic islet transplantation model.

    PubMed

    Plesner, Annette; Soukhatcheva, Galina; Korneluk, Robert G; Verchere, C Bruce

    2010-01-01

    Clinical pancreatic islet transplantation has great promise as a treatment for type 1 diabetes but despite recent advances, it is still limited by the need for lifelong immunosuppression, restricted availability of donor islets, and uncertainty regarding long-term graft survival. Using a syngeneic, suboptimal islet transplantation model, we asked whether adenoviral overexpression of an anti-apoptotic protein, the X-linked inhibitor of apoptosis protein (XIAP) would protect transplanted islet cells from death and reduce the number of islets required for successful transplantation. Transplantation of 100 XIAP-expressing islets into the kidney capsule of syngeneic Balb/c mice restored euglycemia in 86% of recipients, where transplantation of 100 islets transduced with a control adenovirus expressing LacZ restored euglycemia in only 27% of recipients. Analysis of islet grafts by insulin/TUNEL double immunostaining revealed fewer apoptotic beta-cells in recipients of XIAP- compared with LacZ-expressing grafts (0.8±0.5 vs. 2.4±0.8 double-positive cells/graft), suggesting that XIAP enhances graft success by inhibiting β-cell apoptosis in the immediate post-transplant period. In summary, XIAP overexpression inhibits beta cell apoptosis in syngeneic islet transplants, thereby reducing the number of islets and decreasing the number of days required to restore euglycemia. These data raise the possibility that ex vivo XIAP gene transfer in islets prior to transplantation has the potential to increase the number of donor islets available for transplantation and may enhance graft function and long-term transplant success.

  3. Automated digital image analysis of islet cell mass using Nikon's inverted eclipse Ti microscope and software to improve engraftment may help to advance the therapeutic efficacy and accessibility of islet transplantation across centers.

    PubMed

    Gmyr, Valery; Bonner, Caroline; Lukowiak, Bruno; Pawlowski, Valerie; Dellaleau, Nathalie; Belaich, Sandrine; Aluka, Isanga; Moermann, Ericka; Thevenet, Julien; Ezzouaoui, Rimed; Queniat, Gurvan; Pattou, Francois; Kerr-Conte, Julie

    2015-01-01

    Reliable assessment of islet viability, mass, and purity must be met prior to transplanting an islet preparation into patients with type 1 diabetes. The standard method for quantifying human islet preparations is by direct microscopic analysis of dithizone-stained islet samples, but this technique may be susceptible to inter-/intraobserver variability, which may induce false positive/negative islet counts. Here we describe a simple, reliable, automated digital image analysis (ADIA) technique for accurately quantifying islets into total islet number, islet equivalent number (IEQ), and islet purity before islet transplantation. Islets were isolated and purified from n = 42 human pancreata according to the automated method of Ricordi et al. For each preparation, three islet samples were stained with dithizone and expressed as IEQ number. Islets were analyzed manually by microscopy or automatically quantified using Nikon's inverted Eclipse Ti microscope with built-in NIS-Elements Advanced Research (AR) software. The AIDA method significantly enhanced the number of islet preparations eligible for engraftment compared to the standard manual method (p < 0.001). Comparisons of individual methods showed good correlations between mean values of IEQ number (r(2) = 0.91) and total islet number (r(2) = 0.88) and thus increased to r(2) = 0.93 when islet surface area was estimated comparatively with IEQ number. The ADIA method showed very high intraobserver reproducibility compared to the standard manual method (p < 0.001). However, islet purity was routinely estimated as significantly higher with the manual method versus the ADIA method (p < 0.001). The ADIA method also detected small islets between 10 and 50 µm in size. Automated digital image analysis utilizing the Nikon Instruments software is an unbiased, simple, and reliable teaching tool to comprehensively assess the individual size of each islet cell preparation prior to transplantation. Implementation of this

  4. Resistance to adenovirally induced hyperleptinemia in rats. Comparison of ventromedial hypothalamic lesions and mutated leptin receptors.

    PubMed Central

    Koyama, K; Shimabukuro, M; Chen, G; Wang, M Y; Lee, Y; Kalra, P S; Dube, M G; Kalra, S P; Newgard, C B; Unger, R H

    1998-01-01

    Leptin regulates appetite and body weight via hypothalamic targets, but it can act directly on cultured pancreatic islets to regulate their fat metabolism. To obtain in vivo evidence that leptin may act peripherally as well as centrally, we compared the effect of adenovirally induced hyperleptinemia on food intake, body weight, and islet fat content in ventromedial hypothalamic-lesioned (VMHL) rats, sham-lesioned (SL) controls, and Zucker Diabetic Fatty (ZDF) rats in which the leptin receptor is mutated. Infusion with recombinant adenovirus containing the rat leptin cDNA increased plasma leptin by approximately 20 ng/ml in VMHL and ZDF rats but had no effect on their food intake, body weight, or fat tissue weight. Caloric matching of hyperphagic VMHL rats to SL controls did not reduce their resistance to hyperleptinemia. Whereas prediabetic ZDF rats had a fourfold elevation in islet fat, in VMHL rats islet fat was normal and none of them became diabetic. Isolated islets from ZDF rats were completely resistant to the lipopenic action of leptin, while VMHL islets exhibited 50% of the normal response; caloric matching of VMHL rats to SL controls increased leptin responsiveness of their islets to 92% of controls. We conclude that leptin regulation of adipocyte fat requires an intact VMH but that islet fat content is regulated independently of the VMH. PMID:9710441

  5. Real-time, multidimensional in vivo imaging used to investigate blood flow in mouse pancreatic islets

    PubMed Central

    Nyman, Lara R.; Wells, K. Sam; Head, W. Steve; McCaughey, Michael; Ford, Eric; Brissova, Marcela; Piston, David W.; Powers, Alvin C.

    2008-01-01

    The pancreatic islets of Langerhans are highly vascularized micro-organs that play a key role in the regulation of blood glucose homeostasis. The specific arrangement of endocrine cell types in islets suggests a coupling between morphology and function within the islet. Here, we established a line-scanning confocal microscopy approach to examine the relationship between blood flow and islet cell type arrangement by real-time in vivo imaging of intra-islet blood flow in mice. These data were used to reconstruct the in vivo 3D architecture of the islet and time-resolved blood flow patterns throughout the islet vascular bed. The results revealed 2 predominant blood flow patterns in mouse islets: inner-to-outer, in which blood perfuses the core of β cells before the islet perimeter of non–β cells, and top-to-bottom, in which blood perfuses the islet from one side to the other regardless of cell type. Our approach included both millisecond temporal resolution and submicron spatial resolution, allowing for real-time imaging of islet blood flow within the living mouse, which has not to our knowledge been attainable by other methods. PMID:18846254

  6. Imaging islets labeled with magnetic nanoparticles at 1.5 Tesla.

    PubMed

    Tai, Joo Ho; Foster, Paula; Rosales, Alma; Feng, Biao; Hasilo, Craig; Martinez, Violetta; Ramadan, Soha; Snir, Jonatan; Melling, C W James; Dhanvantari, Savita; Rutt, Brian; White, David J G

    2006-11-01

    We have developed a magnetic resonance imaging (MRI) technique for imaging Feridex (superparamagnetic iron oxide [SPIO])-labeled islets of Langerhans using a standard clinical 1.5-Tesla (T) scanner and employing steady-state acquisition imaging sequence (3DFIESTA). Both porcine and rat islets were labeled with SPIO by a transfection technique using a combination of poly-l-lysine and electroporation. Electron microscopy demonstrated presence of SPIO particles within the individual islet cells, including beta-cells and particles trapped between cell membranes. Our labeling method produced a transfection rate of 860 pg to 3.4 ng iron per islet, dependent on the size of the islet. The labeling procedure did not disrupt either the function or viability of the islets. In vitro 3DFIESTA magnetic resonance images of single-labeled islets corresponded with their optical images. In vivo T2*-weighted scan using 1.5 T detected as few as 200 SPIO-labeled islets transplanted under rat kidney capsule, which correlated with immunohistochemistry of the transplant for insulin and iron. Ex vivo 3DFIESTA images of kidneys containing 200, 800 or 2,000 SPIO-labeled islet isografts showed good correlation between signal loss and increasing numbers of islets. These data provide evidence that islets can be labeled with SPIO and imaged using clinically available 1.5- T MRI.

  7. Vascularized tissue-engineered chambers promote survival and function of transplanted islets and improve glycemic control.

    PubMed

    Knight, K R; Uda, Y; Findlay, M W; Brown, D L; Cronin, K J; Jamieson, E; Tai, T; Keramidaris, E; Penington, A J; Rophael, J; Harrison, L C; Morrison, W A

    2006-03-01

    We have developed a chamber model of islet engraftment that optimizes islet survival by rapidly restoring islet-extracellular matrix relationships and vascularization. Our aim was to assess the ability of syngeneic adult islets seeded into blood vessel-containing chambers to correct streptozotocin-induced diabetes in mice. Approximately 350 syngeneic islets suspended in Matrigel extracellular matrix were inserted into chambers based on either the splenic or groin (epigastric) vascular beds, or, in the standard approach, injected under the renal capsule. Blood glucose was monitored weekly for 7 weeks, and an intraperitoneal glucose tolerance test performed at 6 weeks in the presence of the islet grafts. Relative to untreated diabetic animals, glycemic control significantly improved in all islet transplant groups, strongly correlating with islet counts in the graft (P<0.01), and with best results in the splenic chamber group. Glycemic control deteriorated after chambers were surgically removed at week 8. Immunohistochemistry revealed islets with abundant insulin content in grafts from all groups, but with significantly more islets in splenic chamber grafts than the other treatment groups (P<0.05). It is concluded that hyperglycemia in experimental type 1 diabetes can be effectively treated by islets seeded into a vascularized chamber functioning as a "pancreatic organoid."

  8. Size-based separation and collection of mouse pancreatic islets for functional analysis.

    PubMed

    Nam, Ki-Hwan; Yong, Wang; Harvat, Tricia; Adewola, Adeola; Wang, Shesun; Oberholzer, Jose; Eddington, David T

    2010-10-01

    Islet size has recently been demonstrated to be an important factor in determining human islet transplantation outcomes. In this study, a multi-layered microfluidic device was developed and quantified for size-based separation of a heterogeneous population of mouse islets. The device was fabricated using standard soft lithography and polydimethylsiloxane (PDMS). Size-based separation was first demonstrated via injection of a heterogeneous population of glass beads between 50-300 microm in diameter which were separated into five sub-populations based on their diameter. Next, a heterogeneous population of mouse pancreatic islets, between 50-250 microm in diameter was separated into four sub-populations. Throughout this process the islets remained intact without any signs of damage, as indicated by cell viability staining. Islet glucose-stimulated insulin secretion of each sub-population of islets was also evaluated demonstrating that islets smaller than 150 microm have superior stimulation indexes (SI) compared to islets larger than 150 microm. In this study, we found that islets between 100 microm and 150 microm in diameter had the greatest SI value in a heterogeneous population of islets.

  9. Overexpression of Pref-1 in pancreatic islet β-cells in mice causes hyperinsulinemia with increased islet mass and insulin secretion.

    PubMed

    Wang, Yuhui; Lee, Kichoon; Moon, Yang Soo; Ahmadian, Maryam; Kim, Kee-Hong; Roder, Karim; Kang, Chulho; Sul, Hei Sook

    2015-06-12

    Preadipocyte factor-1 (Pref-1) is made as a transmembrane protein containing EGF-repeats at the extracellular domain that can be cleaved to generate a biologically active soluble form. Pref-1 is found in islet β-cells and its level has been reported to increase in neonatal rat islets upon growth hormone treatment. We found here that Pref-1 can promote growth of pancreatic tumor derived AR42J cells. To examine Pref-1 function in pancreatic islets in vivo, we generated transgenic mouse lines overexpressing the Pref-1/hFc in islet β-cells using rat insulin II promoter (RIP). These transgenic mice exhibit an increase in islet mass with higher proportion of larger islets in pancreas compared to wild-type littermates. This is in contrast to pancreas from Pref-1 null mice that show higher proportion of smaller islets. Insulin expression and insulin secretion from pancreatic islets from RIP-Pref-1/hFc transgenic mice are increased also. Thus, RIP-Pref-1/hFc transgenic mice show normal glucose levels but with higher plasma insulin levels in both fasting and fed conditions. These mice show improved glucose tolerance. Taken together, we conclude Pref-1 as a positive regulator of islet β-cells and insulin production.

  10. The morphology of islets within the porcine donor pancreas determines the isolation result: successful isolation of pancreatic islets can now be achieved from young market pigs.

    PubMed

    Krickhahn, Mareike; Bühler, Christoph; Meyer, Thomas; Thiede, Arnulf; Ulrichs, Karin

    2002-01-01

    Clinical islet allotransplantation has become an increasingly efficient "routine" therapy in recent years. Shortage of human donor organs leads to porcine pancreatic islets as a potential source for islet xenotransplantation. Yet it is still very difficult to isolate sufficient numbers of intact porcine islets, particularly from young market pigs. In the following study islets were successfully isolated from retired breeders [4806 +/- 720 islet equivalents per gram organ (IEQ/g); n = 25; 2-3 years old; RB] and also from young hybrid pigs [2868 +/- 260 IEQ/g; n = 65; 4-6 months old; HY] using LiberasePI and a modified version of Ricordi's digestion-filtration technique. As expected, isolations from RB showed significantly better results (p < 0.002). A retrospective histological analysis of almost all donor pancreases showed that the majority of organs from RB (80%) contained mainly large islets (diameter > 200 microm), in contrast to only 35% of all pancreases from HY. Remarkably, the islet size in situ, regardless whether detected in RB or HY, strongly determined the isolation result. A donor organ with predominantly large islets resulted in significantly higher numbers of IEQs compared with a donor organ with predominantly small islets [RB(Large Islets): 5680 +/- 3,318 IEQ/g (n= 20); RB(Small Islets): 1353 +/- 427 IEQ/g (n = 5); p < 0.02]. In addition, isolation results were strongly influenced by the quality of the LiberasePI batch, and therefore single batch testing is invariably required. Purification was performed using Ficoll or OptiPrep density gradient centrifugation manually or in the COBE cell processor. Although islet purity was highest when OptiPrep was used, final islet yields did not differ between the different purification methods. Our study demonstrates that islet size in situ is an extremely critical parameter for highly successful islet isolation; consequently, we are now performing a morphological screening of each donor organ prior to the

  11. Carbohydrate-induced secretion of glucose-dependent insulinotropic polypeptide and glucagon-like peptide-1.

    PubMed

    Seino, Yusuke; Maekawa, Ryuya; Ogata, Hidetada; Hayashi, Yoshitaka

    2016-04-01

    Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are the incretin hormones secreted from enteroendocrine K-cells and L-cells, respectively, by oral ingestion of various nutrients including glucose. K-cells, L-cells and pancreatic β-cells are glucose-responsive cells with similar glucose-sensing machinery including glucokinase and an adenosine triphosphate-sensitive K(+) channel comprising KIR6.2 and sulfonylurea receptor 1. However, the physiological role of the adenosine triphosphate-sensitive K(+) channel in GIP secretion in K-cells and GLP-1 secretion in L-cells is not elucidated. Recently, it was reported that GIP and GLP-1-producing cells are present also in pancreatic islets, and islet-derived GIP and GLP-1 contribute to glucose-induced insulin secretion from pancreatic β-cells. In this short review, we focus on GIP and GLP-1 secretion by monosaccharides, such as glucose or fructose, and the role of the adenosine triphosphate-sensitive K(+) channel in GIP and GLP-1 secretion.

  12. Immunohistochemical analysis of pancreatic islets of platypus (Ornithorhynchus anatinus) and echidna (Tachyglossus aculeatus ssp.).

    PubMed

    He, Chuan; Myers, Mark A; Forbes, Briony E; Grützner, Frank

    2015-04-01

    Monotremes have undergone remarkable changes to their digestive and metabolic control system; however, the monotreme pancreas remains poorly characterized. Previous work in echidna demonstrated the presence of pancreatic islets, but no information is available for platypus and the fine structure has not been described for either monotreme. Based on our recent finding that monotremes lack the ghrelin gene, which is expressed in mouse and human pancreatic islets, we investigated the structure of monotreme islets in more detail. Generally, as in birds, the islets of monotremes were smaller but greater in number compared with mouse. β-cells were the most abundant endocrine cell population in platypus islets and were located peripherally, while α-cells were observed both in the interior and periphery of the islets. δ-cells and pancreatic polypeptide (PP)-cells were mainly found in the islet periphery. Distinct PP-rich (PP-lobe) and PP-poor areas (non-PP-lobe) are present in therian mammals, and we identified these areas in echidna but not platypus pancreas. Interestingly, in some of the echidna islets, α- and β-cells tended to form two poles within the islets, which to our knowledge is the first time this has been observed in any species. Overall, monotreme pancreata share the feature of consisting of distinct PP-poor and PP-rich islets with other mammals. A higher number of islets and α- or β-cell only islets are shared between monotremes and birds. The islets of monotremes were larger than those of birds but smaller compared with therian mammals. This may indicate a trend of having fewer larger islets comprising several endocrine cell types during mammalian evolution.

  13. Effect of micro- and macroencapsulation on oxygen consumption by pancreatic islets.

    PubMed

    Cornolti, Roberta; Figliuzzi, Marina; Remuzzi, Andrea

    2009-01-01

    Immunoisolation of pancreatic islets is extensively investigated for glycemic control in diabetic experimental animals. We previously reported that subcutaneous xenotransplantation of bovine islets protected by a selective polysulfone membrane successfully controlled glycemia in diabetic rats for up to 20 days. We then wondered whether immunoisolated islets have adequate oxygen supply in this device, where only diffusive transport allows cell function and survival. Here we set up an experimental technique to measure oxygen consumption rate (OCR) using a Clark's electrode inserted in a glass thermostated chamber connected to a data recorder and acquisition system. Bovine islets were isolated from 6-month-old calves, encapsulated in sodium alginate microcapsules or inserted in polysulfone hollow fibers. After 1 and 2 days in culture a series of measurements was performed using free islets (at normal or high-glucose concentration), islets encapsulated in microcapsules, or in hollow fibers. In free islets OCR averaged from 2.0 +/- 0.8 pmol/IEQ/min at low-glucose concentration and from 2.5 +/- 1.0 pmol/IEQ/min at high-glucose concentration (p < 0.01). OCR in islets encapsulated in microcapsules and in hollow fibers was comparable, and not significantly different from that measured in free islets. Two days after isolation OCR averaged 2.3 +/- 0.6 in free islets, 2.3 +/- 0.9 in alginate microcapsules, and 2.2 +/- 0.7 pmol/IEQ/min in hollow fibers. These results show that OCR by bovine islets is comparable to that previously reported for other species. OCR increases in islets stimulated with high glucose and may be considered as a functional index. Moreover, islet encapsulation in alginate microcapsule, as well as in hollow fiber membranes, did not significantly affect in vitro OCR, suggesting adequate islet oxygenation in these conditions.

  14. The use of non-heart-beating donors for isolated pancreatic islet transplantation.

    PubMed

    Markmann, James F; Deng, Shaoping; Desai, Niraj M; Huang, Xiaolun; Velidedeoglu, Ergun; Frank, Adam; Liu, Chengyang; Brayman, Kenneth L; Lian, Moh Moh; Wolf, Bryan; Bell, Ewan; Vitamaniuk, Marko; Doliba, Nicolai; Matschinsky, Franz; Markmann, Eileen; Barker, Clyde F; Naji, Ali

    2003-05-15

    Recent improvements in isolated islet transplantation indicate that this therapy may ultimately prove applicable to patients with type I diabetes. An obstacle preventing widespread application of islet transplantation is an insufficient supply of cadaveric pancreata. Non-heart-beating donors (NHBDs) are generally not deemed suitable for whole-organ pancreas donation and could provide a significant source of pancreata for islet transplantation. Isolated pancreatic islets prepared from 10 NHBDs were compared with those procured from 10 brain-dead donors (BDDs). The success of the isolation for the two groups was analyzed for preparation purity, quality, and recovered islet mass. The function of NHBD and BDD islets was evaluated using in vitro and in vivo assays. On the basis of the results of this analysis, an NHBD isolated islet allograft was performed in a type I diabetic. The recovery of islets from NHBDs was comparable to that of control BDDs. In vitro assessment of NHBD islet function revealed function-equivalent BDD islets, and NHBD islets transplanted to non-obese diabetic-severe combined immunodeficient (NOD-SCID) mice efficiently reversed diabetes. Transplantation of 446,320 islet equivalents (IEq) (8,500 IEq/kg of recipient body weight) from a single NHBD successfully reversed the diabetes of a type I diabetic recipient. Normally functioning pancreatic islets can be isolated successfully from NHBDs. A single donor transplant from an NHBD resulted in a state of stable insulin independence in a type I diabetic recipient. These results indicate that NHBDs may provide an as yet untapped source of pancreatic tissue for preparation of isolated islets for clinical transplantation.

  15. Diffusion into human islets is limited to molecules below 10 kDa.

    PubMed

    Williams, S J; Schwasinger-Schmidt, T; Zamierowski, D; Stehno-Bittel, L

    2012-10-01

    Isolated islets are important tools in diabetes research and are used for islet transplantation as a treatment for type 1 diabetes. Yet these cell clusters have a dramatic diffusion barrier that leads to core cell death. Computer modeling has provided theoretical size limitations, but little has been done to measure the actual rate of diffusion in islets. The purpose of this study was to directly measure the diffusion barrier in intact human islets and determine its role in restricting insulin secretion. Impeded diffusion into islets was monitored with fluorescent dextran beads. Dextran beads of 10-70 kDa failed to diffuse into the core of the intact islets, while 0.9 kDa probe was observed within the core of smaller islets. Diffusion of the fluorescent form of glucose, 2-NBDG, had similar diffusion limitations as the beads, with an average intra-islet diffusion rate of 1.5 ± 0.2 μm/min. The poor diffusion properties were associated with core cell death from necrosis, not apoptosis. Short-term exposure to a mild papain/0 Ca(2+) cocktail, dramatically reduced the diffusion barrier so that all cells within islets were exposed to media components. Lowering the diffusion barrier increased the immediate and long-term viability of islet cells, and tended to increase the amount of insulin released, especially in low glucose conditions. However, it failed to improve the large islet's glucose-stimulated insulin secretion. Thus, the islet diffusion barrier leads to low viability and poor survival of large islets, but is not solely responsible for the reduced insulin secretion of large isolated islets.

  16. 1. AERIAL VIEW, NAVAL INACTIVE SHIPS MAINTENANCE FACILITY, SINCLAIR ISLET, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    1. AERIAL VIEW, NAVAL INACTIVE SHIPS MAINTENANCE FACILITY, SINCLAIR ISLET, BREMERTON, KITSAP COUNTY, WASHINGTON WITH EX-USS HORNET CVS-12, THREE MINECRAFT ALONGSIDE TO PORT. OTHER INACTIVE SHIPS IN BACKGROUND. - U.S.S. HORNET, Puget Sound Naval Shipyard, Sinclair Inlet, Bremerton, Kitsap County, WA

  17. Interrelationship of islet metabolism, adenosine triphosphate content and insulin release

    PubMed Central

    Ashcroft, Stephen J. H.; Weerasinghe, L. Chatra C.; Randle, Philip J.

    1973-01-01

    The oxidation of some exogenous substrates and their effects on ATP content and insulin release in mouse pancreatic islets were measured. The ATP concentration of islets incubated without exogenous substrate shows a gradual decrease, which can be prevented by glucose or mannose (20mm) or leucine (2.5mm); d-glyceraldehyde (5mm) is as effective as glucose (5mm); fructose or N-acetylglucosamine (20mm), pyruvate (10mm) and dl-3-hydroxybutyrate (2mm) are less effective; galactose (20mm), acetate (10mm), octanoate (2mm) and succinate (10mm) have no ATP-maintaining ability. Islets oxidize glucose, mannose, glyceraldehyde, leucine and, less readily, N-acetylglucosamine and glucosamine; galactose, however, is poorly metabolized. Mannoheptulose inhibits the oxidation of glucose but not of glyceraldehyde. Insulin release, measured over a 2h incubation, is stimulated by glucose, mannose, leucine, glyceraldehyde or glucosamine but not by fructose or N-acetylglucosamine. The latter, however, potentiates the effects of glucose or glyceraldehyde (5mm) or leucine (2.5mm) on release; the potentiating effects are inhibited by mannoheptulose, which also blocks glucose-, but not glyceraldehyde- or leucine-stimulated release. In the presence of glucose (20mm), metabolic inhibitors depress insulin release and islet ATP content in parallel. However, rates of insulin release and ATP content measured after incubation with various combinations of exogenous substrates do not appear to be correlated. Sulphonylureas stimulate insulin release but decrease islet ATP concentrations. These results provide further evidence of a close association between the metabolic activity of exogenous substrates and their ability to initiate insulin release. Glucoreceptor models are formulated in the light of these observations and discussed. PMID:4199014

  18. Add-on therapy with anagliptin in Japanese patients with type-2 diabetes mellitus treated with metformin and miglitol can maintain higher concentrations of biologically active GLP-1/total GIP and a lower concentration of leptin.

    PubMed

    Osonoi, Takeshi; Saito, Miyoko; Hariya, Natsuyo; Goto, Moritaka; Mochizuki, Kazuki

    2016-12-01

    Metformin, α-glucosidase inhibitors (α-GIs), and dipeptidyl peptidase 4 inhibitors (DPP-4Is) reduce hyperglycemia without excessive insulin secretion, and enhance postprandial plasma concentration of glucagon-like peptide-1 (GLP-1) in type-2 diabetes mellitus (T2DM) patients. We assessed add-on therapeutic effects of DPP-4I anagliptin in Japanese T2DM patients treated with metformin, an α-GI miglitol, or both drugs on postprandial responses of GLP-1 and glucose-dependent insulinotropic polypeptide (GIP), and on plasma concentration of the appetite-suppressing hormone leptin. Forty-two Japanese T2DM patients with inadequately controlled disease (HbA1c: 6.5%-8.0%) treated with metformin (n=14), miglitol (n=14) or a combination of the two drugs (n=14) received additional treatment with anagliptin (100mg, p.o., b.i.d.) for 52 weeks. We assessed glycemic control, postprandial responses of GLP-1 and glucose-dependent insulinotropic polypeptide (GIP), and on plasma concentration of leptin in those patients. Add-on therapy with anagliptin for 52 weeks improved glycemic control and increased the area under the curve of biologically active GLP-1 concentration without altering obesity indicators. Total GIP concentration at 52 weeks was reduced by add-on therapy in groups treated with miglitol compared with those treated with metformin. Add-on therapy reduced leptin concentrations. Add-on therapy with anagliptin in Japanese T2DM patients treated with metformin and miglitol for 52 weeks improved glycemic control and enhanced postprandial concentrations of active GLP-1/total GIP, and reduce the leptin concentration.

  19. Meal feeding improves oral glucose tolerance in male rats and causes adaptations in postprandial islet hormone secretion that are independent of plasma incretins or glycemia

    PubMed Central

    P., Torsten; Aulinger, Benedikt A.; Smith, Eric P.; Drazen, Deborah L.; Ulrich-Lai, Yve; Seeley, Randy J.; Woods, Stephen C.

    2014-01-01

    Meal-fed (MF) rats with access to food for only 4 consecutive hours during the light cycle learn to eat large meals to maintain energy balance. MF animals develop behavioral and endocrine changes that permit glucose tolerance despite increased meal size. We hypothesized that enhanced activity of the enteroinsular axis mediates glucose homeostasis during MF. Cohorts of rats were allocated to MF or ad libitum (AL) regimens for 2–4 wk. Insulin secretion and glucose tolerance were determined after oral carbohydrate and intraperitoneal (ip) and intravenous (iv) glucose. MF rats ate less than AL in the first week but maintained a comparable weight trajectory thereafter. MF rats had decreased glucose excursions after a liquid mixed meal (AUC: MF 75 ± 7, AL 461 ± 28 mmol·l−1·min, P < 0.001), with left-shifted insulin secretion (AUC0–15: MF 31.0 ± 4.9, AL 9.6 ± 4.4 pM·min, P < 0.02), which peaked before a significant rise in blood glucose. Both groups had comparable fasting glucagon levels, but postprandial responses were lower with MF. However, neither intestinal expression of proGIP and proglucagon mRNA nor plasma incretin levels differed between MF and AL groups. There were no differences in the insulin response to ip or iv glucose between MF and AL rats. These findings demonstrate that MF improves oral glucose tolerance and is associated with significant changes in postprandial islet hormone secretion. Because MF enhanced β-cell function during oral but not parenteral carbohydrate administration, and was not accounted for by changes in circulating incretins, these results support a neural mechanism of adaptive insulin secretion. PMID:25159330

  20. Expression of Innate Immunity Genes and Damage of Primary Human Pancreatic Islets by Epidemic Strains of Echovirus: Implication for Post-Virus Islet Autoimmunity

    PubMed Central

    Sarmiento, Luis; Frisk, Gun; Anagandula, Mahesh; Cabrera-Rode, Eduardo; Roivainen, Merja; Cilio, Corrado M.

    2013-01-01

    Three large-scale Echovirus (E) epidemics (E4,E16,E30), each differently associated to the acute development of diabetes related autoantibodies, have been documented in Cuba. The prevalence of islet cell autoantibodies was moderate during the E4 epidemic but high in the E16 and E30 epidemic. The aim of this study was to evaluate the effect of epidemic strains of echovirus on beta-cell lysis, beta-cell function and innate immunity gene expression in primary human pancreatic islets. Human islets from non-diabetic donors (n = 7) were infected with the virus strains E4, E16 and E30, all isolated from patients with aseptic meningitis who seroconverted to islet cell antibody positivity. Viral replication, degree of cytolysis, insulin release in response to high glucose as well as mRNA expression of innate immunity genes (IFN-b, RANTES, RIG-I, MDA5, TLR3 and OAS) were measured. The strains of E16 and E30 did replicate well in all islets examined, resulting in marked cytotoxic effects. E4 did not cause any effects on cell lysis, however it was able to replicate in 2 out of 7 islet donors. Beta-cell function was hampered in all infected islets (P<0.05); however the effect of E16 and E30 on insulin secretion appeared to be higher than the strain of E4. TLR3 and IFN-beta mRNA expression increased significantly following infection with E16 and E30 (P<0.033 and P<0.039 respectively). In contrast, the expression of none of the innate immunity genes studied was altered in E4-infected islets. These findings suggest that the extent of the epidemic-associated islet autoimmunity may depend on the ability of the viral strains to damage islet cells and induce pro-inflammatory innate immune responses within the infected islets. PMID:24223733

  1. Facile mechanical shaking method is an improved isolation approach for islet preparation and transplantation

    PubMed Central

    Yin, Nina; Chen, Tao; Yu, Yuling; Han, Yongming; Yan, Fei; Zheng, Zhou; Chen, Zebin

    2016-01-01

    Successful islet isolation is crucial for islet transplantation and cell treatment for type 1 diabetes. Current isolation methods are able to obtain 500–1,000 islets per rat, which results in a waste of ≥50% of total islets. In the present study, a facile mechanical shaking method for improving islet yield (up to 1,500 per rat) was developed and summarized, which was demonstrated to be more effective than the existing well-established stationary method. The present results showed that isolated islets have a maximum yield of 1,326±152 when shaking for 15 min for the fully-cannulated pancreas. For both fully-cannulated and half-cannulated pancreas in the presence of rat DNAse inhibitor, the optimal shaking time was amended to 20 min with a further increased yield of 1,344±134 and 1,286±124 islets, respectively. Furthermore, the majority of the isolated islets were morphologically intact with a well-defined surface and almost no central necrotic zone, which suggested that the condition of islets obtained via the mechanical shaking method was consistent with the stationary method. Islet size distribution was also calculated and it was demonstrated that islets from the stationary method exhibited the same size distribution as the non-cannulated group, which had more larger islets than the fully-cannulated and half-cannulated groups isolated via the shaking method. In addition, the results of glucose challenge showed that the refraction index of each group was >2.5, which indicated the well-preserved function of isolated islets. Furthermore, the transplanted islets exhibited a therapeutic effect after 1 day of transplantation; however, they failed to control blood glucose levels after ~7 days of transplantation. In conclusion, these results demonstrated that the facile mechanical shaking method may markedly improve the yield of rat islet isolation, and in vitro and in vivo investigation demonstrated the well-preserved function of isolated islets in the control of

  2. Quantitative analysis of cell composition and purity of human pancreatic islet preparations

    PubMed Central

    Pisania, Anna; Weir, Gordon C.; O'Neil, John J.; Omer, Abdulkadir; Tchipashvili, Vaja; Lei, Ji; Colton, Clark K.; Bonner-Weir, Susan

    2010-01-01

    Despite improvements in outcomes for human islet transplantation, characterization of islet preparations remains poorly defined. This study used both light (LM) and electron microscopy (EM) to characterize 33 islet preparations used for clinical transplants. EM allowed accurate identification and quantification of cell types with measured cell number fractions (mean ± SEM) 35.6 ± 2.1% β-cells, 12.6 ± 1.0% non-β-islet cells, (48.3 ± 2.6% total islet cells), 22.7 ± 1.5% duct cells, and 25.3 ± 1.8% acinar cells. Of the islet cells, 73.6 ± 1.7% were β cells. For comparison to the literature, estimates of cell number fraction, cell volume, and extracellular volume were combined to convert number fraction data to volume fractions applicable to cells, islets, and the entire preparation. The mathematical framework for this conversion was developed. By volume, β cells were 86.5 ± 1.1% of the total islet cell volume and 61.2 ± 0.8% of intact islets (including the extracellular volume), which is similar to that of islets in the pancreas. Our estimates gave 1560 ± 20 cells in an islet equivalent (volume of 150-μm diameter sphere), of which 1140 ± 15 were β cells. To test if LM analysis of the same tissue samples could provide reasonable estimates of purity of the islet preparations, volume fraction islet tissue was measured on thin sections available from 27 of the clinical preparations by point counting morphometrics. Islet purity (islet volume fraction) of individual preparations determined by LM and EM analysis correlated linearly with excellent agreement (R2 = 0.95). However, islet purity by conventional dithizone staining was substantially higher with a 20-30% overestimation. Thus, both EM and LM provide accurate methods to determine the cell composition of human islets preparations and can help us understand many of the discrepancies of islet composition in the literature. PMID:20697378

  3. Intrinsic Islet Heterogeneity and Gap Junction Coupling Determine Spatiotemporal Ca2+ Wave Dynamics

    PubMed Central

    Benninger, Richard K.P.; Hutchens, Troy; Head, W. Steven; McCaughey, Michael J.; Zhang, Min; Le Marchand, Sylvain J.; Satin, Leslie S.; Piston, David W.

    2014-01-01

    Insulin is released from the islets of Langerhans in discrete pulses that are linked to synchronized oscillations of intracellular free calcium ([Ca2+]i). Associated with each synchronized oscillation is a propagating calcium wave mediated by Connexin36 (Cx36) gap junctions. A computational islet model predicted that waves emerge due to heterogeneity in β-cell function throughout the islet. To test this, we applied defined patterns of glucose stimulation across the islet using a microfluidic device and measured how these perturbations affect calcium wave propagation. We further investigated how gap junction coupling regulates spatiotemporal [Ca2+]i dynamics in the face of heterogeneous glucose stimulation. Calcium waves were found to originate in regions of the islet having elevated excitability, and this heterogeneity is an intrinsic property of islet β-cells. The extent of [Ca2+]i elevation across the islet in the presence of heterogeneity is gap-junction dependent, which reveals a glucose dependence of gap junction coupling. To better describe these observations, we had to modify the computational islet model to consider the electrochemical gradient between neighboring β-cells. These results reveal how the spatiotemporal [Ca2+]i dynamics of the islet depend on β-cell heterogeneity and cell-cell coupling, and are important for understanding the regulation of coordinated insulin release across the islet. PMID:25468351

  4. Imatinib prevents beta cell death in vitro but does not improve islet transplantation outcome

    PubMed Central

    Griffiths, Lisa A.; Persaud, Shanta J.; Jones, Peter M.; Howell, Simon L.; Welsh, Nils

    2016-01-01

    Introduction Improving islet transplantation outcome could not only bring benefits to individual patients but also widen the patient pool to which this life-changing treatment is available. Imatinib has previously been shown to protect beta cells from apoptosis in a variety of in vitro and in vivo models. The aim of this study was to investigate whether imatinib could be used to improve islet transplantation outcome. Methods Islets were isolated from C57Bl/6 mice and pre-cultured with imatinib prior to exposure to streptozotocin and cytokines in vitro. Cell viability and glucose-induced insulin secretion were measured. For transplantation experiments, islets were pre-cultured with imatinib for either 72 h or 24 h prior to transplantation into streptozotocin-diabetic C57Bl/6 mice. In one experimental series mice were also administered imatinib after islet transplantation. Results Imatinib partially protected islets from beta cell death in vitro. However, pre-culturing islets in imatinib or administering the drug to the mice in the days following islet transplantation did not improve blood glucose concentrations more than control-cultured islets. Conclusion Although imatinib protected against beta cell death from cytokines and streptozotocin in vitro, it did not significantly improve syngeneic islet transplantation outcome. PMID:26953716

  5. The Spleen Is an Ideal Site for Inducing Transplanted Islet Graft Expansion in Mice

    PubMed Central

    Takahashi, Hiroyuki; Kodama, Shohta

    2017-01-01

    Alternative islet transplantation sites have the potential to reduce the marginal number of islets required to ameliorate hyperglycemia in recipients with diabetes. Previously, we reported that T cell leukemia homeobox 1 (Tlx1)+ stem cells in the spleen effectively regenerated into insulin-producing cells in the pancreas of non-obese diabetic mice with end-stage disease. Thus, we investigated the spleen as a potential alternative islet transplantation site. Streptozotocin-induced diabetic C57BL/6 mice received syngeneic islets into the portal vein (PV), beneath the kidney capsule (KC), or into the spleen (SP). The marginal number of islets by PV, KC, or SP was 200, 100, and 50, respectively. Some plasma inflammatory cytokine levels in the SP group were significantly lower than those of the PV group after receiving a marginal number of islets, indicating reduced inflammation in the SP group. Insulin contents were increased 280 days after islet transplantation compared with those immediately following transplantation (p<0.05). Additionally, Tlx1-related genes, including Rrm2b and Pla2g2d, were up-regulated, which indicates that islet grafts expanded in the spleen. The spleen is an ideal candidate for an alternative islet transplantation site because of the resulting reduced inflammation and expansion of the islet graft. PMID:28135283

  6. Human BM stem cells initiate angiogenesis in human islets in vitro.

    PubMed

    Luo, J Z Q; Xiong, F; Al-Homsi, A S; Roy, T; Luo, L G

    2011-08-01

    BM stem cells may have regenerative effects on islet function through angiogenesis. Human islets (100islet equivalent/mL) were cultured alone (control) or co-cultured (experimental group) with whole human BM (1 × 10(6) cells/mL) for 210 days. A protein array measuring angiogenesis factors found upregulated (experimental vs control, day 210) proteins levels of VEGF-a (535 vs 2 pg/mL), PDGF (280.79 vs 0 pg/mL), KGF (939 vs 8 pg/mL), TIMP-1 (4592 vs 4332 pg/mL) and angiogenin (506 vs 97 pg/mL). Lower protein levels of angiopoietin-2 (5 vs 709 pg/mL) were observed. Depletion of pro-angiogenesis factors in co-culture decreased the effects of BM-induced islet vascularization. Depletion of VEGF-a, eKGF and PDGF significantly reduced islet vascularization but individual depletion of KGF and PDGF had less effects overall on vessel formation. BM-induced vascularization showed significant endothelial cell distribution. Islet vascularization was linked to islet growth. A decrease in islet size indicated poor vascularization. Insulin release was evident in the tissues generated from human islet-BM co-culture throughout the entire culture period. Significant increase in insulin (28.66-fold vs control) and glucagon (24.4-fold vs control) gene expression suggest BM can induce endocrine cell regeneration. In conclusion, BM promotes human islet tissue regeneration via regulation of angiogenesis factors.

  7. A novel fluorescence imaging approach for comparative measurements of pancreatic islet function in vitro.

    PubMed

    Corbin, Kathryn L; Hall, Thomas E; Haile, Ruth; Nunemaker, Craig S

    2011-01-01

    Pancreatic islet dysfunction is a key element in the development of type 2 diabetes. Determining possible early warning signs of dysfunction is thus important to determining the underlying causes of diabetes. We describe an improved fluorescent imaging approach to detect potential islet dysfunction. Using Cell Tracker Red (CTR, a mildly thiol-reactive fluorescent probe) to positively label particular islets, we measured intracellular free calcium with fura-2 AM in both CTR-labeled and unlabeled sets of pancreatic islets simultaneously in vitro. This approach enhances sensitivity by controlling for differences in background fluorescence, temperature, and perifusion dynamics. We confirmed that 200 nM CTR produced no spectral overlap with fura-2 and no significant physiological effects in selective tests of islet function. To demonstrate the utility of dual-labeling, we compared untreated islets with islets pretreated with low-dose pro-inflammatory cytokines (IL-6 + IL-1B) to induce mild dysfunction. We alternated CTR-labeling between control and test islets and identified consistent reductions in the amplitude and trajectory of glucose-stimulated calcium responses (GSCa) among cytokine-treated islets that were independent of labeling. Observations were verified using a MATLAB program specifically designed to identify key features in the GSCa. Our findings thus demonstrate the utility of CTR-labeling in identifying islet dysfunction and propose that this technique can be adapted for other cells and tissues.

  8. Leptin- or troglitazone-induced lipopenia protects islets from interleukin 1beta cytotoxicity.

    PubMed Central

    Shimabukuro, M; Koyama, K; Lee, Y; Unger, R H

    1997-01-01

    Interleukin 1beta (IL-1beta)-induced beta cell cytotoxicity has been implicated in the autoimmune cytotoxicity of insulin-dependent diabetes mellitus. These cytotoxic effects may be mediated by nitric oxide (NO). Since long-chain fatty acids (FFA), like IL-1beta, upregulate inducible nitric oxide synthase and enhance NO generation in islets, it seemed possible that islets might be protected from IL-1beta-induced damage by lowering their lipid content. We found that IL-1beta-induced NO production varied directly and islet cell viability inversely with islet triglyceride (TG) content. Fat-laden islets of obese rats were most vulnerable to IL-1beta, while moderately fat-depleted islets of food-restricted normal rats were less vulnerable than those of free-feeding normal rats. Severely lipopenic islets of rats made chronically hyperleptinemic by adenoviral leptin gene transfer resisted IL-1beta cytotoxicity even at 300 pg/ml, the maximal concentration. Troglitazone lowered islet TG in cultured islets from both normal rats and obese, leptin-resistant rats and reduced NO production and enhanced cell survival. We conclude that measures that lower islet TG content protect against IL-1beta-induced NO production and cytotoxicity. Leptin or troglitazone could provide in vivo protection against insulin-dependent diabetes mellitus. PMID:9312173

  9. A Combinatorial Protein Microarray for Probing Materials Interaction with Pancreatic Islet Cell Populations

    PubMed Central

    Delalat, Bahman; Rojas-Canales, Darling M.; Rasi Ghaemi, Soraya; Waibel, Michaela; Harding, Frances J.; Penko, Daniella; Drogemuller, Christopher J.; Loudovaris, Thomas; Coates, Patrick T. H.; Voelcker, Nicolas H.

    2016-01-01

    Pancreatic islet transplantation has become a recognized therapy for insulin-dependent diabetes mellitus. During isolation from pancreatic tissue, the islet microenvironment is disrupted. The extracellular matrix (ECM) within this space not only provides structural support, but also actively signals to regulate islet survival and function. In addition, the ECM is responsible for growth factor presentation and sequestration. By designing biomaterials that recapture elements of the native islet environment, losses in islet function and number can potentially be reduced. Cell microarrays are a high throughput screening tool able to recreate a multitude of cellular niches on a single chip. Here, we present a screening methodology for identifying components that might promote islet survival. Automated fluorescence microscopy is used to rapidly identify islet derived cell interaction with ECM proteins and immobilized growth factors printed on arrays. MIN6 mouse insulinoma cells, mouse islets and, finally, human islets are progressively screened. We demonstrate the capability of the platform to identify ECM and growth factor protein candidates that support islet viability and function and reveal synergies in cell response. PMID:27600088

  10. Graft revascularization is essential for non-invasive monitoring of transplanted islets with radiolabeled exendin

    PubMed Central

    Eter, Wael A.; Bos, Desirée; Frielink, Cathelijne; Boerman, Otto C.; Brom, Maarten; Gotthardt, Martin

    2015-01-01

    Islet transplantation is a novel promising strategy to cure type 1 diabetes. However, the long-term outcome is still poor, because both function and survival of the transplant decline over-time. Non-invasive imaging methods have the potential to enable monitoring of islet survival after transplantation and the effects of immunosuppressive drugs on transplantation outcome. 111In-labeled exendin-3 is a promising tracer to visualize native and transplanted islets by SPECT (Single Photon Emission Computed Tomography). In the present study, we hypothesized that islet microvasculature plays an important role determining the uptake of exendin-3 in islets when monitoring transplant survival. We observed 111In-exendin-3 accumulation in the transplant as early as three days after transplantation and an increase in the uptake up to three weeks post-transplantation. Islet-revascularization correlated with the increase in 111In-exendin-3 uptake, whereas fully re-established islet vasculature coincided with a stabilized uptake of the radiotracer in the transplant. Here, we demonstrate the importance of islet vasculature for in vivo delivery of radiotracers to transplanted islets and we demonstrate that optimal and stable uptake of exendin four weeks after transplantation opens the possibility for long-term monitoring of islet survival by SPECT imaging. PMID:26490110

  11. Graft revascularization is essential for non-invasive monitoring of transplanted islets with radiolabeled exendin.

    PubMed

    Eter, Wael A; Bos, Desirée; Frielink, Cathelijne; Boerman, Otto C; Brom, Maarten; Gotthardt, Martin

    2015-10-22

    Islet transplantation is a novel promising strategy to cure type 1 diabetes. However, the long-term outcome is still poor, because both function and survival of the transplant decline over-time. Non-invasive imaging methods have the potential to enable monitoring of islet survival after transplantation and the effects of immunosuppressive drugs on transplantation outcome. (111)In-labeled exendin-3 is a promising tracer to visualize native and transplanted islets by SPECT (Single Photon Emission Computed Tomography). In the present study, we hypothesized that islet microvasculature plays an important role determining the uptake of exendin-3 in islets when monitoring transplant survival. We observed (111)In-exendin-3 accumulation in the transplant as early as three days after transplantation and an increase in the uptake up to three weeks post-transplantation. Islet-revascularization correlated with the increase in (111)In-exendin-3 uptake, whereas fully re-established islet vasculature coincided with a stabilized uptake of the radiotracer in the transplant. Here, we demonstrate the importance of islet vasculature for in vivo delivery of radiotracers to transplanted islets and we demonstrate that optimal and stable uptake of exendin four weeks after transplantation opens the possibility for long-term monitoring of islet survival by SPECT imaging.

  12. Activated pancreatic stellate cells can impair pancreatic islet function in mice

    PubMed Central

    Zang, Guangxiang; Sandberg, Monica; Carlsson, Per-Ola; Welsh, Nils; Jansson, Leif

    2015-01-01

    Background Pancreatic or islet fibrosis is often associated with activated pancreatic stellate cells (PSCs). PSCs are considered not only to promote fibrosis, but also to be associated with glucose intolerance in some diseases. We therefore evaluated morphological and functional relationships between islets and PSCs in the normal mouse pancreas and transplanted islets. Methods Immunohistochemistry was used to map the presence of PSCs in the normal mouse pancreas and islets implanted under the renal capsule. We isolated and cultured mouse PSCs and characterized them morphologically by immunofluorescence staining. Furthermore, we measured their cytokine production and determined their effects on insulin release from simultaneously cultured islets. Results PSCs were scattered throughout the pancreas, with occasional cells within the islets, particularly in the islet capsule. In islet transplants they were found mainly in the graft periphery. Cultured PSCs became functionally activated and produced several cytokines. Throughout the culture period they linearly increased their production of interleukin-6 and mammalian keratinocyte-derived chemokine. PSC cytokine production was not affected by acute hyperglycemia. Syngeneic islets co-cultured with PSCs for 24–48 h increased their insulin release and lowered their insulin content. However, short-term insulin release in batch-type incubations was unaffected after 48 h of co-culture. Increased islet cell caspase-3 activation and a decreased islet cell replication were consistently observed after co-culture for 2 or 7 days. Conclusion Activated PSCs may contribute to impaired islet endocrine function seen in exocrine pancreatitis and in islet fibrosis associated with some cases of type 2 diabetes. PMID:25854824

  13. Paternal high-fat diet consumption induces common changes in the transcriptomes of retroperitoneal adipose and pancreatic islet tissues in female rat offspring.

    PubMed

    Ng, Sheau-Fang; Lin, Ruby C Y; Maloney, Christopher A; Youngson, Neil A; Owens, Julie A; Morris, Margaret J

    2014-04-01

    We previously showed that paternal high-fat diet (HFD) consumption programs β-cell dysfunction in female rat offspring, together with transcriptome alterations in islets. Here we investigated the retroperitoneal white adipose tissue (RpWAT) transcriptome using gene and pathway enrichment and pathway analysis to determine whether commonly affected network topologies exist between these two metabolically related tissues. In RpWAT, 5108 genes were differentially expressed due to a paternal HFD; the top 5 significantly enriched networks identified by pathway analysis in offspring of HFD fathers compared with those of fathers fed control diet were: mitochondrial and cellular response to stress, telomerase signaling, cell death and survival, cell cycle, cellular growth and proliferation, and cancer. A total of 187 adipose olfactory receptor genes were down-regulated. Interrogation against the islet transcriptome identified specific gene networks and pathways, including olfactory receptor genes that were similarly affected in both tissues (411 common genes, P<0.05). In particular, we highlight a common molecular network, cell cycle and cancer, with the same hub gene, Myc, suggesting early onset developmental changes that persist, shared responses to programmed systemic factors, or crosstalk between tissues. Thus, paternal HFD consumption triggers unique gene signatures, consistent with premature aging and chronic degenerative disorders, in both RpWAT and pancreatic islets of daughters.

  14. Soluble epoxide hydrolase deficiency alters pancreatic islet size and improves glucose homeostasis in a model of insulin resistance.

    PubMed

    Luria, Ayala; Bettaieb, Ahmed; Xi, Yannan; Shieh, Guang-Jong; Liu, Hsin-Chen; Inoue, Hiromi; Tsai, Hsing-Ju; Imig, John D; Haj, Fawaz G; Hammock, Bruce D

    2011-05-31

    Visceral obesity has been defined as an important element of the metabolic syndrome and contributes to the development of insulin resistance and cardiovascular disease. Increasing endogenous levels of epoxyeicosatrienoic acids (EETs) are known for their analgesic, antihypertensive, and antiinflammatory effects. The availability of EETs is limited primarily by the soluble epoxide hydrolase (sEH, EPHX2), which metabolizes EETs to their less active diols. In this study, we tested the hypothesis that EETs are involved in glucose regulation and in retarding the development of insulin resistance. To address the role of EETs in regulating glucose homeostasis and insulin signaling, we used mice with targeted gene deletion of sEH (Ephx2-null mice) and a subsequent study with a selective sEH inhibitor. When wild-type mice are fed a high fat diet, insulin resistance develops. However, knockout or inhibition of sEH activity resulted in a significant decrease in plasma glucose. These findings are characterized by enhancement of tyrosyl phosphorylation of the insulin receptor, insulin receptor substrate 1, and their downstream cascade. In addition, pancreatic islets were larger when sEH was disrupted. This effect was associated with an increase in vasculature. These observations were supported by pharmacological inhibition of sEH. These data suggest that an increase in EETs due to sEH-gene knockout leads to an increase in the size of islets and improved insulin signaling and sensitivity.

  15. Measurement and modeling of glucose-6-phosphatase in pancreatic islets.

    PubMed

    Sweet, I R; Najafi, H; Li, G; Grodberg, J; Matschinsky, F M

    1997-04-01

    In the beta-cells of the pancreas, glucose phosphorylation carried out by glucokinase is the rate-controlling step in glycolysis, and the kinetic characteristics of glucokinase govern to a high degree the dose-response relationship between glucose and insulin release. Because glucose-6-phosphatase (G-6-Pase) opposes the action of glucokinase, it may have a regulatory role in the release of insulin in response to glucose if the enzyme is present in the beta-cells. A number of researchers have reported finding high levels of G-6-Pase in islets, but quantitation of its activity remains controversial, mainly because of difficulties in solubilizing a particulate enzyme. Therefore a method developed to measure functional glucose phosphorylation activity in intact brain was applied (Chi, M. M.-Y., M. E. Pusateri, J. G. Carter, B. J. Norris, D. B. McDougal, Jr., and O. H. Lowry. Anal. Biochem. 161: 508-513, 1987), and the rates of accumulation and disappearance of 2-deoxyglucose 6-phosphate (DG-6-P) in freshly harvested islets were determined as a measure of glucose cycling. Islets were incubated in the presence of 30 mM 2-deoxyglucose (DG) for 60 min, and subsequently the incubation medium was replaced with medium containing no DG, but instead high levels of mannoheptulose as a blocker of phosphorylation. The content of DG-6-P in the islets was measured at strategic times during the protocol. As predicted by a mathematical model, DG-6-P accumulated in the presence of DG and decayed after its washout. Both of these results are consistent with islets containing dephosphorylation activity for this substrate. The kinetic curves were fit using a mathematical model, and the maximal G-6-Pase activity was estimated to be 0.13 +/- 0.005 micromol x g(-1) x min(-1). However, when the physiological effect of this amount of G-6-Pase activity was assessed by use of a model of glycolysis, it was found that the impact on glucose cycling and usage was insignificant. It was concluded that

  16. Validation of Islet Transport From a Geographically Distant Isolation Center Enabling Equitable Access and National Health Service Funding of a Clinical Islet Transplant Program for England

    PubMed Central

    Aldibbiat, Ali; Huang, Guo Cai; Zhao, Min; Holliman, Graham N.; Ferguson, Linda; Hughes, Stephen; Brigham, Ken; Wardle, Julie; Williams, Rob; Dickinson, Anne; White, Steven A.; Johnson, Paul R. V.; Manas, Derek; Amiel, Stephanie A.; Shaw, James A. M.

    2012-01-01

    Islet transplantation has become established as a successful treatment for type 1 diabetes complicated by recurrent severe hypoglycemia. In the UK access has been limited to a few centrally located units. Our goal was to validate a quality-assured system for safe/effective transport of human islets in the UK and to successfully undertake the first transplants with transported islets. Pancreases were retrieved from deceased donors in the north of England and transported to King’s College London using two-layer method (TLM) or University of Wisconsin solution alone. Islets were isolated and transported back to Newcastle in standard blood transfusion or gas-permeable bags with detailed evaluation pre- and posttransport. In the preclinical phase, islets were isolated from 10 pancreases with mean yield of 258,000 islet equivalents. No significant differences were seen between TLM and University of Wisconsin solution organ preservation. A significant loss of integrity was demonstrated in islets shipped in gas-permeable bags, whereas sterility, number, purity, and viability were maintained in blood transfusion bags. Maintenance of secretory granules and glucose-stimulated insulin secretion was confirmed following transport. A Standard Operating Procedure enabling final pretransplant quality control from a simple side-arm sample was validated. Moreover, levels of insulin and cytokines in transport medium were low, enabling transplant without centrifugation/resuspension at the recipient site. Six clinical transplants of transported islets were undertaken in five recipients with 100% primary graft function and resolution of severe hypoglycemia. Safe and clinically effective islet transport has been established facilitating sustainable NHS funding of a clinical islet transplant program for the UK. PMID:27004135

  17. Pancreatic islet cells express a family of inwardly rectifying K+ channel subunits which interact to form G-protein-activated channels.

    PubMed

    Ferrer, J; Nichols, C G; Makhina, E N; Salkoff, L; Bernstein, J; Gerhard, D; Wasson, J; Ramanadham, S; Permutt, A

    1995-11-03

    Insulin secretion is associated with changes in pancreatic beta-cell K+ permeability. A degenerate polymerase chain reaction strategy based on the conserved features of known inwardly rectifying K+ (KIR) channel genes was used to identify members of this family expressed in human pancreatic islets and insulinoma. Three related human KIR transcript sequences were found: CIR (also known as cardiac KATP-1), GIRK1, and GIRK2 (KATP-2). The pancreatic islet CIR and GIRK2 full-length cDNAs were cloned, and their genes were localized to human chromosomes 11q23-ter and 21, respectively. Northern blot analysis detected CIR mRNA at similar levels in human islets and exocrine pancreas, while the abundance of GIRK2 mRNA in the two tissues was insufficient for detection by this method. Using competitive reverse-transcription polymerase chain reaction, CIR was found to be present at higher levels than GIRK2 mRNA in native purified beta-cells. Xenopus oocytes injected with M2 muscarinic receptor (M2) plus either GIRK2 or CIR cRNA expressed only very small carbachol-induced currents, while co-injection of CIR plus GIRK2 along with M2 resulted in expression of carbachol-activated strong inwardly rectifying currents. Activators of KATP channels failed to elicit currents in the presence or absence of co-expressed sulfonylurea receptor. These results show that two components of islet cell KIR channels, CIR and GIRK2, may interact to form heteromeric G-protein-activated inwardly rectifying K+ channels that do not possess the typical properties of KATP channels.

  18. Ischaemia is linked to inflammation and induction of angiogenesis in pancreatic islets

    PubMed Central

    Linn, T; Schmitz, J; Hauck-Schmalenberger, I; Lai, Y; Bretzel, R G; Brandhorst, H; Brandhorst, D

    2006-01-01

    β-cell replacement is the only way to restore euglycaemia in patients with type-1 diabetes. Pancreatic tissue, processed for subsequent clinical islet transplantation, is exposed to ischaemia causing injury and death in a large number of islets before and after transplantation. In this review we summarize what is known on the sources of environmental stress for pancreatic islets, such as insufficient oxygen supply during pancreas procurement and in culture prior to intraportal transplantation, nutritional and oxygen deprivation during the isolation process, and the consequences of hyperglycaemia. An increasingly recognized role in the modulation of β-cell function and these environmental stress factors plays the vascular network of the pancreatic islets. Islet revascularization by angiogenesis is relevant for the survival of the graft subsequent to transplantation. Potential strategies offered by therapeutic induction of revascularization to ameliorate the detrimental impact of these factors on the quality of islet transplants are discussed. PMID:16634789

  19. Minireview: Emerging Concepts in Islet Macrophage Biology in Type 2 Diabetes

    PubMed Central

    2015-01-01

    Chronic systemic inflammation is a hallmark feature of obesity and type 2 diabetes. Both resident and recruited islet macrophages contribute to the proinflammatory milieu of the diabetic islet. However, macrophages also appear to be critical for β-cell formation during development and support β-cell replication in experimental models of pancreas regeneration. In light of these findings, perhaps macrophages in the islet need to be viewed more as a fulcrum where deleterious inflammatory activation is balanced with beneficial tissue repair processes. Undoubtedly, defining the factors that contribute to the ontogeny, heterogeneity, and functionality of macrophages in normal, diseased, and regenerating islets will be necessary to determine whether that fulcrum can be moved to preserve functional β-cell mass in persons with diabetes. The intent of this review is to introduce the reader to emerging concepts of islet macrophage biology that may challenge the perception that macrophage accumulation in islets is merely a pathological feature of type 2 diabetes. PMID:26001058

  20. Insulin biosynthesis: studies of Islet polyribosomes (nascent peptides-sucrose gradient analysis-gel filtration).

    PubMed

    Permutt, M A; Kipnis, D M

    1972-02-01

    A method is described for separation of polyribosomes from as few as 25 isolated Islets of Langerhans, representing about 250 mug of pancreatic tissue. Islets are labeled with [(3)H]leucine and polysomes are isolated with liver polyribosomes, which serve as carrier and inhibitor of ribonuclease activity. Islets incubated at 37 degrees C for 45 min in 15.5 mM glucose, then pulsed with [(3)H]leucine, incorporated about 2-3 times more label into nascent peptides on islet polysomes than islets incubated in 2.8 mM glucose. Sucrose gradient analysis of the labeled polysomes indicated that raising the glucose concentration preferentially stimulated synthesis of peptides on trisomes and larger polyribosomes. Islets incubated with [(3)H]leucine for 15 min incorporated two-thirds of the label into proteins on membrane-bound polysomes. At least 85% of the proinsulin synthesis during this time occurs on membrane-bound polysomes.

  1. Functional imaging of glucose-evoked rat islet activities using transient intrinsic optical signals

    NASA Astrophysics Data System (ADS)

    Yao, Xin-Cheng; Cui, Wan-Xing; Li, Yi-Chao; Zhang, Wei; Lu, Rong-Wen; Thompson, Anthony; Amthor, Franklin; Wang, Xu-Jing

    2012-05-01

    We demonstrate intrinsic optical signal (IOS) imaging of intact rat islet, which consists of many endocrine cells working together. A near-infrared digital microscope was employed for optical monitoring of islet activities evoked by glucose stimulation. Dynamic NIR images revealed transient IOS responses in the islet activated by low-dose (2.75 mM) and high-dose (5.5 mM) glucose stimuli. Comparative experiments and quantitative analysis indicated that both glucose metabolism and calcium/insulin dynamics might contribute to the observed IOS responses. Further investigation of the IOS imaging technology may provide a high resolution method for ex vivo functional examination of the islet, which is important for advanced study of diabetes associated islet dysfunctions and for improved quality control of donor islets for transplantation.

  2. Standardized transportation of human islets: an islet cell resource center study of more than 2,000 shipments.

    PubMed

    Kaddis, John S; Hanson, Matthew S; Cravens, James; Qian, Dajun; Olack, Barbara; Antler, Martha; Papas, Klearchos K; Iglesias, Itzia; Barbaro, Barbara; Fernandez, Luis; Powers, Alvin C; Niland, Joyce C

    2013-01-01

    Preservation of cell quality during shipment of human pancreatic islets for use in laboratory research is a crucial, but neglected, topic. Mammalian cells, including islets, have been shown to be adversely affected by temperature changes in vitro and in vivo, yet protocols that control for thermal fluctuations during cell transport are lacking. To evaluate an optimal method of shipping human islets, an initial assessment of transportation conditions was conducted using standardized materials and operating procedures in 48 shipments sent to a central location by eight pancreas-processing laboratories using a single commercial airline transporter. Optimization of preliminary conditions was conducted, and human islet quality was then evaluated in 2,338 shipments pre- and postimplementation of a finalized transportation container and standard operating procedures. The initial assessment revealed that the outside temperature ranged from a mean of -4.6 ± 10.3°C to 20.9 ± 4.8°C. Within-container temperature drops to or below 15°C occurred in 16 shipments (36%), while the temperature was found to be stabilized between 15°C and 29°C in 29 shipments (64%). Implementation of an optimized transportation container and operating procedure reduced the number of within-container temperature drops (≤ 15°C) to 13% (n = 37 of 289 winter shipments), improved the number desirably maintained between 15°C and 29°C to 86% (n = 250), but also increased the number reaching or exceeding 29°C to 1% (n = 2; overall p < 0.0001). Additionally, postreceipt quality ratings of excellent to good improved pre- versus postimplantation of the standardized protocol, adjusting for preshipment purity/viability levels (p < 0.0001). Our results show that extreme temperature fluctuations during transport of human islets, occurring when using a commercial airline transporter for long distance shipping, can be controlled using standardized containers, materials, and operating procedures. This cost

  3. The determination of membrane permeability coefficients of canine pancreatic islet cells and their application to islet cryopreservation.

    PubMed

    Liu, J; Zieger, M A; Lakey, J R; Woods, E J; Critser, J K

    1997-08-01

    Sufficient numbers of pancreatic islets for successful allotransplantation can be achieved by storing and then pooling islets from several donors. Optimal MHC matching and infectious disease screening also require long-term storage of islets, and cryopreservation is currently the only practical approach. Cryopreservation protocols may be optimized by modeling the changes in cell volume and the associated damage incurred during cryoprotectant addition and dilution and during cooling and warming. The objective of the present work was to determine the following biophysical parameters of canine islet cells; the osmotically inactive cell volume (Vb), hydraulic conductivity (Lp), cryoprotectant permeability coefficient (Ps), and the reflection coefficient sigma. A determination of these parameters allows the simulation of cell responses using computer models. Islets were isolated by collagenase digestion and Euro-Ficoll purification. After 24 h culture, islets were dissociated into single cells using trypsin and 2 mM EGTA. The kinetic change in cell volume as a function of time after exposure to 2 M dimethyl sulfoxide (Me2SO) was measured using an electronic particle counter at 22, 5, and -3 degrees C. At -11 degrees C, cells were preloaded with 1 M Me2SO and exposed to 4 M Me2SO to prevent the formation of ice in the working solution. Kedem-Katchalsky theory was used to describe the cell volume change kinetics, and a three-parameter curve fitting was performed using the Marquardt-Levenberg method to determine Lp, Ps, and sigma values. The Lp was determined to be 0.19 +/- 0.05, 0.037 +/- 0.005, 0.020 +/- 0.003, and 0.013 +/- 0.005 micron.min-1.atm-1 (mean +/- SD) at 22, 5, -3, and -11 degrees C, respectively. The Ps values were 1.05 +/- 0.50, 0.15 +/- 0.04, 0.096 +/- 0.028, and 0.067 +/- 0.029 x 10(-3) cm.min-1 at 22, 5, -3, and -11 degrees C, respectively. The sigma values were 0.81 +/- 0.16, 0.91 +/- 0.09, 0.80 +/- 0.21, and 0.98 +/- 0.04 at 22, 5, -3, and -11 degrees

  4. Autologous and Allogenous Antibodies in Lung and Islet Cell Transplantation

    PubMed Central

    Nayak, Deepak Kumar; Saravanan, Prathab Balaji; Bansal, Sandhya; Naziruddin, Bashoo; Mohanakumar, Thalachallour

    2016-01-01

    The field of organ transplantation has undoubtedly made great strides in recent years. Despite the advances in donor–recipient histocompatibility testing, improvement in transplantation procedures, and development of aggressive immunosuppressive regimens, graft-directed immune responses still pose a major problem to the long-term success of organ transplantation. Elicitation of immune responses detected as antibodies to mismatched donor antigens (alloantibodies) and tissue-restricted self-antigens (autoantibodies) are two major risk factors for the development of graft rejection that ultimately lead to graft failure. In this review, we describe current understanding on genesis and pathogenesis of antibodies in two important clinical scenarios: lung transplantation and transplantation of islet of Langerhans. It is evident that when compared to any other clinical solid organ or cellular transplant, lung and islet transplants are more susceptible to rejection by combination of allo- and autoimmune responses. PMID:28066448

  5. Insulin secretion from beta cells within intact islets: location matters.

    PubMed

    Hoang Do, Oanh; Thorn, Peter

    2015-04-01

    The control of hormone secretion is central to body homeostasis, and its dysfunction is important in many diseases. The key cellular steps that lead to hormone secretion have been identified, and the stimulus-secretion pathway is understood in outline for many endocrine cells. In the case of insulin secretion from pancreatic beta cells, this pathway involves the uptake of glucose, cell depolarization, calcium entry, and the triggering of the fusion of insulin-containing granules with the cell membrane. The wealth of information on the control of insulin secretion has largely been obtained from isolated single-cell studies. However, physiologically, beta cells exist within the islets of Langerhans, with structural and functional specializations that are not preserved in single-cell cultures. This review focuses on recent work that is revealing distinct aspects of insulin secretion from beta cells within the islet.

  6. Tissue dissociation enzymes for isolating human islets for transplantation: factors to consider in setting enzyme acceptance criteria.

    PubMed

    McCarthy, Robert C; Breite, Andrew G; Green, Michael L; Dwulet, Francis E

    2011-01-27

    Tissue dissociation enzymes are critical reagents that affect the yield and quality of human pancreatic islets required for islet transplantation. The United States Food and Drug Administration's oversight of this procedure recommends laboratories to set acceptance criteria for enzymes used in the manufacture of islet products for transplantation. Currently, many laboratories base this selection on personal experience because biochemical analysis is not predictive of success of the islet isolation procedure. This review identifies the challenges of correlating results from enzyme biochemical analysis to their effectiveness in human islet isolation and suggests a path forward to address these challenges to improve control of the islet manufacturing process.

  7. Collagen IV-modified scaffolds improve islet survival and function and reduce time to euglycemia.

    PubMed

    Yap, Woon Teck; Salvay, David M; Silliman, Michael A; Zhang, Xiaomin; Bannon, Zachary G; Kaufman, Dixon B; Lowe, William L; Shea, Lonnie D

    2013-11-01

    Islet transplantation on extracellular matrix (ECM) protein-modified biodegradable microporous poly(lactide-co-glycolide) scaffolds is a potential curative treatment for type 1 diabetes mellitus (T1DM). Collagen IV-modified scaffolds, relative to control scaffolds, significantly decreased the time required to restore euglycemia from 17 to 3 days. We investigated the processes by which collagen IV-modified scaffolds enhanced islet function and mediated early restoration of euglycemia post-transplantation. We characterized the effect of collagen IV-modified scaffolds on islet survival, metabolism, and insulin secretion in vitro and early- and intermediate-term islet mass and vascular density post-transplantation and correlated these with early restoration of euglycemia in a syngeneic mouse model. Control scaffolds maintained native islet morphologies and architectures as well as collagen IV-modified scaffolds in vivo. The islet size and vascular density increased, while β-cell proliferation decreased from day 16 to 113 post-transplantation. Collagen IV-modified scaffolds promoted islet cell viability and decreased early-stage apoptosis in islet cells in vitro-phenomena that coincided with enhanced islet metabolic function and glucose-stimulated insulin secretion. These findings suggest that collagen IV-modified scaffolds promote the early restoration of euglycemia post-transplantation by enhancing islet metabolism and glucose-stimulated insulin secretion. These studies of ECM proteins, in particular collagen IV, and islet function provide key insights for the engineering of a microenvironment that would serve as a platform for enhancing islet transplantation as a viable clinical therapy for T1DM.

  8. Efficient Gene Transduction of Dispersed Islet Cells in Culture Using Fiber-Modified Adenoviral Vectors

    PubMed Central

    Hanayama, Hiroyuki; Ohashi, Kazuo; Utoh, Rie; Shimizu, Hirofumi; Ise, Kazuya; Sakurai, Fuminori; Mizuguchi, Hiroyuki; Tsuchiya, Hiroyuki; Okano, Teruo; Gotoh, Mitsukazu

    2015-01-01

    To establish novel islet-based therapies, our group has recently developed technologies for creating functional neo-islet tissues in the subcutaneous space by transplanting monolithic sheets of dispersed islet cells (islet cell sheets). Improving cellular function and viability are the next important challenges for enhancing the therapeutic effects. This article describes the adenoviral vector-mediated gene transduction of dispersed islet cells under culture conditions. Purified pancreatic islets were obtained from Lewis rats and dissociated into single islet cells. Cells were plated onto laminin-5-coated temperature-responsive polymer poly(N-isopropylacrylamide)-immobilized plastic dishes. At 0 h, islet cells were infected for 1 h with either conventional type 5 adenoviral vector (Ad-CA-GFP) or fiber-modified adenoviral vector (AdK7-CA-GFP) harboring a polylysine (K7) peptide in the C terminus of the fiber knob. We investigated gene transduction efficiency at 48 h after infection and found that AdK7-CA-GFP yielded higher transduction efficiencies than Ad-CA-GFP at a multiplicity of infection (MOI) of 5 and 10. For AdK7-CA-GFP at MOI = 10, 84.4 ± 1.5% of islet cells were found to be genetically transduced without marked vector infection-related cellular damage as determined by viable cell number and lactate dehydrogenase (LDH) release assay. After AdK7-CA-GFP infection at MOI = 10, cells remained attached and expanded to nearly full confluency, showing that this adenoviral infection protocol is a feasible approach for creating islet cell sheets. We have shown that dispersed and cultured islet cells can be genetically modified efficiently using fiber-modified adenoviral vectors. Therefore, this gene therapy technique could be used for cellular modification or biological assessment of dispersed islet cells. PMID:26858906

  9. Lack of evidence for recipient precursor cells replenishing β-cells in transplanted islets.

    PubMed

    Hamamoto, Yoshiyuki; Akashi, Tomoyuki; Inada, Akari; Bonner-Weir, Susan; Weir, Gordon C

    2010-01-01

    Bone marrow and tissue precursor cells have been postulated to replenish grafts of transplanted islets. Several investigators have reported that bone marrow cells can promote the regeneration of injured islets. In this study, we investigated the potential of recipient-derived precursor cells to form new pancreatic endocrine cells in islet grafts transplanted under the kidney capsule. Mouse insulin promoter (MIP)-green fluorescence protein (GFP) mice, which express GFP only in β-cells, or β-actin GFP mice, which express GFP ubiquitously, were used to determine if the recipient-derived cells differentiate into β-cells or other types of endocrine cells. We transplanted MIP-GFP islets into wild-type mice, wild-type islets into MIP-GFP mice, β-actin GFP islets into wild-type mice, and wild-type islets into β-actin GFP mice. β-Actin GFP bone marrow cells were then injected into wild-type mice to evaluate the potential role of bone marrow stem cells to provide new islet cells to the graft. No β-cells with green fluorescence were seen in the graft when wild-type islets were transplanted into MIP-GFP mice. When wild-type islets were transplanted into β-actin GFP mice, no β-cells with GFP staining could be identified in the grafts. Similarly, no endocrine cells with GFP staining could be identified in the grafts after injection of β-actin GFP bone marrow cells into wild-type islet-transplanted wild-type mice. This study provides further support for the concept that recipient precursor cells do not produce new β-cells in grafts of transplanted islets.

  10. Beneficial Effect of Insulin Treatment on Islet Transplantation Outcomes in Akita Mice

    PubMed Central

    Kikawa, Kazuhide; Sakano, Daisuke; Shiraki, Nobuaki; Tsuyama, Tomonori; Kume, Kazuhiko; Endo, Fumio; Kume, Shoen

    2014-01-01

    Islet transplantation is a promising potential therapy for patients with type 1 diabetes. The outcome of islet transplantation depends on the transplantation of a sufficient amount of β-cell mass. However, the initial loss of islets after transplantation is problematic. We hypothesized the hyperglycemic status of the recipient may negatively affect graft survival. Therefore, in the present study, we evaluated the effect of insulin treatment on islet transplantation involving a suboptimal amount of islets in Akita mice, which is a diabetes model mouse with an Insulin 2 gene missense mutation. Fifty islets were transplanted under the left kidney capsule of the recipient mouse with or without insulin treatment. For insulin treatment, sustained-release insulin implants were implanted subcutaneously into recipient mice 2 weeks before transplantation and maintained for 4 weeks. Islet transplantation without insulin treatment did not reverse hyperglycemia. In contrast, the group that received transplants in combination with insulin treatment exhibited improved fasting blood glucose levels until 18 weeks after transplantation, even after insulin treatment was discontinued. The group that underwent islet transplantation in combination with insulin treatment had better glucose tolerance than the group that did not undergo insulin treatment. Insulin treatment improved graft survival from the acute phase (i.e., 1 day after transplantation) to the chronic phase (i.e., 18 weeks after transplantation). Islet apoptosis increased with increasing glucose concentration in the medium or blood in both the in vitro culture and in vivo transplantation experiments. Expression profile analysis of grafts indicated that genes related to immune response, chemotaxis, and inflammatory response were specifically upregulated when islets were transplanted into mice with hyperglycemia compared to those with normoglycemia. Thus, the results demonstrate that insulin treatment protects islets from

  11. Decomplementation with cobra venom factor prolongs survival of xenografted islets in a rat to mouse model

    PubMed Central

    OBERHOLZER, J; YU, D; TRIPONEZ, F; CRETIN, N; ANDEREGGEN, E; MENTHA, G; WHITE, D; BUEHLER, L; MOREL, P; LOU, J

    1999-01-01

    Although the involvement of complement in hyperacute rejection of xenotransplants is well recognized, its role in rejection of devascularized xenografts, such as pancreatic islets, is not completely understood. In this study, we investigated whether complement participates in the immunopathology of xeno-islet transplantation in a concordant rat to mouse model. Rat pancreatic islets were implanted under the kidney capsule of normal and cobra venom factor (CVF)-decomplementized diabetic C57BL/6 mice. Graft survival was monitored by blood glucose levels. Deposition of IgM and C3 on grafted islets in vivo or on isolated islets in vitro (after incubation with normal and decomplementized mouse serum), as well as CD4- and CD8-positive leucocyte infiltration of grafts, was checked by immunohistochemistry. In addition, complement-mediated cytotoxicity on rat islet cells was evaluated by a 3-(4,5-dimethythiazolyl)-2.5-diphenyl-2H-tetrazolium-bromide (MTT) assay. A significant C3 deposition was found on grafted islets from the first day after transplantation in vivo, as well as on isolated islets after incubation with mouse serum in vitro. By MTT assay, complement-mediated cytotoxicity for islet cells was found. Decomplementation by CVF decreased C3 deposition on either isolated or grafted islets, delayed CD4- and CD8-positive leucocyte infiltration, led to significant inhibition of complement-mediated cytotoxicity for islet cells, and prolonged graft survival (mean survival time 21·3 versus 8·5 days; P <0·01). Our results indicate that decomplementation can prolong the survival time of devascularized xenografts across concordant species. The deposition of complement on transplanted islets may contribute to xenograft rejection by direct cytotoxicity and by promoting leucocyte infiltration. PMID:10447729

  12. In Vivo Magnetic Resonance Imaging of Small Interfering RNA Nanodelivery to Pancreatic Islets.

    PubMed

    Wang, Ping; Moore, Anna

    2016-01-01

    Pancreatic islet transplantation is a promising therapeutic approach for type 1 diabetes.However, recent advances in islet transplantation are limited by significant graft loss after transplantation. Multiple immunological and nonimmunological factors contribute to this loss. Novel therapies that could target the core reasons for the islet graft loss are desperately needed. Small interfering RNA can be used to inhibit the expression of virtually any gene with single-nucleotide specificity including genes responsible for islet damage. Applying adequate delivery of siRNA molecules to pancreatic islets prior to transplantation holds a great potential for improving the survival of islet grafts. Noninvasive imaging provides means for monitoring the survival of transplanted islets in real time. Here, we summarize the approach that has been developed to deliver siRNA to pancreatic islets in conjunction with tracking of the graft outcome by in vivo magnetic resonance imaging (MRI). We synthesize a nano-sized theranostic agent consisting of magnetic nanoparticles (MN), a reporter for MRI, labeled with Cy5.5 dye for near-infrared fluorescence (NIRF) imaging, and conjugated to siRNA molecule targeting genes that are harmful to islet grafts. Pre-labeling of islets by MN-Cy5.5-siRNA allowed us to monitor the survival of transplanted islet grafts by MRI and NIRF imaging and resulted in efficient silencing of the target genes in vivo. This novel approach combines a therapeutic effect provided by RNA interference technology with in vivo MR imaging and is expected to significantly improve the outcome of islet transplantation in patients with type 1 diabetes.

  13. Diazoxide, a K(ATP) channel opener, prevents ischemia-reperfusion injury in rodent pancreatic islets.

    PubMed

    Wang, Yong; Wang, Shusen; Harvat, Tricia; Kinzer, Katie; Zhang, Lisa; Feng, Feng; Qi, Meirigeng; Oberholzer, Jose

    2015-01-01

    Diazoxide (DZ) is a pharmacological opener of ATP-sensitive K(+) channels that has been used for mimicking ischemic preconditioning and shows protection against ischemic damage. Here we investigated whether diazoxide supplementation to University of Wisconsin (UW) solution has cellular protection during islet isolation and improves in vivo islet transplant outcomes in a rodent ischemia model. C57/B6 mice pancreata were flushed with UW or UW + DZ solution and cold preserved for 6 or 10 h prior to islet isolation. Islet yield, in vitro and in vivo function, mitochondrial morphology, and apoptosis were evaluated. Significantly higher islet yields were observed in the UW + DZ group than in the UW group (237.5 ± 25.6 vs. 108.7 ± 49.3, p < 0.01). The islets from the UW + DZ group displayed a significantly higher glucose-induced insulin secretion (0.97 ng/ml ± 0.15 vs. 0.758 ng/ml ± 0.21, p = 0.009) and insulin content (60.96 ng/islet ± 13.94 vs. 42.09 ng/islet ± 8.15, p = 0.002). The DZ-treated islets had well-preserved mitochondrial morphology with superior responses of mitochondrial potentials, and calcium influx responded to glucose. A higher number of living cells and less late apoptotic cells were observed in the UW + DZ group (p < 0.05). Additionally, the islets from the UW + DZ group had a significantly higher cure rate and improved glucose tolerance. This study is the first to report mitoprotective effects of DZ for pancreas preservation and islet isolation. In the future, it will be necessary to further understand the underlying mechanism for the mitoprotection and to test this promising approach for pancreas preservation and the islet isolation process in nonhuman primates and ultimately humans.

  14. Can pancreatic duct-derived progenitors be a source of islet regeneration?

    SciTech Connect

    Xia, Bing; Zhan, Xiao-Rong; Yi, Ran; Yang, Baofeng

    2009-06-12

    The regenerative process of the pancreas is of interest because the main pathogenesis of diabetes mellitus is an inadequate number of insulin-producing {beta}-cells. The functional mass of {beta}-cells is decreased in type 1 diabetes, so replacing missing {beta}-cells or triggering their regeneration may allow for improved type 1 diabetes treatment. Therefore, expansion of the {beta}-cell mass from endogenous sources, either in vivo or in vitro, represents an area of increasing interest. The mechanism of islet regeneration remains poorly understood, but the identification of islet progenitor sources is critical for understanding {beta}-cell regeneration. One potential source is the islet proper, via the dedifferentiation, proliferation, and redifferentiation of facultative progenitors residing within the islet. Neogenesis, or that the new pancreatic islets can derive from progenitor cells present within the ducts has been reported, but the existence and identity of the progenitor cells have been debated. In this review, we focus on pancreatic ductal cells, which are islet progenitors capable of differentiating into islet {beta}-cells. Islet neogenesis, seen as budding of hormone-positive cells from the ductal epithelium, is considered to be one mechanism for normal islet growth after birth and in regeneration, and has suggested the presence of pancreatic stem cells. Numerous results support the neogenesis hypothesis, the evidence for the hypothesis in the adult comes primarily from morphological studies that have in common the production of damage to all or part of the pancreas, with consequent inflammation and repair. Although numerous studies support a ductal origin for new islets after birth, lineage-tracing experiments are considered the 'gold standard' of proof. Lineage-tracing experiments show that pancreatic duct cells act as progenitors, giving rise to new islets after birth and after injury. The identification of differentiated pancreatic ductal cells as

  15. Assessment of islet quality following international shipping of more than 10,000 km.

    PubMed

    Ikemoto, Tetsuya; Matsumoto, Shinichi; Itoh, Takeshi; Noguchi, Hirofumi; Tamura, Yoshiko; Jackson, Andrew M; Shimoda, Masayuki; Naziruddin, Bashoo; Onaca, Nicholas; Yasunami, Yohichi; Levy, Marlon F

    2010-01-01

    Islet transplantation is an attractive therapy for type 1 diabetes, although some issues remain. One of them is the severe donor shortage in some countries. In this study, we investigated the possibility of international islet shipping beyond 10,000 km to supply islets to countries with donor shortages. Human islets were isolated from six cadaver donors and cultured until shipment. Islets were packed in either gas-permeable bags or in non-gas-permeable bags and shipped from Baylor Research Institute (Dallas, TX, USA) to Fukuoka University (Fukuoka, Japan). Pre- and postshipment islet number, purity, viability, and stimulation index (by glucose stimulation test) were assessed. Shipped 1,500 IE islets were transplanted into streptozotocin-induced diabetic nude mice for in vivo assay. The distance of our shipment was 11,148.4 km, and the mean duration of the shipments was 48.2 ± 8.2 h. The islet number recovery rate (postshipment/preshipment) was significantly higher in gas-permeable bags (56.4 ± 10.1% vs. 20.5 ± 20.6%, p < 0.01). Islet purity was significantly reduced during shipment in non-gas-permeable bags (from 47.7 ± 18.6% to 40.2 ± 28.2 in gas-permeable bags vs. from 50.4 ± 6.4% to 25.9 ± 15.6% in non-gas-permeable bags, p < 0.05). Islet viability and stimulation index did not change significantly between pre- and postshipping, in either gas-permeable bags or in non-gas-permeable bags. One of three diabetic nude mice (33.3%) converted to normoglycemia. It is feasible to ship human islet cells internationally in gas-permeable bags. This strategy would promote basic and preclinical research for countries with donor shortages, even though the research centers are remote (over 10,000 km from the islet isolation center).

  16. Virus-induced alterations in insulin release in hamster islets of Langerhans.

    PubMed

    Rayfield, E J; Seto, Y; Walsh, S; McEvoy, R C

    1981-11-01

    After the inoculation of Golden Syrian hamsters with the TC-83 vaccine strain of Venezuelan encephalitis (VE) virus, a sustained diminution in glucose-stimulated insulin release and glucose intolerance of shorter duration develops. To understand better the mechanism of this defect in insulin release, we examined insulin secretion in response to several test agents in isolated perifused islets from control and 24-d post-VE virus-infected hamsters. 50 islets were used in all perifusion experiments, and data were expressed as total insulin released as well as peak response for each test agent during a 30-min perifusion period from control and VE-infected islets. After perifusion with 20 mM glucose, a 45% diminution of insulin release was noted in VE-infected islets in comparison with control islets, which in turn was similar to in vivo findings. However, following 1-mM tolbutamide stimulation, insulin release was similar in control and VE-infected islets. In separate studies, 1 mM tolbutamide, 10 mM theophilline, 1 mM dibutyryl cyclic (c)AMP, and 1 mM 8-bromo-cAMP resulted in statistically similar insulin-release curves in control and VE-infected islets. Additional experiments assessing [5-3H]glucose use in control and infected islets after 20 min of perifusion with 20 mM glucose revealed virtually identical values (239 +/- 30-control; and 222 +/- 27-VE-infected islets). Morphological and morphometric evaluation of VE-infected islets (21 d following virus inoculation) showed no changes in islet volume density, beta cell density, and beta cell granulation. Thus, VE virus induces a defect in glucose-stimulated insulin release from hamster beta cells that can be corrected by cAMP analogues and does not alter islet glucose use.

  17. Diazoxide, a KATP Channel Opener, Prevents Ischemia-Reperfusion Injury in Rodent Pancreatic Islets

    PubMed Central

    Wang, Yong; Wang, Shusen; Harvat, Tricia; Kinzer, Katie; Zhang, Lisa; Feng, Feng; Qi, Meirigeng; Oberholzer, Jose

    2014-01-01

    Objective Diazoxide (DZ) is a pharmacological opener of ATP-sensitive K+ channels and has been used for mimicking ischemic preconditioning and shows protection against ischemic damage. Here, we investigated whether Diazoxide supplementation to University of Wisconsin (UW) solution has cellular protection during islet isolation and improves in vivo islet transplant outcomes in rodent ischemia model. Research Design and Methods C57/B6 mice pancreata were flushed with UW or UW+DZ solution and cold preserved for 6 or 10 hrs prior to islet isolation. Islet yield, in vitro and in vivo function, mitochondrial morphology, and apoptosis were evaluated. Results Significantly higher islet yields were observed in the UW+DZ group than in the UW group (237.5 ± 25.6 vs. 108.7 ± 49.3, p < 0.01). The islets from the UW+DZ group displayed a significantly higher glucose-induced insulin secretion (0.97 ng/ml ± 0.15 vs. 0.758 ng/ml ± 0.21, p = 0.009) and insulin content (6095.6 ng/islet ± 1394.5 vs. 4209.2 ng/islet ± 815.1, p = 0.002). The DZ-treated islets had well-preserved mitochondrial morphology with superior responses of mitochondrial potentials and calcium influx responded to glucose. Higher living cells and less late apoptotic cells were observed in the UW+DZ group (p < 0.05). Additionally, the transplanted islets from the UW+DZ group had a significantly higher cure rate and improved glucose tolerance. Conclusion This study is the first to report mitoprotective effects of DZ for pancreas preservation and islet isolation. It remains to be tested whether these findings can be replicated in human islet isolation and transplantation. PMID:24070013

  18. 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.

  19. A cistrome roadmap for understanding pancreatic islet biology

    PubMed Central

    Avrahami, Dana; Kaestner, Klaus H

    2017-01-01

    Although dozens of common variants have been associated with increased risk of type 2 diabetes (T2D), the mechanisms by which these variants increase disease susceptibility are largely unknown. A new study mapping the human pancreatic islet cistrome provides a roadmap for exploring the effects of these variants and suggests that altered enhancer function might be a common contributor to the genetic risk of T2D. PMID:24473321

  20. Islet preconditioning via multimodal microfluidic modulation of intermittent hypoxia.

    PubMed

    Lo, Joe F; Wang, Yong; Blake, Alexander; Yu, Gene; Harvat, Tricia A; Jeon, Hyojin; Oberholzer, Jose; Eddington, David T

    2012-02-21

    Simultaneous stimulation of ex vivo pancreatic islets with dynamic oxygen and glucose is a critical technique for studying how hypoxia alters glucose-stimulated response, especially in transplant environments. Standard techniques using a hypoxic chamber cannot provide both oxygen and glucose modulations, while monitoring stimulus-secretion coupling factors in real-time. Using novel microfluidic device with integrated glucose and oxygen modulations, we quantified hypoxic impairment of islet response by calcium influx, mitochondrial potentials, and insulin secretion. Glucose-induced calcium response magnitude and phase were suppressed by hypoxia, while mitochondrial hyperpolarization and insulin secretion decreased in coordination. More importantly, hypoxic response was improved by preconditioning islets to intermittent hypoxia (IH, 1 min/1 min 5-21% cycling for 1 h), translating to improved insulin secretion. Moreover, blocking mitochondrial K(ATP) channels removed preconditioning benefits of IH, similar to mechanisms in preconditioned cardiomyocytes. Additionally, the multimodal device can be applied to a variety of dynamic oxygen-metabolic studies in other ex vivo tissues.

  1. IL-10 Induction from Implants Delivering Pancreatic Islets and Hyaluronan

    PubMed Central

    Bollyky, Paul L.; Vernon, Robert B.; Falk, Ben A.; Preisinger, Anton; Gooden, Michel D.; Nepom, Gerald T.; Gebe, John A.

    2013-01-01

    Local induction of pro-tolerogenic cytokines, such as IL-10, is an appealing strategy to help facilitate transplantation of islets and other tissues. Here, we describe a pair of implantable devices that capitalize on our recent finding that hyaluronan (HA) promotes IL-10 production by activated T cells. The first device is an injectable hydrogel made of crosslinked HA and heparan sulfate loaded with anti-CD3/anti-CD28 antibodies and IL-2. T cells embedded within this hydrogel prior to polymerization go on to produce IL-10 in vivo. The second device is a bioengineered implant consisting of a polyvinyl alcohol sponge scaffold, supportive collagen hydrogel, and alginate spheres mediating sustained release of HA in fluid form. Pancreatic islets that expressed ovalbumin (OVA) antigen were implanted within this device for 14 days into immunodeficient mice that received OVA-specific DO.11.10 T cells and a subsequent immunization with OVA peptide. Splenocytes harvested from these mice produced IL-10 upon re-challenge with OVA or anti-CD3 antibodies. Both of these devices represent model systems that will be used, in future studies, to further evaluate IL-10 induction by HA, with the objective of improving the survival and function of transplanted islets in the setting of autoimmune (type 1) diabetes. PMID:23971054

  2. IL-10 induction from implants delivering pancreatic islets and hyaluronan.

    PubMed

    Bollyky, Paul L; Vernon, Robert B; Falk, Ben A; Preisinger, Anton; Gooden, Michel D; Nepom, Gerald T; Gebe, John A

    2013-01-01

    Local induction of pro-tolerogenic cytokines, such as IL-10, is an appealing strategy to help facilitate transplantation of islets and other tissues. Here, we describe a pair of implantable devices that capitalize on our recent finding that hyaluronan (HA) promotes IL-10 production by activated T cells. The first device is an injectable hydrogel made of crosslinked HA and heparan sulfate loaded with anti-CD3/anti-CD28 antibodies and IL-2. T cells embedded within this hydrogel prior to polymerization go on to produce IL-10 in vivo. The second device is a bioengineered implant consisting of a polyvinyl alcohol sponge scaffold, supportive collagen hydrogel, and alginate spheres mediating sustained release of HA in fluid form. Pancreatic islets that expressed ovalbumin (OVA) antigen were implanted within this device for 14 days into immunodeficient mice that received OVA-specific DO.11.10 T cells and a subsequent immunization with OVA peptide. Splenocytes harvested from these mice produced IL-10 upon re-challenge with OVA or anti-CD3 antibodies. Both of these devices represent model systems that will be used, in future studies, to further evaluate IL-10 induction by HA, with the objective of improving the survival and function of transplanted islets in the setting of autoimmune (type 1) diabetes.

  3. Pancreatic islets and their roles in metabolic programming.

    PubMed

    Barella, Luiz Felipe; de Oliveira, Júlio Cezar; Mathias, Paulo Cezar de Freitas

    2014-04-01

    Experimental and epidemiologic data have confirmed that undernutrition or overnutrition during critical periods of life can result in metabolic dysfunction, leading to the development of obesity, hypertension, and type 2 diabetes, later in life. These studies have contributed to the concept of the developmental origins of health and disease (DOHaD), which involves metabolic programming patterns. Beyond the earlier phases of development, puberty can be an additional period of plasticity, during which any insult can lead to changes in metabolism. Impaired brain development, associated with imbalanced autonomous nervous system activity due to metabolic programming, is pivotal to the creation of pathophysiology. Excess glucocorticoid exposure, due to hypothalamic-pituitary-adrenal axis deregulation, is also involved in malprogramming in early life. Additionally, the pancreatic islets appear to play a decisive role in the setup and maintenance of these metabolic dysfunctions as key targets of metabolic programming, and epigenetic mechanisms may underlie these changes. Moreover, studies have indicated the possibility that deprogramming renders the islets able to recover their functioning after malprogramming. In this review, we discuss the key roles of the pancreatic islets as targets of malprogramming; however, we also discuss their roles as important targets for the treatment and prevention of metabolic diseases.

  4. Control of IBMIR in Neonatal Porcine Islet Xenotransplantation in Baboons

    PubMed Central

    Hawthorne, W J; Salvaris, E J; Phillips, P; Hawkes, J; Liuwantara, D; Burns, H; Barlow, H; Stewart, A B; Peirce, S B; Hu, M; Lew, A M; Robson, S C; Nottle, M B; D'Apice, A J F; O'Connell, P J; Cowan, P J

    2014-01-01

    The instant blood-mediated inflammatory reaction (IBMIR) is a major obstacle to the engraftment of intraportal pig islet xenografts in primates. Higher expression of the galactose-α1,3-galactose (αGal) xenoantigen on neonatal islet cell clusters (NICC) than on adult pig islets may provoke a stronger reaction, but this has not been tested in the baboon model. Here, we report that WT pig NICC xenografts triggered profound IBMIR in baboons, with intravascular clotting and graft destruction occurring within hours, which was not prevented by anti-thrombin treatment. In contrast, IBMIR was minimal when recipients were immunosuppressed with a clinically relevant protocol and transplanted with NICC from αGal-deficient pigs transgenic for the human complement regulators CD55 and CD59. These genetically modified (GM) NICC were less susceptible to humoral injury in vitro than WT NICC, inducing significantly less complement activation and thrombin generation when incubated with baboon platelet-poor plasma. Recipients of GM NICC developed a variable anti-pig antibody response, and examination of the grafts 1 month after transplant revealed significant cell-mediated rejection, although scattered insulin-positive cells were still present. Our results indicate that IBMIR can be attenuated in this model, but long-term graft survival may require more effective immunosuppression or further donor genetic modification. PMID:24842781

  5. The lizard fauna of Guam's fringing islets: Island biogeography, phylogenetic history, and conservation implications

    USGS Publications Warehouse

    Perry, G.; Rodda, G.H.; Fritts, T.H.; Sharp, T.R.

    1998-01-01

    We sampled the lizard fauna of twenty-two small islets fringing the Pacific island of Guam and used these data to shed light on the processes responsible for present-day diversity. Habitat diversity, measured by islet area and vegetation complexity, was significantly correlated with the number of species found on an islet. However, islet distance and elevation were not significant predictors of diversity. Distribution patterns were slightly different for the two major families in our sample, Scincidae and Gekkonidae: skinks needed larger islets to maintain a population than did geckos. Presence/absence patterns were highly and significantly nested, and population density was correlated with the number of islets on which a species was found. An area cladogram was poorly supported and showed no faunal similarity between nearby islands. These patterns indicate that extinctions on most islets were due mostly to non-catastrophic, long-acting biological causes. The presence on the islets of species extirpated on Guam and the lack of significant nestedness on islands with greater maximum elevation highlight the impact that predators (primarily brown treesnakes) can have. Our findings also show that small reserves will not suffice to protect endangered lizard faunas, and that the islets may serve as a short-term repository of such species until snake-free areas can be established on Guam.

  6. Improved survival and function of rat cryopreserved islets by coculture with sertoli cells.

    PubMed

    Li, Yang; Xue, Wujun; Tian, Xiaohui; Ding, Xiaoming; Tian, Puxun; Feng, Xinshun; Song, Yong; Luo, Xiaohui; Liu, Hongbao; Wang, Xiaohong; Ding, Chenguang

    2011-06-01

    In order to investigate how to improve the function and survival of cryopreserved islets, we cocultured cryopreserved thawed rat islets with rat Sertoli cells. After thawing, the islets were divided into the Sertoli cell coculture group and the control group. Using light and transmission electron microscopes, we examined the morphology of islets and measured their apoptosis index (AI) and insulin release stimulation index (SI). Moreover, we measured apoptosis protein and mRNA by western-blot and reverse transcription polymerase chain reaction and cytokine concentrations in supernatant by ELISA. We examined islet graft survival time in diabetic mice and detected insulin in grafts by immunohistochemistry. We found that the morphology, AI, and SI of the coculture group were all significantly improved. The relative expression levels of cleaved caspase-3 P20, P11, and caspase-7 in the coculture group were lower than those in the control group. Compared with the control group, the expression level of Bax was decreased, but that of Bcl-2 was increased. After transplantation, islet survival in the coculture group was similar to that of fresh islets but longer than that in the control group. These results suggest that coculture with rat Sertoli cells significantly improves the yield and function of rat cryopreserved thawed islets by effectively reducing islet apoptosis.

  7. Simulated Microgravity Combined with Polyglycolic Acid Scaffold Culture Conditions Improves the Function of Pancreatic Islets

    PubMed Central

    Song, Yimin; Wei, Zheng; Song, Chun; Xie, Shanshan; Feng, Jinfa; Fan, Jiehou; Zhang, Zengling; Shi, Yubo

    2013-01-01

    The in vitro culture of pancreatic islets reduces their immunogenicity and prolongs their availability for transplantation. Both simulated microgravity (sMG) and a polyglycolic acid scaffold (PGA) are believed to confer advantages to cell culture. Here, we evaluated the effects of sMG combined with a PGA on the viability, insulin-producing activity and morphological alterations of pancreatic islets. Under PGA-sMG conditions, the purity of the islets was ≥85%, and the islets had a higher survival rate and an increased ability to secrete insulin compared with islets cultured alone in the static, sMG, or PGA conditions. In addition, morphological analysis under scanning electron microscopy (SEM) revealed that the PGA-sMG treatment preserved the integral structure of the islets and facilitated islet adhesion to the scaffolds. These results suggest that PGA-sMG coculture has the potential to improve the viability and function of islets in vitro and provides a promising method for islet transplantation. PMID:24024182

  8. Angiopoietin-like peptide 4 regulates insulin secretion and islet morphology.

    PubMed

    Kim, Hyun-Kyong; Kwon, Obin; Park, Kyeong-Han; Lee, Kyung Jin; Youn, Byung-Soo; Kim, Seung-Whan; Kim, Min-Seon

    2017-02-07

    Insulin secretion from pancreatic islet β-cells is primarily regulated by the blood glucose level, and also modulated by a number of biological factors produced inside the islets or released from remote organs. Previous studies have shown that angiopoietin-like protein 4 (Angptl4) controls glucose and lipid metabolism through its actions in the liver, adipose tissue, and skeletal muscles. In this present study, we investigated the possible role of Angptl4 in the regulation of insulin secretion from pancreatic islets. Angptl4 was found to be highly expressed in the α-cells but not β-cells of rodent islets. Moreover, treatment of rodent islets with Angptl4 peptide potentiated glucose-stimulated insulin secretion through a protein kinase A-dependent mechanism. Consistently, Angptl4 knockout mice showed impaired glucose tolerance. In the cultured islets from Angptl4 knockout mice, glucose-stimulated insulin secretion was significantly lower than in islets from wild type mice. Angptl4 peptide replacement partially reversed this reduction. Moreover, Angptl4 knockout mice had dysmorphic islets with abnormally distributed α-cells. In contrast, the β-cell mass and distribution were not significantly altered in these knockout mice. Our current data collectively suggest that Angptl4 may play a critical role in the regulation of insulin secretion and islet morphogenesis.

  9. Anti-diabetic and neuroprotective effects of pancreatic islet transplantation into the central nervous system.

    PubMed

    Lazard, Daniel; Vardi, Pnina; Bloch, Konstantin

    2016-01-01

    During the last decades, the central nervous system (CNS) was intensively tested as a site for islet transplantation in different animal models of diabetes. Immunoprivilege properties of intracranial and intrathecal sites were found to delay and reduce rejection of transplanted allo-islets and xeno-islets, especially in the form of dispersed single cells. Insulin released from islets grafted in CNS was shown to cross the blood-brain barrier and to act as a regulator of peripheral glucose metabolism. In diabetic animals, sufficient nutrition and oxygen supply to islets grafted in the CNS provide adequate insulin response to increase glucose level resulting in rapid normoglycemia. In addition to insulin, pancreatic islets produce and secrete several other hormones, as well as neurotrophic and angiogenic factors with potential neuroprotective properties. Recent experimental studies and clinical trials provide a strong support for delivery of islet-derived macromolecules to CNS as a promising strategy to treat various brain disorders. This review article focuses mainly on analysis of current status of intracranial and intrathecal islet transplantations for treatment of experimental diabetes and discusses the possible neuroprotective properties of grafted islets into CNS as a novel therapeutic approach to brain disorders with cognitive dysfunctions characterized by impaired brain insulin signalling. Copyright © 2015 John Wiley & Sons, Ltd.

  10. α-1 Antitrypsin Enhances Islet Engraftment by Suppression of Instant Blood-Mediated Inflammatory Reaction.

    PubMed

    Wang, Jingjing; Sun, Zhen; Gou, Wenyu; Adams, David B; Cui, Wanxing; Morgan, Katherine A; Strange, Charlie; Wang, Hongjun

    2017-04-01

    Islet cell transplantation has limited effectiveness because of an instant blood-mediated inflammatory reaction (IBMIR) that occurs immediately after cell infusion and leads to dramatic β-cell death. In intraportal islet transplantation models using mouse and human islets, we demonstrated that α-1 antitrypsin (AAT; Prolastin-C), a serine protease inhibitor used for the treatment of AAT deficiency, inhibits IBMIR and cytokine-induced inflammation in islets. In mice, more diabetic recipients reached normoglycemia after intraportal islet transplantation when they were treated with AAT compared with mice treated with saline. AAT suppressed blood-mediated coagulation pathways by diminishing tissue factor production, reducing plasma thrombin-antithrombin complex levels and fibrinogen deposition on islet grafts, which correlated with less graft damage and apoptosis. AAT-treated mice showed reduced serum tumor necrosis factor-α levels, decreased lymphocytic infiltration, and decreased nuclear factor (NF)-κB activation compared with controls. The potent anti-inflammatory effect of AAT is possibly mediated by suppression of c-Jun N-terminal kinase (JNK) phosphorylation. Blocking JNK activation failed to further reduce cytokine-induced apoptosis in β-cells. Taken together, AAT significantly improves islet graft survival after intraportal islet transplantation by mitigation of coagulation in IBMIR and suppression of cytokine-induced JNK and NF-κB activation. AAT-based therapy has the potential to improve graft survival in human islet transplantation and other cellular therapies on the horizon.

  11. Characterization of succinate dehydrogenase and alpha-glycerophosphate dehydrogenase in pancreatic islets.

    PubMed

    Lenzen, S; Panten, U

    1983-12-01

    Succinate dehydrogenase activities in homogenates of rat and ob/ob mouse pancreatic islets were only 13% of the activities in homogenates of liver and were also several times lower than in homogenates of pancreatic acinar tissue. This indicates that the content of mitochondria in pancreatic islet cells is very low. The very low activity of succinate dehydrogenase is in agreement with the low mitochondrial volume in the cytoplasmic ground substance of pancreatic islet cells as observed in morphometric studies. This may represent the poor equipment of pancreatic islet cells with electron transport chains and thus provide a regulatory role for the generation of reducing equivalents and chemical energy for the regulation of insulin secretion. The activities of succinate dehydrogenase in tissue homogenates of pancreatic islets, pancreatic acinar tissue, and liver were significantly inhibited by malonate and diazoxide but not by glucose, mannoheptulose, streptozotocin, or verapamil. Tolbutamide inhibited only pancreatic islet succinate dehydrogenase significantly, providing evidence for a different behavior of pancreatic islet cell mitochondria. Therefore diazoxide and tolbutamide may affect pancreatic islet function through their effects on succinate dehydrogenase activity. The activities of alpha-glycerophosphate dehydrogenase in homogenates of pancreatic islets and liver from rats and ob/ob mice were in the same range, while activities in homogenates of pancreatic acinar tissue were lower. None of the test agents affected alpha-glycerophosphate dehydrogenase activity. Thus the results provide no support for the recent contention that alpha-glycerophosphate dehydrogenase activity may be critical for the regulation of insulin secretion.

  12. Engineering of microscale three-dimensional pancreatic islet models in vitro and their biomedical applications.

    PubMed

    Gao, Bin; Wang, Lin; Han, Shuang; Pingguan-Murphy, Belinda; Zhang, Xiaohui; Xu, Feng

    2016-08-01

    Diabetes now is the most common chronic disease in the world inducing heavy burden for the people's health. Based on this, diabetes research such as islet function has become a hot topic in medical institutes of the world. Today, in medical institutes, the conventional experiment platform in vitro is monolayer cell culture. However, with the development of micro- and nano-technologies, several microengineering methods have been developed to fabricate three-dimensional (3D) islet models in vitro which can better mimic the islet of pancreases in vivo. These in vitro islet models have shown better cell function than monolayer cells, indicating their great potential as better experimental platforms to elucidate islet behaviors under both physiological and pathological conditions, such as the molecular mechanisms of diabetes and clinical islet transplantation. In this review, we present the state-of-the-art advances in the microengineering methods for fabricating microscale islet models in vitro. We hope this will help researchers to better understand the progress in the engineering 3D islet models and their biomedical applications such as drug screening and islet transplantation.

  13. An effective purification method using large bottles for human pancreatic islet isolation.

    PubMed

    Shimoda, Masayuki; Itoh, Takeshi; Iwahashi, Shuichi; Takita, Morihito; Sugimoto, Koji; Kanak, Mazhar A; Chujo, Daisuke; Naziruddin, Bashoo; Levy, Marlon F; Grayburn, Paul A; Matsumoto, Shinichi

    2012-01-01

    The purification process is one of the most difficult procedures in pancreatic islet isolation. It was demonstrated that the standard purification method using a COBE 2991 cell processor with Ficoll density gradient solution harmed islets mechanically by high shear force. We reported that purification using large bottles with a lower viscosity gradient solution could improve the efficacy of porcine islet purification. In this study, we examined whether the new bottle purification method could improve the purification of human islets. Nine human pancreata from brain-dead donors were used. After pancreas digestion, the digested tissue was divided into three groups. Each group was purified by continuous density gradient using ET-Kyoto and iodixanol gradient solution with either the standard COBE method (COBE group) or the top loading (top group) or bottom loading (bottom group) bottle purification methods. Islet yield, purity, recovery rate after purification, and in vitro and in vivo viability were compared. Islet yield per pancreas weight (IE/g) and the recovery rate in the top group were significantly higher than in the COBE and bottom groups. Furthermore, the average size of purified islets in the top group was significantly larger than in the COBE group, which indicated that the bottle method could reduce the shear force to the islets. In vivo viability was also significantly higher in the top group compared with the COBE group. In conclusion, the top-loading bottle method could improve the quality and quantity of human islets after purification.

  14. Matching CHAMP and C/NOFS Climatology at Low Latitudes with the ionosphere-thermosphere TIEGCM and the plasmasphere GIP Models

    NASA Astrophysics Data System (ADS)

    Emery, B. A.; Bhaneja, P.; Bilitza, D.; Chau, J. L.; Coley, W. R.; Drob, D. P.; Fang, T.; Fejer, B. G.; Foster, B. T.; Haeusler, K.; Jones, M.; Klenzing, J.; Luhr, H.; Retterer, J. M.; Richmond, A. D.; Roddy, P. A.; Stoneback, R.; Su, Y.; Wu, Q.; Zhang, X.

    2013-05-01

    The equatorial orbiting Communications/Navigation Outage Forecasting System (C/NOFS) satellite observations from 400-800km started in mid-2008 in a period of deep solar minimum where the 10.7 cm solar radio flux flattened out around 67. By 2012, the solar flux increased to about 135. The polar orbiting CHAllenging Minisatellite Payload (CHAMP) satellite observations between about 410-310km above the equator are available from 2000 to 2010 from high to very low solar flux conditions. CHAMP Planar Langmuir Probe (PLP) observations of the electron density and temperature and the instrument measuring neutral cross-track winds (mostly zonal) are used. C/NOFS PLP, Ion Velocity Meter (IVM), and Vector Electric Field Instrument (VEFI) observations are used. The IVM package gives cross-track ExB ion drifts (meridional/upward and perpendicular) which can be mapped to the magnetic equator, along with a Retarding Potential Analyzer (RPA) for along-track ion drifts (zonal), plus [O+] composition and ion temperatures. VEFI also gives meridional/upward and zonal ion drifts. We compare climatological observations in equinox and winter with empirical models, and with the first-principles model estimates from the Thermosphere-Ionosphere-Electrodynamics General Circulation Model (TIEGCM), coupled with the Global Ionosphere-Plasmasphere (GIP) model, with various tidal forcings from the lower atmosphere. We compare longitude variations as well as local time zonal averages between the observations and model results. We also compare with ground observations of the meridional/upward and zonal ion drifts from the magnetic equator at Jicamarca, Peru.

  15. Pancreatic islet-specific overexpression of Reg3β protein induced the expression of pro-islet genes and protected the mice against streptozotocin-induced diabetes mellitus.

    PubMed

    Xiong, Xiaoquan; Wang, Xiao; Li, Bing; Chowdhury, Subrata; Lu, Yarong; Srikant, Coimbatore B; Ning, Guang; Liu, Jun-Li

    2011-04-01

    Reg family proteins have been implicated in islet β-cell proliferation, survival, and regeneration. The expression of Reg3β (pancreatitis-associated protein) is highly induced in experimental diabetes and acute pancreatitis, but its precise role has not been established. Through knockout studies, this protein was shown to be mitogenic, antiapoptotic, and anti-inflammatory in the liver and pancreatic acinars. To test whether it can promote islet cell growth or survival against experimental damage, we developed β-cell-specific overexpression using rat insulin I promoter, evaluated the changes in normal islet function, gene expression profile, and the response to streptozotocin-induced diabetes. Significant and specific overexpression of Reg3β was achieved in the pancreatic islets of RIP-I/Reg3β mice, which exhibited normal islet histology, β-cell mass, and in vivo and in vitro insulin secretion in response to high glucose yet were slightly hyperglycemic and low in islet GLUT2 level. Upon streptozotocin treatment, in contrast to wild-type littermates that became hyperglycemic in 3 days and lost 15% of their weight, RIP-I/Reg3β mice were significantly protected from hyperglycemia and weight loss. To identify specific targets affected by Reg3β overexpression, a whole genome DNA microarray on islet RNA isolated from the transgenic mice revealed more than 45 genes significantly either up- or downregulated. Among them, islet-protective osteopontin/SPP1 and acute responsive nuclear protein p8/NUPR1 were significantly induced, a result further confirmed by real-time PCR, Western blots, and immunohistochemistry. Our results suggest that Reg3β is unlikely an islet growth factor but a putative protector that prevents streptozotocin-induced damage by inducing the expression of specific genes.

  16. A novel long-acting glucose-dependent insulinotropic peptide analogue: enhanced efficacy in normal and diabetic rodents

    PubMed Central

    Tatarkiewicz, K; Hargrove, D M; Jodka, C M; Gedulin, B R; Smith, P A; Hoyt, J A; Lwin, A; Collins, L; Mamedova, L; Levy, O E; D’Souza, L; Janssen, S; Srivastava, V; Ghosh, S S; Parkes, D G

    2014-01-01

    Aim Glucose-dependent insulinotropic peptide (GIP) is an incretin hormone that is released from intestinal K cells in response to nutrient ingestion. We aimed to investigate the therapeutic potential of the novel N- and C-terminally modified GIP analogue AC163794. Methods AC163794 was synthesized by solid-phase peptide synthesis. Design involved the substitution of the C-terminus tail region of the dipeptidyl peptidase IV (DPP-IV)-resistant GIP analogue [d-Ala2]GIP(1–42) with the unique nine amino acid tail region of exenatide. The functional activity and binding of AC163794 to the GIP receptor were evaluated in RIN-m5F β-cells. In vitro metabolic stability was tested in human plasma and kidney membrane preparations. Acute insulinotropic effects were investigated in isolated mouse islets and during an intravenous glucose tolerance test in normal and diabetic Zucker fatty diabetic (ZDF) rats. The biological actions of AC163794 were comprehensively assessed in normal, ob/ob and high-fat-fed streptozotocin (STZ)-induced diabetic mice. Acute glucoregulatory effects of AC163794 were tested in diet-induced obese mice treated subchronically with AC3174, the exendatide analogue [Leu14] exenatide. Human GIP or [d-Ala2]GIP(1–42) were used for comparison. Results AC163794 exhibited nanomolar functional GIP receptor potency in vitro similar to GIP and [d-Ala2]GIP(1–42). AC163794 was metabolically more stable in vitro and displayed longer duration of insulinotropic action in vivo versus GIP and [d-Ala2]GIP(1–42). In diabetic mice, AC163794 improved HbA1c through enhanced insulinotropic action, partial restoration of pancreatic insulin content and improved insulin sensitivity with no adverse effects on fat storage and metabolism. AC163794 provided additional baseline glucose-lowering when injected to mice treated with AC3174. Conclusions These studies support the potential use of a novel GIP analogue AC163794 for the treatment of type 2 diabetes. PMID:23859463

  17. Clock-controlled output gene Dbp is a regulator of Arnt/Hif-1β gene expression in pancreatic islet β-cells

    SciTech Connect

    Nakabayashi, Hiroko; Ohta, Yasuharu Yamamoto, Masayoshi; Susuki, Yosuke; Taguchi, Akihiko; Tanabe, Katsuya; Kondo, Manabu; Hatanaka, Masayuki; Nagao, Yuko; Tanizawa, Yukio

    2013-05-03

    Highlights: •Arnt mRNA expressed in a circadian manner in mouse pancreatic islets. •Expressions of Dbp and Arnt damped in the islets of a diabetic model mouse. •DBP and E4BP4 regulate Arnt promoter activity by direct binding. •Arnt may have a role in connecting circadian rhythm and metabolism. -- Abstract: Aryl hydrocarbon receptor nuclear translocator (ARNT)/hypoxia inducible factor-1β (HIF-1β) has emerged as a potential determinant of pancreatic β-cell dysfunction and type 2 diabetes in humans. An 82% reduction in Arnt expression was observed in islets from type 2 diabetic donors as compared to non-diabetic donors. However, few regulators of Arnt expression have been identified. Meanwhile, disruption of the clock components CLOCK and BMAL1 is known to result in hypoinsulinemia and diabetes, but the molecular details remain unclear. In this study, we identified a novel molecular connection between Arnt and two clock-controlled output genes, albumin D-element binding protein (Dbp) and E4 binding protein 4 (E4bp4). By conducting gene expression studies using the islets of Wfs1{sup −/−} A{sup y}/a mice that develop severe diabetes due to β-cell apoptosis, we demonstrated clock-related gene expressions to be altered in the diabetic mice. Dbp mRNA decreased by 50%, E4bp4 mRNA increased by 50%, and Arnt mRNA decreased by 30% at Zeitgever Time (ZT) 12. Mouse pancreatic islets exhibited oscillations of clock gene expressions. E4BP4, a D-box negative regulator, oscillated anti-phase to DBP, a D-box positive regulator. We also found low-amplitude circadian expression of Arnt mRNA, which peaked at ZT4. Over-expression of DBP raised both mRNA and protein levels of ARNT in HEK293 and MIN6 cell lines. Arnt promoter-driven luciferase reporter assay in MIN6 cells revealed that DBP increased Arnt promoter activity by 2.5-fold and that E4BP4 competitively inhibited its activation. In addition, on ChIP assay, DBP and E4BP4 directly bound to D-box elements within the

  18. Human islet preparations distributed for research exhibit a variety of insulin-secretory profiles.

    PubMed

    Kayton, Nora S; Poffenberger, Gregory; Henske, Joseph; Dai, Chunhua; Thompson, Courtney; Aramandla, Radhika; Shostak, Alena; Nicholson, Wendell; Brissova, Marcela; Bush, William S; Powers, Alvin C

    2015-04-01

    Human islet research is providing new insights into human islet biology and diabetes, using islets isolated at multiple US centers from donors with varying characteristics. This creates challenges for understanding, interpreting, and integrating research findings from the many laboratories that use these islets. In what is, to our knowledge, the first standardized assessment of human islet preparations from multiple isolation centers, we measured insulin secretion from 202 preparations isolated at 15 centers over 11 years and noted five distinct patterns of insulin secretion. Approximately three quarters were appropriately responsive to stimuli, but one quarter were dysfunctional, with unstable basal insulin secretion and/or an impairment in stimulated insulin secretion. Importantly, the patterns of insulin secretion by responsive human islet preparations (stable Baseline and Fold stimulation of insulin secretion) isolated at different centers were similar and improved slightly over the years studied. When all preparations studied were considered, basal and stimulated insulin secretion did not correlate with isolation center, biological differences of the islet donor, or differences in isolation, such as Cold Ischemia Time. Dysfunctional islet preparations could not be predicted from the information provided by the isolation center and had altered expression of genes encoding components of the glucose-sensing pathway, but not of insulin production or cell death. These results indicate that insulin secretion by most preparations from multiple centers is similar but that in vitro responsiveness of human islets cannot be predicted, necessitating preexperimental human islet assessment. These results should be considered when one is designing, interpreting, and integrating experiments using human islets.

  19. Pregnancy restores insulin secretion from pancreatic islets in cafeteria diet-induced obese rats.

    PubMed

    Vanzela, E C; Ribeiro, R A; de Oliveira, C A Machado; Rodrigues, F B; Bonfleur, M L; Carneiro, E M; Souza, K L A; Boschero, A C

    2010-02-01

    Insulin resistance during pregnancy is counteracted by enhanced insulin secretion. This condition is aggravated by obesity, which increases the risk of gestational diabetes. Therefore, pancreatic islet functionality was investigated in control nonpregnant (C) and pregnant (CP), and cafeteria diet-fed nonpregnant (Caf), and pregnant (CafP) obese rats. Isolated islets were used for measurements of insulin secretion (RIA), NAD(P)H production (MTS), glucose oxidation ((14)CO(2) production), intracellular Ca(2+) levels (fura-2 AM), and gene expression (real-time PCR). Impaired glucose tolerance was clearly established in Caf and CafP rats at the 14th wk on a diet. Insulin secretion induced by direct depolarizing agents such as KCl and tolbutamide and increasing concentrations of glucose was significantly reduced in Caf, compared with C islets. This reduction was not observed in islets from CP and CafP rats. Accordingly, the glucose oxidation and production of reduced equivalents were increased in CafP islets. The glucose-induced Ca(2+) increase was significantly lower in Caf and higher in CafP, compared with all other groups. CP and CafP islets demonstrated an increased Ca(2+) oscillation frequency, compared with both C and Caf islets, and the amplitude of oscillations was augmented in CafP, compared with Caf islets. In addition, Ca(v)alpha1.2 and SERCA2a mRNA levels were reduced in Caf islets. Ca(v)alpha1.2, but not SERCA2a, mRNA was normalized in CafP islets. In conclusion, cafeteria diet-induced obesity impairs insulin secretion. This alteration is related to the impairment of Ca(2+) handling in pancreatic islets, in especial Ca(2+) influx, a defect that is reversed during pregnancy allowing normalization of insulin secretion.

  20. Biologic and immunomodulatory properties of mesenchymal stromal cells derived from human pancreatic islets

    PubMed Central

    KIM, JAEHYUP; BREUNIG, MELISSA J.; ESCALANTE, LEAH E.; BHATIA, NEEHAR; DENU, RYAN A.; DOLLAR, BRIDGET A.; STEIN, ANDREW P.; HANSON, SUMMER E.; NADERI, NADIA; RADEK, JAMES; HAUGHY, DERMOT; BLOOM, DEBRA D.; ASSADI-PORTER, FARIBA M.; HEMATTI, PEIMAN

    2012-01-01

    Background aims Mesenchymal stromal cells (MSC) have now been shown to reside in numerous tissues throughout the body, including the pancreas. Ex vivo culture-expanded MSC derived from many tissues display important interactions with different types of immune cells in vitro and potentially play a significant role in tissue homeostasis in vivo. In this study, we investigated the biologic and immunomodulatory properties of human pancreatic islet-derived MSC. Methods We culture-expanded MSC from cadaveric human pancreatic islets and characterized them using flow cytometry, differentiation assays and nuclear magnetic resonance-based metabolomics. We also investigated the immunologic properties of pancreatic islet-derived MSC compared with bone marrow (BM) MSC. Results Pancreatic islet and BM-derived MSC expressed the same cell-surface markers by flow cytometry, and both could differentiate into bone, fat and cartilage. Metabolomics analysis of MSC from BM and pancreatic islets also showed a similar set of metabolic markers but quantitative polymerase chain reactions showed that pancreatic islet MSC expressed more interleukin(IL)-1b, IL-6, STAT3 and FGF9 compared with BM MSC, and less IL-10. However, similar to BM MSC, pancreatic islet MSC were able to suppress proliferation of allogeneic T lymphocytes stimulated with anti-CD3 and anti-CD28 antibodies. Conclusions Our in vitro analysis shows pancreatic islet-derived MSC have phenotypic, biologic and immunomodulatory characteristics similar, but not identical, to BM-derived MSC. We propose that pancreatic islet-derived MSC could potentially play an important role in improving the outcome of pancreatic islet transplantation by promoting engraftment and creating a favorable immune environment for long-term survival of islet allografts. PMID:22571381

  1. Transplanted human bone marrow progenitor subtypes stimulate endogenous islet regeneration and revascularization.

    PubMed

    Bell, Gillian I; Broughton, Heather C; Levac, Krysta D; Allan, David A; Xenocostas, Anargyros; Hess, David A

    2012-01-01

    Transplanted murine bone marrow (BM) progenitor cells recruit to the injured pancreas and induce endogenous beta cell proliferation to improve islet function. To enrich for analogous human progenitor cell types that stimulate islet regeneration, we purified human BM based on high-aldehyde dehydrogenase activity (ALDH(hi)), an enzymatic function conserved in hematopoietic, endothelial, and mesenchymal progenitor lineages. We investigated the contributions of ALDH(hi) mixed progenitor cells or culture-expanded, ALDH-purified multipotent stromal cell (MSC) subsets to activate endogenous programs for islet regeneration after transplantation into streptozotocin-treated NOD/SCID mice. Intravenous injection of uncultured BM ALDH(hi) cells improved systemic hyperglycemia and augmented insulin secretion by increasing islet size and vascularization, without increasing total islet number. Augmented proliferation within regenerated endogenous islets and associated vascular endothelium indicated the induction of islet-specific proliferative and pro-angiogenic programs. Although cultured MSC from independent human BM samples showed variable capacity to improve islet function, and prolonged expansion diminished hyperglycemic recovery, transplantation of ALDH-purified regenerative MSC reduced hyperglycemia and augmented total beta cell mass by stimulating the formation of small beta cell clusters associated with the ductal epithelium, without evidence of increased islet vascularization or Ngn3(+) endocrine precursor activation. Thus, endogenous islet recovery after progenitor cell transplantation can occur via distinct regenerative mechanisms modulated by subtypes of progenitor cells administered. Further, understanding of how these islet regenerative and pro-angiogenic programs are activated by specific progenitor subsets may provide new approaches for combination cellular therapies to combat diabetes.

  2. Isles within islets: The lattice origin of small-world networks in pancreatic tissues

    NASA Astrophysics Data System (ADS)

    Barua, Amlan K.; Goel, Pranay

    2016-02-01

    The traditional computational model of the pancreatic islets of Langerhans is a lattice of β-cells connected with gap junctions. Numerous studies have investigated the behavior of networks of coupled β-cells and have shown that gap junctions synchronize bursting strongly. This simplistic architecture of islets, however, seems increasingly untenable at the face of recent experimental advances. In a microfluidics experiment on isolated islets, Rocheleau et al. (2004) showed a failure of penetration of excitation when one end received high glucose and other end was not excited sufficiently; this suggested that gap junctions may not be efficient at inducing synchrony throughout the islet. Recently, Stozer et al. (2013) have argued that the functional networks of β-cells in an islet are small world. Their results implicate the existence of a few long-range connections among cells in the network. The physiological reason underlying this claim is not well understood. These studies cast doubt on the original lattice model that largely predict an all-or-none synchrony among the cells. Here we have attempted to reconcile these observations in a unified framework. We assume that cells in the islet are coupled randomly to their nearest neighbors with some probability, p. We simulated detailed β-cell bursting in such islets. By varying p systematically we were led to network parameters similar to those obtained by Stozer et al. (2013). We find that the networks within islets break up into components giving rise to smaller isles within the super structure-isles-within-islets, as it were. This structure can also account for the partial excitation seen by Rocheleau et al. (2004). Our updated view of islet architecture thus explains the paradox how islets can have strongly synchronizing gap junctions, and be weakly coordinated at the same time.

  3. Selection of polymers for application in scaffolds applicable for human pancreatic islet transplantation.

    PubMed

    Smink, Alexandra M; de Haan, Bart J; Paredes-Juarez, Genaro A; Wolters, Anouk H G; Kuipers, Jeroen; Giepmans, Ben N G; Schwab, Leendert; Engelse, Marten A; van Apeldoorn, Aart A; de Koning, Eelco; Faas, Marijke M; de Vos, Paul

    2016-05-13

    The liver is currently the site for transplantation of islets in humans. This is not optimal for islets, but alternative sites in humans are not available. Polymeric scaffolds in surgically accessible areas are a solution. As human donors are rare, the polymers should not interfere with functional survival of human-islets. We applied a novel platform to test the adequacy of polymers for application in scaffolds for human-islet transplantation. Viability, functionality, and immune parameters were included to test poly(D,L-lactide-co-ε-caprolactone) (PDLLCL), poly(ethylene oxide terephthalate)/polybutylene terephthalate (PEOT/PBT) block copolymer, and polysulfone. The type of polymer influenced the functional survival of human islets. In islets cultured on PDLLCL the glucagon-producing α-cells and insulin-producing β-cells contained more hormone granules than in islets in contact with PEOT/PBT or polysulfone. This was studied with ultrastructural analysis by electron microscopy (nanotomy) during 7 d of culture. PDLLCL was also associated with statistically significant lower release of double-stranded DNA (dsDNA, a so called danger-associate molecular pattern (DAMP)) from islets on PDLLCL when compared to the other polymers. DAMPs support undesired immune responses. Hydrophilicity of the polymers did not influence dsDNA release. Islets on PDLLCL also showed less cellular outgrowth. These outgrowing cells were mainly fibroblast and some β-cells undergoing epithelial to mesenchymal cell transition. None of the polymers influenced the glucose-stimulated insulin secretion. As PDLLCL was associated with less release of DAMPs, it is a promising candidate for creating a scaffold for human islets. Our study demonstrates that for sensitive, rare cadaveric donor tissue such as pancreatic islets it might be necessary to first select materials that do not influence functionality before proposing the biomaterial for in vivo application. Our presented platform may facilitate

  4. Oral administration of the high-chromium yeast improve blood plasma variables and pancreatic islet tissue in diabetic mice.

    PubMed

    Liu, Lu; Jin, Wen; Lv, Jia-Ping

    2010-12-01

    The in vivo effects of oral administration of the high-chromium yeast to healthy and diabetic mice are described. Given that these complexes are proposed to function by potentiating the actions of insulin and activating the insulin receptor kinase, changes in lipid and carbohydrate metabolism would be expected. After 15 weeks administration (500 μg Cr/kg body mass) to healthy mice, abnormal metabolism and pathological change were not observed. After 15 weeks of treatment (0-1,000 μg Cr/kg body mass) of diabetic mice, the effect of high-chromium yeast on blood lipids and blood glycosylated hemoglobin (GHb) of diabetes are not consistent. High-chromium yeast results in a lowering (P < 0.05) of GHb and triglyceride, lowering (P < 0.01) of total cholesterol, and restoration (P < 0.01) of insulin; these results are in stark contrast to those of diabetic mice of administration of normal yeast, which have no effect on these parameters and serve as control group. The histopathological analysis of pancreas islet shows that high-chromium yeast could profoundly protect the impaired pancreatic islet and β-cells from inflammatory infiltration and fibrosis.

  5. Inhibition of instant blood-mediated inflammatory responses by co-immobilization of sCR1 and heparin on islets.

    PubMed

    Luan, Nguyen Minh; Iwata, Hiroo

    2013-07-01

    Intraportal transplantation of islets of Langerhans is followed by marked islet loss, mainly caused by instant blood-mediated inflammatory responses (IBMIR). We previously developed a method of co-immobilizing sCR1 and heparin on islets. Here we examined whether this process could reduce islet loss following intraportal islet transplantation in a syngeneic mouse model. sCR1-heparin islets or unmodified islet controls were transplanted into the livers of streptozotocin-induced diabetic mice. Transplantation of 100 and 125 sCR1-heparin islets normalized blood glucose levels in 8 of 9 (88.9%) and 9 of 9 diabetic mice (100%), respectively, whereas transplantation of 100 and 125 non-treated islets induced normoglycemia in 0 of 9 and 2 of 9 diabetic mice, respectively. Fibrin staining and plasma insulin measurements indicated that, compared to non-treated islets, sCR1-heparin islet transplantation was associated with fewer blood clots around islets, and significantly less insulin leakage from damaged islets at 1 h post-transplantation. Long-term follow-up of the sCR1-heparin islet group showed islet cells in the livers and insulin expression. In conclusion, co-immobilization of sCR1 and heparin on islets could effectively reduce islet damage by IBMIR, and might be useful to enable transplantation with only one donor and one recipient.

  6. Whole organ and islet of Langerhans dosimetry for calculation of absorbed doses resulting from imaging with radiolabeled exendin

    PubMed Central

    van der Kroon, Inge; Woliner-van der Weg, Wietske; Brom, Maarten; Joosten, Lieke; Frielink, Cathelijne; Konijnenberg, Mark W.; Visser, Eric P.; Gotthardt, Martin

    2017-01-01

    Radiolabeled exendin is used for non-invasive quantification of beta cells in the islets of Langerhans in vivo. High accumulation of radiolabeled exendin in the islets raised concerns about possible radiation-induced damage to these islets in man. In this work, islet absorbed doses resulting from exendin-imaging were calculated by combining whole organ dosimetry with small scale dosimetry for the islets. Our model contains the tissues with high accumulation of radiolabeled exendin: kidneys, pancreas and islets. As input for the model, data from a clinical study (radiolabeled exendin distribution in the human body) and from a preclinical study with Biobreeding Diabetes Prone (BBDP) rats (islet-to-exocrine uptake ratio, beta cell mass) were used. We simulated 111In-exendin and 68Ga-exendin absorbed doses in patients with differences in gender, islet size, beta cell mass and radiopharmaceutical uptake in the kidneys. In all simulated cases the islet absorbed dose was small, maximum 1.38 mGy for 68Ga and 66.0 mGy for 111In. The two sources mainly contributing to the islet absorbed dose are the kidneys (33–61%) and the islet self-dose (7.5–57%). In conclusion, all islet absorbed doses are low (<70 mGy), so even repeated imaging will hardly increase the risk on diabetes. PMID:28067253

  7. Alpha 1-antitrypsin reduces inflammation and enhances mouse pancreatic islet transplant survival.

    PubMed

    Koulmanda, Maria; Bhasin, Manoj; Fan, Zhigang; Hanidziar, Dusan; Goel, Nipun; Putheti, Prabhakar; Movahedi, Babak; Libermann, Towia A; Strom, Terry B

    2012-09-18

    The promise of islet cell transplantation cannot be fully realized in the absence of improvements in engraftment of resilient islets. The marginal mass of islets surviving the serial peritransplant insults may lead to exhaustion and thereby contribute to an unacceptably high rate of intermediate and long-term graft loss. Hence, we have studied the effects of treatment with alpha 1-antitrypsin (AAT) in a syngeneic nonautoimmune islet graft model. A marginal number of syngeneic mouse islets were transplanted into nonautoimmune diabetic hosts and islet function was analyzed in control and AAT treated hosts. In untreated controls, marginal mass islet transplants did not restore euglycemia. Outcomes were dramatically improved by short-term AAT treatment. Transcriptional profiling identified 1,184 differentially expressed transcripts in AAT-treated hosts at 3 d posttransplantation. Systems-biology-based analysis revealed AAT down-regulated regulatory hubs formed by inflammation-related molecules (e.g., TNF-α, NF-κB). The conclusions yielded by the systems-biology analysis were rigorously confirmed by QRT-PCR and immunohistology. These data suggest that short-term AAT treatment of human islet transplant recipients may be worthy of a clinical trial.

  8. Preserving Mafa expression in diabetic islet β-cells improves glycemic control in vivo.

    PubMed

    Matsuoka, Taka-aki; Kaneto, Hideaki; Kawashima, Satoshi; Miyatsuka, Takeshi; Tochino, Yoshihiro; Yoshikawa, Atsushi; Imagawa, Akihisa; Miyazaki, Jun-ichi; Gannon, Maureen; Stein, Roland; Shimomura, Iichiro

    2015-03-20

    The murine Mafa transcription factor is a key regulator of postnatal islet β-cell activity, affecting insulin transcription, insulin secretion, and β-cell mass. Human MAFA expression is also markedly decreased in islet β-cells of type 2 diabetes mellitus (T2DM) patients. Moreover, levels are profoundly reduced in db/db islet β-cells, a mouse model of T2DM. To examine the significance of this key islet β-cell-enriched protein to glycemic control under diabetic conditions, we generated transgenic mice that conditionally and specifically produced Mafa in db/db islet β-cells. Sustained expression of Mafa resulted in significantly lower plasma glucose levels, higher plasma insulin, and augmented islet β-cell mass. In addition, there was increased expression of insulin, Slc2a2, and newly identified Mafa-regulated genes involved in reducing β-cell stress, like Gsta1 and Gckr. Importantly, the levels of human GSTA1 were also compromised in T2DM islets. Collectively, these results illustrate how consequential the reduction in Mafa activity is to islet β-cell function under pathophysiological conditions.

  9. The Evaluation of Islet Purification Methods That Use Large Bottles to Create a Continuous Density Gradient

    PubMed Central

    Miyagi-Shiohira, Chika; Kobayashi, Naoya; Saitoh, Issei; Watanabe, Masami; Noguchi, Yasufumi; Matsushita, Masayuki; Noguchi, Hirofumi

    2017-01-01

    Islet purification is one of the most important steps of islet isolation for pancreatic islet transplantation. The most common method of islet purification is density gradient centrifugation using a COBE 2991 cell processor. However, this method can damage islets mechanically through its high shearing force. We recently reported that a new purification method using large plastic bottles effectively achieves a high yield of islets from the porcine pancreas. In the present study, we evaluated the methods of making a continuous density gradient. The gradient was produced with a gradient maker and two types of candy cane-shaped stainless steel pipes. One method was to use a “bent-tipped” stainless steel pipe and to load from a high-density solution to a low-density solution, uploading the stainless steel pipe. The other method was to use a regular stainless steel pipe and to load from a low-density solution to a high-density solution, leaving the stainless steel pipe in place. There were no significant differences between the two solutions in terms of the islet yield, rate of viability or purity, score, or the stimulation index after purification. Furthermore, there were no differences in the attainability or suitability of posttransplantation normoglycemia. Our study shows the equivalency of these two methods of islet purification. PMID:28174674

  10. Single-Cell Sequencing of Human Pancreatic Islets-New Kids on the Block.

    PubMed

    Prasad, Rashmi B; Groop, Leif

    2016-10-11

    RNA sequencing of human pancreatic islets has provided important insights into the islet transcriptome but little information on the specific cells. In this issue, Segerstolpe et al. (2016) and Xin et al. (2016b) report on the transcriptome of single pancreatic cells from non-diabetic and type 2 diabetic donors.

  11. Bone marrow mesenchymal stem cells promote the repair of islets from diabetic mice through paracrine actions.

    PubMed

    Gao, Xiaodong; Song, Lujun; Shen, Kuntang; Wang, Hongshan; Qian, Mengjia; Niu, Weixin; Qin, Xinyu

    2014-05-05

    Transplantation of bone marrow mesenchymal stem cells (MSCs) has been shown to effectively lower blood glucose levels in diabetic individuals, but the mechanism has not been adequately explained. We hypothesized that MSCs exert beneficial paracrine actions on the injured islets by releasing biologically active factors. To prove our hypothesis, we tested the cytoprotective effect of conditioned medium from cultured MSCs on isolated islets exposed to STZ in vitro and on mice islets after the experimental induction of diabetes in vivo. We assessed islet regeneration in the presence of conditioned medium and explored the possible mechanisms involved. Transplantation of MSCs can ameliorate hyperglycemia in diabetic mice by promoting the regeneration of β cells. Both β cell replication and islet progenitors differentiation contribute to β cell regeneration. MSC transplantation resulted in increases in pAkt and pErk expression by islets in vivo. Treatment with MSC-CM promoted islet cell proliferation and resulted in increases in pAkt and pErk expression by islets in vitro. The MSC-CM-mediated induction of β cell proliferation was completely blocked by the PI3K/Akt inhibitor LY294002 but not by the MEK/Erk inhibitor PD98059. Together, these data suggest that the PI3K/Akt signal pathway plays a critical role in β cell proliferation after MSC transplantation.

  12. 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 ...

  13. The Efficacy of an Immunoisolating Membrane System for Islet Xenotransplantation in Minipigs

    PubMed Central

    Weir, Gordon C.; Colton, Clark K.; Evron, Yoav; Balyura, Maria; Yavriyants, Karina; Zimermann, Baruch; Azarov, Dmitri; Maimon, Shiri; Shabtay, Noa; Rozenshtein, Tania; Lorber, Dana; Steffen, Anja; Willenz, Udi; Bloch, Konstantine; Vardi, Pnina; Taube, Ran; de Vos, Paul; Lewis, Eli C.; Bornstein, Stefan R.; Rotem, Avi

    2013-01-01

    Developing a device that protects xenogeneic islets to allow treatment and potentially cure of diabetes in large mammals has been a major challenge in the past decade. Using xenogeneic islets for transplantation is required in light of donor shortage and the large number of diabetic patients that qualify for islet transplantation. Until now, however, host immunoreactivity against the xenogeneic graft has been a major drawback for the use of porcine islets. Our study demonstrates the applicability of a novel immunoprotective membrane that allows successful xenotransplantation of rat islets in diabetic minipigs without immunosuppressive therapy. Rat pancreatic islets were encapsulated in highly purified alginate and integrated into a plastic macrochamber covered by a poly-membrane for subcutaneous transplantation. Diabetic Sinclair pigs were transplanted and followed for up to 90 days. We demonstrated a persistent graft function and restoration of normoglycemia without the need for immunosuppressive therapy. This concept could potentially offer an attractive strategy for a more widespread islet replacement therapy that would restore endogenous insulin secretion in diabetic patients without the need for immunosuppressive drugs and may even open up an avenue for safe utilization of xenogeneic islet donors. PMID:23936385

  14. Characterization of the Human Pancreatic Islet Proteome by Two-Dimensional LC/MS/MS

    SciTech Connect

    Metz, Thomas O.; Jacobs, Jon M.; Gritsenko, Marina A.; Fontes, Ghislaine; Qian, Weijun; Camp, David G.; Poitout, Vincent J.; Smith, Richard D.

    2006-12-01

    Research to elucidate the pathogenesis of type 1 diabetes mellitus has traditionally focused on the genetic and immunological factors associated with the disease, and, until recently, has not considered the target cell. While there have been reports detailing proteomic analyses of established islet cell lines or isolated rodent islets, the information gained is not always easily extrapolated to humans. Therefore, extensive characterization of the human islet proteome could result in better understanding of islet biology and lead to more effective treatment strategies. We have applied a two-dimensional LC-MS/MS-based analysis to the characterization of the human islet proteome, resulting in the detection of 29,021 unique peptides corresponding to 4,925 proteins. As expected, major islet hormones (insulin, glucagon, somatostatin), beta-cell enriched secretory products (IAPP), ion channels (K-ATP channel), and transcription factors (PDX-1, Nkx 6.1, HNF-1 beta) were detected. In addition, significant proteome coverage of metabolic enzymes and cellular pathways was obtained, including the insulin signaling cascade and the MAP kinase, NF-κβ, and JAK/STAT pathways. This work represents the most extensive characterization of the human islet proteome to date and provides a peptide reference library that may be utilized in future studies of islet biology and type 1 diabetes.

  15. Phase transitions in pancreatic islet cellular networks and implications for type-1 diabetes

    NASA Astrophysics Data System (ADS)

    Stamper, I. J.; Jackson, Elais; Wang, Xujing

    2014-01-01

    In many aspects the onset of a chronic disease resembles a phase transition in a complex dynamic system: Quantitative changes accumulate largely unnoticed until a critical threshold is reached, which causes abrupt qualitative changes of the system. In this study we examine a special case, the onset of type-1 diabetes (T1D), a disease that results from loss of the insulin-producing pancreatic islet β cells. Within each islet, the β cells are electrically coupled to each other via gap-junctional channels. This intercellular coupling enables the β cells to synchronize their insulin release, thereby generating the multiscale temporal rhythms in blood insulin that are critical to maintaining blood glucose homeostasis. Using percolation theory we show how normal islet function is intrinsically linked to network connectivity. In particular, the critical amount of β-cell death at which the islet cellular network loses site percolation is consistent with laboratory and clinical observations of the threshold loss of β cells that causes islet functional failure. In addition, numerical simulations confirm that the islet cellular network needs to be percolated for β cells to synchronize. Furthermore, the interplay between site percolation and bond strength predicts the existence of a transient phase of islet functional recovery after onset of T1D and introduction of treatment, potentially explaining the honeymoon phenomenon. Based on these results, we hypothesize that the onset of T1D may be the result of a phase transition of the islet β-cell network.

  16. Impact of an autologous oxygenating matrix culture system on rat islet transplantation outcome.

    PubMed

    Schaschkow, A; Mura, C; Bietiger, W; Peronet, C; Langlois, A; Bodin, F; Dissaux, C; Bruant-Rodier, C; Pinget, M; Jeandidier, N; Juszczak, M T; Sigrist, S; Maillard, E

    2015-06-01

    Disruption of the pancreatic islet environment combined with the decrease in oxygen supply that occurs during isolation leads to poor islet survival. The aim of this study was to validate the benefit of using a plasma-based scaffold supplemented with perfluorodecalin to improve islet transplantation outcome. Rat islets were cultured in three conditions: i) control group, ii) plasma based-matrix (P-matrix), and iii) P-matrix supplemented with emulsified perfluorodecalin. After 24 h culture, matrix/cell contacts (Integrinβ1, p-FAK/FAK, p-Akt/Akt), survival (caspase 3, TUNEL, FDA/PI), function, and HIF-1α translocation were assessed. Afterwards, P-matrices were dissolved and the islets were intraportally transplanted. Graft function was monitored for 31 days with glycaemia and C-peptide follow up. Inflammation was assessed by histology (macrophage and granulocyte staining) and thrombin/anti-thrombin complex measurement. Islet survival correlated with an increase in integrin, FAK, and Akt activation in P-matrices and function was maintained. Perfluorodecalin supplementation decreased translocation of HIF-1α in the nucleus and post-transplantation islet structure was better preserved in P-matrices, but a quicker activation of IBMIR resulted in early loss of graft function. "Oxygenating" P-matrices provided a real benefit to islet survival and resistance in vivo. However, intraportal transplantation is not suitable for this kind of culture due to IBMIR; thus, alternative sites must be explored.

  17. Advanced Glycation End-Products Induce Apoptosis in Pancreatic Islet Endothelial Cells via NF-κB-Activated Cyclooxygenase-2/Prostaglandin E2 Up-Regulation

    PubMed Central

    Lan, Kuo-Cheng; Chiu, Chen-Yuan; Kao, Chia-Wei; Huang, Kuo-How; Wang, Ching-Chia; Huang, Kuo-Tong; Tsai, Keh-Sung

    2015-01-01

    Microvascular complications eventually affect nearly all patients with diabetes. Advanced glycation end-products (AGEs) resulting from hyperglycemia are a complex and heterogeneous group of compounds that accumulate in the plasma and tissues in diabetic patients. They are responsible for both endothelial dysfunction and diabetic vasculopathy. The aim of this study was to investigate the cytotoxicity of AGEs on pancreatic islet microvascular endothelial cells. The mechanism underlying the apoptotic effect of AGEs in pancreatic islet endothelial cell line MS1 was explored. The results showed that AGEs significantly decreased MS1 cell viability and induced MS1 cell apoptosis in a dose-dependent manner. AGEs dose-dependently increased the expressions of cleaved caspase-3, and cleaved poly (ADP-ribose) polymerase in MS1 cells. Treatment of MS1 cells with AGEs also resulted in increased nuclear factor (NF)-κB-p65 phosphorylation and cyclooxygenase (COX)-2 expression. However, AGEs did not affect the expressions of endoplasmic reticulum (ER) stress-related molecules in MS1 cells. Pretreatment with NS398 (a COX-2 inhibitor) to inhibit prostaglandin E2 (PGE2) production reversed the induction of cleaved caspase-3, cleaved PARP, and MS1 cell viability. Moreover, AGEs significantly increased the receptor for AGEs (RAGE) protein expression in MS1 cells, which could be reversed by RAGE neutralizing antibody. RAGE Neutralizing antibody could also reverse the induction of cleaved caspase-3 and cleaved PARP and decreased cell viability induced by AGEs. These results implicate the involvement of NF-κB-activated COX-2/PGE2 up-regulation in AGEs/RAGE-induced islet endothelial cell apoptosis and cytotoxicity. These findings may provide insight into the pathological processes within the pancreatic islet microvasculature induced by AGEs accumulation. PMID:25898207

  18. Encapsulated islets for diabetes therapy: history, current progress, and critical issues requiring solution.

    PubMed

    Scharp, David W; Marchetti, Piero

    2014-04-01

    Insulin therapy became a reality in 1921 dramatically saving lives of people with diabetes, but not protecting them from long-term complications. Clinically successful free islet implants began in 1989 but require life long immunosuppression. Several encapsulated islet approaches have been ongoing for over 30 years without defining a clinically relevant product. Macro-devices encapsulating islet mass in a single device have shown long-term success in large animals but human trials have been limited by critical challenges. Micro-capsules using alginate or similar hydrogels encapsulate individual islets with many hundreds of promising rodent results published, but a low incidence of successful translation to large animal and human results. Reduction of encapsulated islet mass for clinical transplantation is in progress. This review covers the status of both early and current studies including the presentation of corporate efforts involved. It concludes by defining the critical items requiring solution to enable a successful clinical diabetes therapy.

  19. Gene expression profiles for the human pancreas and purified islets in Type 1 diabetes: new findings at clinical onset and in long-standing diabetes

    PubMed Central

    Planas, R; Carrillo, J; Sanchez, A; Ruiz de Villa, M C; Nuñez, F; Verdaguer, J; James, R F L; Pujol-Borrell, R; Vives-Pi, M

    2010-01-01

    Type 1 diabetes (T1D) is caused by the selective destruction of the insulin-producing β cells of the pancreas by an autoimmune response. Due to ethical and practical difficulties, the features of the destructive process are known from a small number of observations, and transcriptomic data are remarkably missing. Here we report whole genome transcript analysis validated by quantitative reverse transcription–polymerase chain reaction (qRT–PCR) and correlated with immunohistological observations for four T1D pancreases (collected 5 days, 9 months, 8 and 10 years after diagnosis) and for purified islets from two of them. Collectively, the expression profile of immune response and inflammatory genes confirmed the current views on the immunopathogenesis of diabetes and showed similarities with other autoimmune diseases; for example, an interferon signature was detected. The data also supported the concept that the autoimmune process is maintained and balanced partially by regeneration and regulatory pathway activation, e.g. non-classical class I human leucocyte antigen and leucocyte immunoglobulin-like receptor, subfamily B1 (LILRB1). Changes in gene expression in islets were confined mainly to endocrine and neural genes, some of which are T1D autoantigens. By contrast, these islets showed only a few overexpressed immune system genes, among which bioinformatic analysis pointed to chemokine (C-C motif) receptor 5 (CCR5) and chemokine (CXC motif) receptor 4) (CXCR4) chemokine pathway activation. Remarkably, the expression of genes of innate immunity, complement, chemokines, immunoglobulin and regeneration genes was maintained or even increased in the long-standing cases. Transcriptomic data favour the view that T1D is caused by a chronic inflammatory process with a strong participation of innate immunity that progresses in spite of the regulatory and regenerative mechanisms. PMID:19912253

  20. Islet Endothelial Cells Derived From Mouse Embryonic Stem Cells.

    PubMed

    Jain, Neha; Lee, Eun Jung

    2016-01-01

    The islet endothelium comprises a specialized population of islet endothelial cells (IECs) expressing unique markers such as nephrin and α-1 antitrypsin (AAT) that are not found in endothelial cells in surrounding tissues. However, due to difficulties in isolating and maintaining a pure population of these cells, the information on these islet-specific cells is currently very limited. Interestingly, we have identified a large subpopulation of endothelial cells exhibiting IEC phenotype, while deriving insulin-producing cells from mouse embryonic stem cells (mESCs). These cells were identified by the uptake of low-density lipoprotein (LDL) and were successfully isolated and subsequently expanded in endothelial cell culture medium. Further analysis demonstrated that the mouse embryonic stem cell-derived endothelial cells (mESC-ECs) not only express classical endothelial markers, such as platelet endothelial cell adhesion molecule (PECAM1), thrombomodulin, intercellular adhesion molecule-1 (ICAM-1), and endothelial nitric oxide synthase (eNOS) but also IEC-specific markers such as nephrin and AAT. Moreover, mESC-ECs secrete basement membrane proteins such as collagen type IV, laminin, and fibronectin in culture and form tubular networks on a layer of Matrigel, demonstrating angiogenic activity. Further, mESC-ECs not only express eNOS, but also its eNOS expression is glucose dependent, which is another characteristic phenotype of IECs. With the ability to obtain highly purified IECs derived from pluripotent stem cells, it is possible to closely examine the function of these cells and their interaction with pancreatic β-cells during development and maturation in vitro. Further characterization of tissue-specific endothelial cell properties may enhance our ability to formulate new therapeutic angiogenic approaches for diabetes.

  1. Suppression of islet homeostasis protein thwarts diabetes mellitus progression.

    PubMed

    Oh, Seh-Hoon; Jorgensen, Marda L; Wasserfall, Clive H; Gjymishka, Altin; Petersen, Bryon E

    2017-02-20

    During progression to type 1 diabetes, insulin-producing β-cells are lost through an autoimmune attack resulting in unrestrained glucagon expression and secretion, activation of glycogenolysis, and escalating hyperglycemia. We recently identified a protein, designated islet homeostasis protein (IHoP), which specifically co-localizes within glucagon-positive α-cells and is overexpressed in the islets of both post-onset non-obese diabetic (NOD) mice and type 1 diabetes patients. Here we report that in the αTC1.9 mouse α-cell line, IHoP was released in response to high-glucose challenge and was found to regulate secretion of glucagon. We also show that in NOD mice with diabetes, major histocompatibility complex class II was upregulated in islets. In addition hyperglycemia was modulated in NOD mice via suppression of IHoP utilizing small interfering RNA (IHoP-siRNA) constructs/approaches. Suppression of IHoP in the pre-diabetes setting maintained normoglycemia, glyconeolysis, and fostered β-cell restoration in NOD mice 35 weeks post treatment. Furthermore, we performed adoptive transfer experiments using splenocytes from IHoP-siRNA-treated NOD/ShiLtJ mice, which thwarted the development of hyperglycemia and the extent of insulitis seen in recipient mice. Last, IHoP can be detected in the serum of human type 1 diabetes patients and could potentially serve as an early novel biomarker for type 1 diabetes in patients.Laboratory Investigation advance online publication, 20 February 2017; doi:10.1038/labinvest.2017.15.

  2. Modeling K,ATP-Dependent Excitability in Pancreatic Islets

    PubMed Central

    Silva, Jonathan R.; Cooper, Paige; Nichols, Colin G.

    2014-01-01

    In pancreatic β-cells, K,ATP channels respond to changes in glucose to regulate cell excitability and insulin release. Confirming a high sensitivity of electrical activity to K,ATP activity, mutations that cause gain of K,ATP function cause neonatal diabetes. Our aim was to quantitatively assess the contribution of K,ATP current to the regulation of glucose-dependent bursting by reproducing experimentally observed changes in excitability when K,ATP conductance is altered by genetic manipulation. A recent detailed computational model of single cell pancreatic β-cell excitability reproduces the β-cell response to varying glucose concentrations. However, initial simulations showed that the model underrepresents the significance of K,ATP activity and was unable to reproduce K,ATP conductance-dependent changes in excitability. By altering the ATP and glucose dependence of the L-type Ca2+ channel and the Na-K ATPase to better fit experiment, appropriate dependence of excitability on K,ATP conductance was reproduced. Because experiments were conducted in islets, which contain cell-to-cell variability, we extended the model from a single cell to a three-dimensional model (10×10×10 cell) islet with 1000 cells. For each cell, the conductance of the major currents was allowed to vary as was the gap junction conductance between cells. This showed that single cell glucose-dependent behavior was then highly variable, but was uniform in coupled islets. The study highlights the importance of parameterization of detailed models of β-cell excitability and suggests future experiments that will lead to improved characterization of β-cell excitability and the control of insulin secretion. PMID:25418087

  3. The tick (Acari: Ixodidae) fauna of Herald's Beacon Islet, Australia.

    PubMed

    Kwak, Mackenzie L; Mintram, Kate

    2017-01-01

    A rare opportunity to travel to Herald's Beacon Islet with permission from the Australian government to collect ticks allowed for a survey of the tick fauna of the island to be undertaken for the first time. The avian fauna of the island, which serve as hosts, was also recorded and includes one new species record for the island. The seabird soft tick Ornithodoros capensis Neumann and the seabird hard tick Amblyomma loculosum Neumann were found to be present on the island. Images of the ticks present on the island are presented along with morphological characters for their identification.

  4. Preservation of beta cell function after pancreatic islet autotransplantation: University of Chicago experience.

    PubMed

    Savari, Omid; Golab, Karolina; Wang, Ling-Jia; Schenck, Lindsay; Grose, Randall; Tibudan, Martin; Ramachandran, Sabarinathan; Chon, W James; Posner, Mitchell C; Millis, J Michael; Matthews, Jeffrey B; Gelrud, Andres; Witkowski, Piotr

    2015-04-01

    The aim of the study was to assess the rate of insulin independence in patients after total pancreatectomy (TP) and islet autotransplantation in our center. TP followed by islet autotransplantation was performed in 10 patients. Severe unrelenting pain associated with chronic pancreatitis was the major indication for surgery. Islets were isolated using the modified Ricordi method and infused through the portal vein. Exogenous insulin therapy was implemented for at least two months posttransplant to support islet engraftment and was subsequently weaned off, if possible. Median follow-up was 26 months (range, 2 to 60 months). Median islet yield was 158,860 islet equivalents (IEQ) (range, 40,203 to 330,472 IEQ) with an average islet yield of 2,478 IEQ/g (range, 685 to 6,002 IEQ/g) of processed pancreas. One patient developed transient partial portal vein thrombosis, which resolved without sequela. Five (50%) patients are currently off insulin with excellent glucose control and HbA1c below 6. Patients who achieved and maintained insulin independence were transplanted with significantly more islets (median, 202,291 IEQ; range, 145,000 to 330,474 IEQ) than patients who required insulin support (64,348 IEQ; range, 40,203 to 260,476 IEQ; P < 0.05). Patient body mass index and time of chronic pancreatitis prior transplant procedure did not correlate with the outcome. The remaining five patients, who require insulin support, had present C-peptide in blood and experience good glucose control without incidence of severe hypoglycemic episodes. Islet autotransplantation efficiently preserved beta cell function in selected patients with chronic pancreatitis and the outcome correlated with transplanted islet mass.

  5. Assessment of DNA synthesis in Islet-1{sup +} cells in the adult murine heart

    SciTech Connect

    Weinberger, Florian Mehrkens, Dennis Starbatty, Jutta Nicol, Philipp Eschenhagen, Thomas

    2015-01-02

    Highlights: • Islet-1 was expressed in the adult heart. • Islet-1-positive cells did not proliferate in the adult heart. • Sinoatrial node cells did not proliferate in the adult heart. - Abstract: Rationale: Islet-1 positive (Islet-1{sup +}) cardiac progenitor cells give rise to the right ventricle, atria and outflow tract during murine cardiac development. In the adult heart Islet-1 expression is limited to parasympathetic neurons, few cardiomyocytes, smooth muscle cells, within the proximal aorta and pulmonary artery and sinoatrial node cells. Its role in these cells is unknown. Here we tested the hypothesis that Islet-1{sup +} cells retain proliferative activity and may therefore play a role in regenerating specialized regions in the heart. Methods and results: DNA synthesis was analyzed by the incorporation of tritiated thymidine ({sup 3}H-thymidine) in Isl-1-nLacZ mice, a transgenic model with an insertion of a nuclear beta-galactosidase in the Islet-1 locus. Mice received daily injections of {sup 3}H-thymidine for 5 days. DNA synthesis was visualized throughout the heart by dipping autoradiography of cryosections. Colocalization of an nLacZ-signal and silver grains would indicate DNA synthesis in Islet-1{sup +} cells. Whereas Islet{sup −} non-myocyte nuclei were regularly marked by accumulation of silver grains, colocalization with nLacZ-signals was not detected in >25,000 cells analyzed. Conclusions: Islet-1{sup +} cells are quiescent in the adult heart, suggesting that, under normal conditions, even pacemaking cells do not proliferate at higher rates than normal cardiac myocytes.

  6. Gamma irradiation of isolated rat islets pretransplantation produces indefinite allograft survival in cyclosporine-treated recipients

    SciTech Connect

    James, R.F.; Lake, S.P.; Chamberlain, J.; Thirdborough, S.; Bassett, P.D.; Mistry, N.; Bell, P.R.

    1989-06-01

    In this study we have examined the use of low-dose gamma-irradiation for the reduction of islet immunogenicity in the strong allogeneic combination of WAG rat islets transplanted into diabetic AUG recipients. First, we determined that gamma-irradiation reduced immunogenicity in vitro by use of a modified MLR with WAG islets as stimulators and AUG splenocytes as responders. We then determined the maximum dose of gamma-irradiation that could be used (250 rads) before islet function was affected. As 250 rads islet pretreatment alone was ineffective in prolonging allograft survival, we combined the pretreatment with a short course (days 0, 1, 2; 30 mg/kg) of cyclosporine. We found that CsA was only effective in significantly prolonging allograft survival when given subcutaneously in olive oil. The CsA treatment alone gave a significantly prolonged survival time for the islet allografts (median, 37 days vs. 6 days for controls), but when combined with the 250 rads islet pretreatment a synergistic effect was seen with 100% becoming long-term survivors (greater than 100 days). The long-term surviving AUG rats from both the CsA alone group and the CsA plus 250 rads pretreated islets group were challenged with WAG dendritic cells (DC). The islets from the 250 rads pretreated group were subsequently rejected (day 6) while the CsA alone group were not affected. The role of low dose gamma-irradiation when combined with CsA treatment of islet graft recipients in inducing specific unresponsiveness will be discussed.

  7. Measurements of oxygen tension in native and transplanted rat pancreatic islets.

    PubMed

    Carlsson, P O; Liss, P; Andersson, A; Jansson, L

    1998-07-01

    This study was performed to measure the oxygen tension before and after revascularization of pancreatic islets transplanted beneath the renal capsule and to investigate to what extent this was affected by acute and chronic hyperglycemia. In addition, the oxygen tension in islets within the pancreas was determined. PO2 was measured with a modified Clark electrode (tip 2-6 microm o.d.). Within native pancreatic islets, the mean PO2 was higher (31-37 mmHg) than within the exocrine pancreas (20-23 mmHg). The mean oxygen tension in the transplanted islets the day after implantation was half of that recorded in native islets (14-19 mmHg) and did not differ between normoglycemic and diabetic recipients. At 1 month after transplantation, when revascularization had occurred, the mean PO2 in the islet grafts was 9-15 mmHgf in normoglycemic animals but was lower (6-8 mmHg) in diabetic animals, whereas the blood perfusion of the transplants, as measured with laser-Doppler flowmetry (probe diameter 0.45 mm), was similar in both groups. The mean oxygen tension in the superficial renal cortex surrounding the implanted islets was similar in all groups and remained stable at 13-21 mmHg. Intravenous administration of D-glucose (1 g/kg) did not affect the oxygen tension in any of the investigated tissues. We conclude that the mean PO2 in islets implanted under the renal capsule is markedly lower than in native islets, not only in the immediate posttransplantation period but also 1 month after implantation, i.e., when revascularization has occurred. Furthermore, persistent hyperglycemia in the recipient leads to a further decrease in graft oxygen tension. To what extent this may contribute to islet graft failure is at present unknown.

  8. Two distinct mechanisms mediate the involvement of bone marrow cells in islet remodeling: neogenesis of insulin-producing cells and support of islet recovery.

    PubMed

    Iskovich, Svetlana; Goldenberg-Cohen, Nitza; Sadikov, Tamila; Yaniv, Isaac; Stein, Jerry; Askenasy, Nadir

    2015-01-01

    We have recently reported that small-sized bone marrow cells (BMCs) isolated by counterflow centrifugal elutriation and depleted of lineage markers (Fr25lin(-)) have the capacity to differentiate and contribute to regeneration of injured islets. In this study, we assess some of the characteristics of these cells compared to elutriated hematopoietic progenitors (R/O) and whole BMCs in a murine model of streptozotocin-induced chemical diabetes. The GFP(bright)CD45(+) progeny of whole BMCs and R/O progenitors progressively infiltrate the pancreas with evolution of donor chimerism; are found at islet perimeter, vascular, and ductal walls; and have a modest impact on islet recovery from injury. In contrast, Fr25lin(-) cells incorporate in the islets, convert to GFP(dim)CD45(-)PDX-1(+) phenotypes, produce proinsulin, and secrete insulin with significant contribution to stabilization of glucose homeostasis. The elutriated Fr25lin(-) cells express low levels of CD45 and are negative for SCA-1 and c-kit, as removal of cells expressing these markers did not impair conversion to produce insulin. BMCs mediate two synergistic mechanisms that contribute to islet recovery from injury: support of islet remodeling by hematopoietic cells and neogenesis of insulin-producing cells from stem cells.

  9. Novel sulfated glucomannan-barium-alginate microcapsules in islet transplantation: significantly decreased the secretion of monocyte chemotactic protein 1 and improved the activity of islet in rats.

    PubMed

    Chen, X; Zhang, L; Qi, Z; Guo, B; Zhong, L; Shen, B; Yan, Z; Zhang, J

    2009-12-01

    The sulfated glucomannan can be used to filter the heparin-binding properties of cytokines. In this study, novel sulfated glucomannan-barium-alginate (SGA) microcapsules were prepared to encapsulate islets with barium-alginate (ABa) and calcium alginate-poly-l-lysine (APA) microcapsules as controls. SD rat islets were purified as donor cells to Lewis rats that had been treated with streptozotocin. Intraperitoneal transplantation was performed with about 3000 islet equivalent (IEQ) rat. At week three after transplantation, the concentrations of monocyte chemotactic protein-1 (MCP-1), interleukin (IL)-1 beta, interferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha in intraperitoneal fluid were determined using ELISA. At week 8, the islet cell mass in the abdominal microcapsules was excised to test insulin release. The EB-FDA fluorescence staining method was used to observe the functional activity of the islet cells. Compared with ABa and APA microcapsules, SGA microcapsules showed significantly decreased MCP-1 secretion by beta-cells. Also, the concentrations of cytokines IL-1beta, IFN-gamma, and TNF-alpha were decreased significantly. The activity of the transplanted islets was significantly improved in SGA microcapsules, which shielded against cytokines better than ABa or APA microcapsules and may serve as novel method.

  10. QUANTITATIVE ASSESSMENT OF BETA CELL APOPTOSIS AND CELL COMPOSITION OF ISOLATED, UNDISRUPTED HUMAN ISLETS BY LASER SCANNING CYTOMETRY

    PubMed Central

    Todorov, Ivan; Nair, Indu; Avakian-Mansoorian, Alina; Rawson, Jeffrey; Omori, Keiko; Ito, Taihei; Valiente, Luis; Iglesias-Meza, Itzia; Orr, Chris; Shiang, Keh D.; Ferreri, Kevin; Al-Abdullah, Ismail H.; Mullen, Yoko; Kandeel, Fouad

    2010-01-01

    Background Assays for assessing human islet cell quality which provide results prior to transplantation would be very beneficial to improving outcomes for islet transplantation therapy. Parameters such as percent beta cell apoptosis and cell composition are found to vary markedly between different islet preparations, and may serve as markers of islet quality. We have developed fluorescence-based assays using laser scanning cytometry (LSC) for assessing beta cell apoptosis and islet cell composition on serial sections of intact isolated islets. Methods Isolated human islets were fixed in formalin and embedded in paraffin. Serial sections were immunostained for the pancreatic hormones, acinar and ductal cell markers. DNA fragmentation was used to label apoptotic cells. Stained cells were quantified using an iCys laser scanning cytometer. Results Islet preparations from 102 human pancreatic islet isolations were analyzed. For the whole set of islet preparations we found a mean islet cell composition of 54.5±1.2% insulin positive; 33.9±1.2% glucagon; 12.1±0.7% somatostatin and 1.5±0.2% pancreatic polypeptide positive cells. The apoptotic beta cells were 2.85±0.4% with a range of 0.27% to 18.3%. The percentage of apoptotic beta cells correlated well (p<0.0001, n=59) with results obtained in vivo by transplantation of the corresponding islets in diabetic NODscid mice. Conclusions The analysis of whole, non-dissociated islets for cell composition and beta cell apoptosis using LSC is giving reliable and reproducible results and could be done both before islet transplantation, as well as on preserved cell blocks at any future time. Thus, they can be a powerful tool for islet quality assessment. PMID:20697327

  11. A novel glucagon-like peptide-1 (GLP-1)/glucagon hybrid peptide with triple-acting agonist activity at glucose-dependent insulinotropic polypeptide, GLP-1, and glucagon receptors and therapeutic potential in high fat-fed mice.

    PubMed

    Gault, Victor A; Bhat, Vikas K; Irwin, Nigel; Flatt, Peter R

    2013-12-06

    Glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), and glucagon bind to related members of the same receptor superfamily and exert important effects on glucose homeostasis, insulin secretion, and energy regulation. The present study assessed the biological actions and therapeutic utility of novel GIP/glucagon/GLP-1 hybrid peptides. Nine novel peptides were synthesized and exhibited complete DPP-IV resistance and enhanced in vitro insulin secretion. The most promising peptide, [dA(2)]GLP-1/GcG, stimulated cAMP production in GIP, GLP-1, and glucagon receptor-transfected cells. Acute administration of [dA(2)]GLP-1/GcG in combination with glucose significantly lowered plasma glucose and increased plasma insulin in normal and obese diabetic (ob/ob) mice. Furthermore, [dA(2)]GLP-1/GcG elicited a protracted glucose-lowering and insulinotropic effect in high fat-fed mice. Twice daily administration of [dA(2)]GLP-1/GcG for 21 days decreased body weight and nonfasting plasma glucose and increased circulating plasma insulin concentrations in high fat-fed mice. Furthermore, [dA(2)]GLP-1/GcG significantly improved glucose tolerance and insulin sensitivity by day 21. Interestingly, locomotor activity was increased in [dA(2)]GLP-1/GcG mice, without appreciable changes in aspects of metabolic rate. Studies in knock-out mice confirmed the biological action of [dA(2)]GLP-1/GcG via multiple targets including GIP, GLP-1, and glucagon receptors. The data suggest significant promise for novel triple-acting hybrid peptides as therapeutic options for obesity and diabetes.

  12. Statistical approaches and software for clustering islet cell functional heterogeneity

    PubMed Central

    Wills, Quin F.; Boothe, Tobias; Asadi, Ali; Ao, Ziliang; Warnock, Garth L.; Kieffer, Timothy J.

    2016-01-01

    ABSTRACT Worldwide efforts are underway to replace or repair lost or dysfunctional pancreatic β-cells to cure diabetes. However, it is unclear what the final product of these efforts should be, as β-cells are thought to be heterogeneous. To enable the analysis of β-cell heterogeneity in an unbiased and quantitative way, we developed model-free and model-based statistical clustering approaches, and created new software called TraceCluster. Using an example data set, we illustrate the utility of these approaches by clustering dynamic intracellular Ca2+ responses to high glucose in ∼300 simultaneously imaged single islet cells. Using feature extraction from the Ca2+ traces on this reference data set, we identified 2 distinct populations of cells with β-like responses to glucose. To the best of our knowledge, this report represents the first unbiased cluster-based analysis of human β-cell functional heterogeneity of simultaneous recordings. We hope that the approaches and tools described here will be helpful for those studying heterogeneity in primary islet cells, as well as excitable cells derived from embryonic stem cells or induced pluripotent cells. PMID:26909740

  13. Islet-1 is required for ventral neuron survival in Xenopus

    SciTech Connect

    Shi, Yu; Zhao, Shuhua; Li, Jiejing; Mao, Bingyu

    2009-10-23

    Islet-1 is a LIM domain transcription factor involved in several processes of embryonic development. Xenopus Islet-1 (Xisl-1) has been shown to be crucial for proper heart development. Here we show that Xisl-1 and Xisl-2 are differentially expressed in the nervous system in Xenopus embryos. Knock-down of Xisl-1 by specific morpholino leads to severe developmental defects, including eye and heart failure. Staining with the neuronal markers N-tubulin and Xisl-1 itself reveals that the motor neurons and a group of ventral interneurons are lost in the Xisl-1 morphants. Terminal dUTP nick-end labeling (TUNEL) analysis shows that Xisl-1 morpholino injection induces extensive apoptosis in the ventral neural plate, which can be largely inhibited by the apoptosis inhibitor M50054. We also find that over-expression of Xisl-1 is able to promote cell proliferation and induce Xstat3 expression in the injected side, suggesting a potential role for Xisl-1 in the regulation of cell proliferation in co-operation with the Jak-Stat pathway.

  14. Functional identification of islet cell types by electrophysiological fingerprinting

    PubMed Central

    Zhang, Quan; Vergari, Elisa; Kellard, Joely A.; Rodriguez, Blanca; Ashcroft, Frances M.; Rorsman, Patrik

    2017-01-01

    The α-, β- and δ-cells of the pancreatic islet exhibit different electrophysiological features. We used a large dataset of whole-cell patch-clamp recordings from cells in intact mouse islets (N = 288 recordings) to investigate whether it is possible to reliably identify cell type (α, β or δ) based on their electrophysiological characteristics. We quantified 15 electrophysiological variables in each recorded cell. Individually, none of the variables could reliably distinguish the cell types. We therefore constructed a logistic regression model that included all quantified variables, to determine whether they could together identify cell type. The model identified cell type with 94% accuracy. This model was applied to a dataset of cells recorded from hyperglycaemic βV59M mice; it correctly identified cell type in all cells and was able to distinguish cells that co-expressed insulin and glucagon. Based on this revised functional identification, we were able to improve conductance-based models of the electrical activity in α-cells and generate a model of δ-cell electrical activity. These new models could faithfully emulate α- and δ-cell electrical activity recorded experimentally. PMID:28275121

  15. Quantifying the carboxylation of pyruvate in pancreatic islets.

    PubMed

    Khan, A; Ling, Z C; Landau, B R

    1996-02-02

    Pyruvate has been estimated to enter the citric acid cycle in islets by carboxylation to the same extent or more than by decarboxylation. Those estimates were made assuming the dimethyl esters of [1,4-14C]succinate and [2-3-14C]succinate, incubated with islets at a concentration of 10 mM, gave the same ratio of 14CO2 yields as if [1-14C]acetate and [2-14C]acetate had been incubated. The labeled succinates, at 10 mM, but not 1 mM, are now shown to give ratios higher than the labeled acetates at those concentrations and therefore higher estimates when related to yields from [2-14C]glucose and [6-14C]glucose. Using the labeled acetate ratios in paired incubations, the rate of pyruvate carboxylation is still estimated to be about two-thirds the rate of pyruvate decarboxylation. Participation of the malic enzyme-catalyzed reaction explains the greater ratio of yields of 14CO2 from the succinates at 10 mM than 1 mM and increases in those ratios on glucose addition and can account for the removal from the citric acid cycle of oxaloacetate carbon formed in the carboxylation.

  16. Phosphatidylcholine biosynthesis and insulin release in rat islets of Langerhans

    SciTech Connect

    Hoffman, J.M.

    1988-01-01

    Turnover of phosphatidylcholine (PC) has been demonstrated to play a role in glucose stimulation of insulin release by pancreatic islets of Langerhans. The activity of the islet CDP-choline pathway of PC synthesis was determined by measuring the incorporation of radiolabeled choline or {sup 32}PO{sub 4} into PC, phosphorylcholine and CDP-choline. Concurrently, insulin release was measured by radioimmunoassay to correlate insulin release and PC synthesis. Glucose concentrations greater than 8.5 mM stimulated CDP-choline pathway activity. However, measurement of PC lipid phosphorus tended to decrease, suggesting that stimulation of the CDP-choline pathway was a means of replenishing PC pools diminished by hydrolysis of PC. Inhibition of glucose oxidation by mannoheptulose or incubations under hypoxic conditions prevented stimulation of the CDP-choline pathway, while inhibition of phospholipase A{sub 2} (PLA{sub 2}) and secretion by the removal of extracellular Ca{sup 2+} potentiated the stimulation seen with glucose.

  17. Gene transfer of manganese superoxide dismutase extends islet graft function in a mouse model of autoimmune diabetes.

    PubMed

    Bertera, Suzanne; Crawford, Megan L; Alexander, Angela M; Papworth, Glenn D; Watkins, Simon C; Robbins, Paul D; Trucco, Massimo

    2003-02-01

    Islet transplantation is a promising cure for diabetes. However, inflammation, allorejection, and recurrent autoimmune damage all may contribute to early graft loss. Pancreatic islets express lower levels of antioxidant genes than most other tissues of the body, and beta-cells in particular are sensitive to oxidative damage. Therefore, damage from oxidative stress may pose a major obstacle to islet replacement therapy in that both the islet isolation and transplantation processes generate oxygen radicals. To determine whether antioxidant gene overexpression in isolated pancreatic islets can prevent oxidative damage and prolong islet function after transplantation, we used the NOD mouse model to study oxidative stress encountered during both transplantation and autoimmune attack. We transferred an antioxidant gene, manganese superoxide dismutase (MnSOD), by adenoviral infection into isolated islets that were transplanted into streptozotocin-treated NODscid recipient mice. Functioning islet grafts were subsequently exposed to diabetogenic spleen cells and monitored until graft failure. The results show that islet grafts overexpressing MnSOD functioned approximately 50% longer than control grafts. This significant prolongation of graft function suggests that the antioxidant activity of MnSOD is beneficial to transplanted islet survival and may be used in combination with other strategies aimed at islet graft protection.

  18. Islet Endothelial Cells Induce Glycosylation and Increase Cell-surface Expression of Integrin β1 in β Cells*

    PubMed Central

    Spelios, Michael G.; Olsen, John A.; Kenna, Lauren A.; Akirav, Eitan M.

    2015-01-01

    The co-culturing of insulinoma and islet-derived endothelial cell (iEC) lines results in the spontaneous formation of free-floating pseudoislets (PIs). We previously showed that iEC-induced PIs display improved insulin expression and secretion in response to glucose stimulation. This improvement was associated with a de novo deposition of extracellular matrix (ECM) proteins by iECs in and around the PIs. Here, iEC-induced PIs were used to study the expression and posttranslational modification of the ECM receptor integrin β1. A wide array of integrin β subunits was detected in βTC3 and NIT-1 insulinomas as well as in primary islets, with integrin β1 mRNA and protein detected in all three cell types. Interestingly, the formation of iEC-induced PIs altered the glycosylation patterns of integrin β1, resulting in a higher molecular weight form of the receptor. This form was found in native pancreas but was completely absent in monolayer β-cells. Fluorescence-activated cell sorting analysis of monolayers and PIs revealed a higher expression of integrin β1 in PIs. Antibody-mediated blocking of integrin β1 led to alterations in β-cell morphology, reduced insulin gene expression, and enhanced glucose secretion under baseline conditions. These results suggest that iEC-induced PI formation may alter integrin β1 expression and posttranslational modification by enhancing glycosylation, thereby providing a more physiological culture system for studying integrin-ECM interactions in β cells. PMID:25911095

  19. Secretion of neurotensin from a human pancreatic islet cell carcinoma cell line (QGP-1N).

    PubMed

    Tateishi, K; Funakoshi, A; Kitayama, N; Matsuoka, Y

    1993-12-10

    Effects of various secretagogues on secretion of neurotensin from a pancreatic islet cell carcinoma cell line (QGP-1N) were examined. Carbachol stimulated secretion of neurotensin concentration-dependently in the range of 10(-6) - 10(-4) M. The neurotensin secretion stimulated with 10(-5) M carbachol was completely inhibited by atropine at 10(-5) M. Phorbol ester and calcium ionophore (A23187) stimulated secretion of neurotensin. The removal of extracellular Ca2+ suppressed the secretion through the stimulation with 10(-5) M carbachol. Fluoride, an activator of guanine nucleotide-binding (G) protein, stimulated secretion of neurotensin. Neurotensin released into culture medium through stimulation with carbachol coeluted with neurotensin 1-13 on a gel-chromatography. Our results suggest that secretion of neurotensin from QGP-1N cells is mainly regulated by acetylcholine through muscarinic receptors coupled to G protein and that an increase in intracellular Ca2+ and protein kinase C play an important role in stimulus-secretion coupling.

  20. Islet neogenesis associated protein (ingap): structural and dynamical properties of its active pentadecapeptide.

    PubMed

    McCarthy, Andrés N; Mogilner, Inés G; Grigera, J Raúl; Borelli, M Inés; Del Zotto, Hector; Gagliardino, Juan José

    2009-02-01

    We have studied the structural and dynamical properties of the biologically active pentadecapeptide of the islet neogenesis associated protein (INGAP-PP) and of two other pentadecapeptides with the same amino acid composition but randomly scrambled primary sequences, using molecular dynamic simulations. Our data demonstrates that whilst the peptides with scrambled sequences show no definite prevalent structure in solution, INGAP-PP maintains a notably stable tertiary fold, namely, a conformer with a central beta-sheet and closed C-terminal. Such structure resembles the one corresponding to the amino acid sequence of human pancreatitis associated protein-1 (PAP-1), which presents 85% sequence homology with INGAP. These results could reasonably explain why the two scrambled sequences tested showed no biological activity, while INGAP-PP significantly increases beta-cells function and mass both in vitro and in vivo conditions. The capability of INGAP-PP to temporarily adopt other closely related conformations offers also a plausible explanation for the 50 fold experimental difference in potency between the active pentadecapeptide and the whole protein. They also suggest that the C-terminal region of INGAP-PP may plausibly be the locus for its interaction with the cell receptor. Consequently, the knowledge gathered through our data can help to obtain more potent INGAP-PP analogs, suitable for the prevention and treatment of diabetes.

  1. PAX6 maintains β cell identity by repressing genes of alternative islet cell types

    PubMed Central

    Swisa, Avital; Avrahami, Dana; Eden, Noa; Zhang, Jia; Feleke, Eseye; Dahan, Tehila; Cohen-Tayar, Yamit; Stolovich-Rain, Miri; Kaestner, Klaus H.; Glaser, Benjamin; Ashery-Padan, Ruth

    2016-01-01

    Type 2 diabetes is thought to involve a compromised β cell differentiation state, but the mechanisms underlying this dysfunction remain unclear. Here, we report a key role for the TF PAX6 in the maintenance of adult β cell identity and function. PAX6 was downregulated in β cells of diabetic db/db mice and in WT mice treated with an insulin receptor antagonist, revealing metabolic control of expression. Deletion of Pax6 in β cells of adult mice led to lethal hyperglycemia and ketosis that were attributed to loss of β cell function and expansion of α cells. Lineage-tracing, transcriptome, and chromatin analyses showed that PAX6 is a direct activator of β cell genes, thus maintaining mature β cell function and identity. In parallel, we found that PAX6 binds promoters and enhancers to repress alternative islet cell genes including ghrelin, glucagon, and somatostatin. Chromatin analysis and shRNA-mediated gene suppression experiments indicated a similar function of PAX6 in human β cells. We conclude that reduced expression of PAX6 in metabolically stressed β cells may contribute to β cell failure and α cell dysfunction in diabetes. PMID:27941241

  2. A microfluidic array for real-time live-cell imaging of human and rodent pancreatic islets.

    PubMed

    Nourmohammadzadeh, Mohammad; Xing, Yuan; Lee, Jin Wuk; Bochenek, Matthew A; Mendoza-Elias, Joshua E; McGarrigle, James J; Marchese, Enza; Chun-Chieh, Yeh; Eddington, David T; Oberholzer, José; Wang, Yong

    2016-04-21

    In this study, we present a microfluidic array for high-resolution imaging of individual pancreatic islets. The device is based on hydrodynamic trapping principle and enables real-time analysis of islet cellular responses to insulin secretagogues. This device has significant advantages over our previously published perifusion chamber device including significantly increased analytical power and assay sensitivity, as well as improved spatiotemporal resolution. The islet array, with live-cell multiparametric imaging integration, provides a better tool to understand the physiological and pathophysiological changes of pancreatic islets through the analysis of single islet responses. This platform demonstrates the feasibility of array-based islet cellular analysis and opens up a new modality to conduct informative and quantitive evaluation of islets and cell-based screening for new diabetes treatments.

  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. Th1-Like ICOS+ Foxp3+ Treg Cells Preferentially Express CXCR3 and Home to β-Islets during Pre-Diabetes in BDC2.5 NOD Mice.

    PubMed

    Kornete, Mara; Mason, Edward S; Girouard, Julien; Lafferty, Erin I; Qureshi, Salman; Piccirillo, Ciriaco A

    2015-01-01

    Type 1 diabetes (T1D) occurs through a breakdown of self-tolerance resulting in the autoimmune destruction of the insulin producing β-islets of the pancreas. A numerical and functional waning of CD4+ Foxp3+ regulatory T (Treg) cells, prompted by a pancreatic IL-2 deficiency, accompanies Th1 autoimmunity and T1D progression in non-obese diabetic (NOD) mice. Recently, we identified a dominant subset of intra-islet Treg cells that expresses the ICOS costimulatory receptor and promotes self-tolerance delaying the onset of T1D. ICOS co-stimulation potently enhances IL-2 induced survival and proliferation, and suppressive activity of Treg cells in situ. Here, we propose an ICOS-dependent mechanism of Treg cell homing to the β-islets during pre-diabetes in the NOD model via upregulation of the CXCR3 chemokine receptor. The islet-specific ICOS+ Treg cell subset preferentially expresses CXCR3 in the pancreatic lymph nodes (pLN) in response to Teff cell-mediated pancreatic inflammation, an expression correlating with the onset and magnitude of IFN-γ production by Teff cells in pancreatic sites. We also reveal that intra-pancreatic APC populations and insulin-producing β, but not α nor δ, islet cells secrete the CXCR3 chemokines, CXCL9, 10 and 11, and selectively promote ICOS+ CXCR3+ Treg cell chemotaxis in vitro. Strikingly, islet-derived Treg cells also produce these chemokines suggesting an auto-regulation of homing by this subset. Unlike ICOS- cells, ICOS+ Treg cells adopt a Th1-like Treg phenotype while maintaining their suppressive capacity, characterized by expression of T-bet and CXCR3 and production of IFN-γ in the draining pLNs. Finally, in vivo neutralization of IFN-γ blocked Treg cell CXCR3 upregulation evincing its role in regulating expression of this chemokine receptor by Treg cells. Thus, CXCR3-mediated trafficking of Treg cells could represent a mechanism of homeostatic immunoregulation during diabetogeneesis.

  5. Transplantation of Heterospheroids of Islet Cells and Mesenchymal Stem Cells for Effective Angiogenesis and Antiapoptosis

    PubMed Central

    Shin, Jung-Youn; Jeong, Jee-Heon; Han, Jin; Bhang, Suk Ho; Jeong, Gun-Jae; Haque, Muhammad R.; Al-Hilal, Taslim A.; Noh, Myungkyung

    2015-01-01

    Although islet transplantation has been suggested as an alternative therapy for type 1 diabetes, there are efficiency concerns that are attributed to poor engraftment of transplanted islets. Hypoxic condition and delayed vasculogenesis induce necrosis and apoptosis of the transplanted islets. To overcome these limitations in islet transplantation, heterospheroids (HSs), which consist of rat islet cells (ICs) and human bone marrow-derived mesenchymal stem cells (hMSCs), were transplanted to the kidney and liver. The HSs cultured under the hypoxic condition system exhibited a significant increase in antiapoptotic gene expression in ICs. hMSCs in the HSs secreted angiogenic and antiapoptotic proteins. With the HS system, ICs and hMSCs were successfully located in the same area of the liver after transplantation of HSs through the portal vein, whereas the transplantation of islets and the dissociated hMSCs did not result in localization of transplanted ICs and hMSCs in the same area. HS transplantation resulted in an increase in angiogenesis at the transplantation area and a decrease in the apoptosis of transplanted ICs after transplantation into the kidney subcapsule compared with transplantation of islet cell clusters (ICCs). Insulin production levels of ICs were higher in the HS transplantation group compared with the ICC transplantation group. The HS system may be a more efficient transplantation method than the conventional methods for the treatment of type 1 diabetes. PMID:25344077

  6. Single-donor islet transplantation in type 1 diabetes: patient selection and special considerations

    PubMed Central

    Tatum, Jacob A; Meneveau, Max O; Brayman, Kenneth L

    2017-01-01

    Type 1 diabetes mellitus is an autoimmune disorder of the endocrine pancreas that currently affects millions of people in the United States. Although the disease can be managed with exogenous insulin administration, the ultimate cure for the condition lies in restoring a patient’s ability to produce their own insulin. Islet cell allotransplantation provides a means of endogenous insulin production. Though far from perfected, islet transplants are now a proven treatment for type 1 diabetics. However, proper patient selection is critical for achieving optimal outcomes. Given the shortage of transplantable organs, selecting appropriate candidates for whom the procedure will be of greatest benefit is essential. Although many of those who receive islets do not retain insulin independence, grafts do play a significant role in preventing hypoglycemic episodes that can be quite detrimental to quality of life and potentially fatal. Additionally, islet transplant requires lifelong immunosuppression. Antibodies, both preformed and following islet infusion, may play important roles in graft outcomes. Finally, no procedure is without inherent risk and islet transfusions can have serious consequences for recipients’ livers in the form of both vascular and metabolic complications. Therefore, patient-specific factors that should be taken into account before islet transplantation include aims of therapy, sensitization, and potential increased risk for hepatic and portal-venous sequelae. PMID:28280376

  7. Vicariance divergence and gene flow among islet populations of an endemic lizard.

    PubMed

    Runemark, Anna; Hey, Jody; Hansson, Bengt; Svensson, Erik I

    2012-01-01

    Allopatry and allopatric speciation can arise through two different mechanisms: vicariance or colonization through dispersal. Distinguishing between these different allopatric mechanisms is difficult and one of the major challenges in biogeographical research. Here, we address whether allopatric isolation in an endemic island lizard is the result of vicariance or dispersal. We estimated the amount and direction of gene flow during the divergence of isolated islet populations and subspecies of the endemic Skyros wall lizard Podarcis gaigeae, a phenotypically variable species that inhabits a major island and small islets in the Greek archipelago. We applied isolation-with-migration models to estimate population divergence times, population sizes and gene flow between islet-mainland population pairs. Divergence times were significantly correlated with independently estimated geological divergence times. This correlation strongly supports a vicariance scenario where islet populations have sequentially become isolated from the major island. We did not find evidence for significant gene flow within P. g. gaigeae. However, gene-flow estimates from the islet to the mainland populations were positively affected by islet area and negatively by distance between the islet and mainland. We also found evidence for gene flow from one subspecies (P. g. weigandi) into another (P. g. gaigeae), but not in the other direction. Ongoing gene flow between the subspecies suggests that even in this geographically allopatric scenario with the sea posing a strong barrier to dispersal, divergence with some gene flow is still feasible.

  8. Endoscopic gastric submucosal transplantation of islets (ENDO-STI): technique and initial results in diabetic pigs.

    PubMed

    Echeverri, G J; McGrath, K; Bottino, R; Hara, H; Dons, E M; van der Windt, D J; Ekser, B; Casu, A; Houser, S; Ezzelarab, M; Wagner, R; Trucco, M; Lakkis, F G; Cooper, D K C

    2009-11-01

    The results of transplantation of human donor islets into the portal vein (PV) in patients with diabetes are encouraging. However, there are complications, for example, hemorrhage, thrombosis and an immediate loss of islets through the 'instant blood-mediated inflammatory reaction' (IBMIR). The gastric submucosal space (GSMS) offers potential advantages. Islets were isolated from adult pigs. Recipient pigs were made diabetic by streptozotocin. Donor islets were injected into the GSMS through a laparotomy (Group 1A, n = 4) or endoscopically (Group 1B, n = 8) or into the PV through a laparotomy (Group 2, n = 3). The pigs were followed for a maximum of 28 days. Monitoring of C-peptide in Group 1 indicated that there was minimal immediate loss of islets whereas in Group 2 there was considerable loss from IBMIR. In Group 1, there were significant reductions in mean blood glucose and mean exogenous insulin requirement between pretransplantation and 20 days posttransplantation. In Group 2, there was no significant reduction in either parameter. Insulin-positive cells were seen in the GSMS in Group 1, but not in the liver in Group 2. Endoscopic gastric submucosal transplantation of islets (ENDO-STI) offers a minimally invasive and quick approach to islet transplantation, avoids IBMIR and warrants further exploration.

  9. Novel Stable Isotope Analyses Demonstrate Significant Rates of Glucose Cycling in Mouse Pancreatic Islets

    PubMed Central

    Pound, Lynley D.; Trenary, Irina; O’Brien, Richard M.

    2015-01-01

    A polymorphism located in the G6PC2 gene, which encodes an islet-specific glucose-6-phosphatase catalytic subunit, is the most important common determinant of variations in fasting blood glucose (FBG) levels in humans. Studies of G6pc2 knockout (KO) mice suggest that G6pc2 represents a negative regulator of basal glucose-stimulated insulin secretion (GSIS) that acts by hydrolyzing glucose-6-phosphate (G6P), thereby reducing glycolytic flux. However, this conclusion conflicts with the very low estimates for the rate of glucose cycling in pancreatic islets, as assessed using radioisotopes. We have reassessed the rate of glucose cycling in pancreatic islets using a novel stable isotope method. The data show much higher levels of glucose cycling than previously reported. In 5 mmol/L glucose, islets from C57BL/6J chow-fed mice cycled ∼16% of net glucose uptake. The cycling rate was further increased at 11 mmol/L glucose. Similar cycling rates were observed using islets from high fat–fed mice. Importantly, glucose cycling was abolished in G6pc2 KO mouse islets, confirming that G6pc2 opposes the action of the glucose sensor glucokinase by hydrolyzing G6P. The demonstration of high rates of glucose cycling in pancreatic islets explains why G6pc2 deletion enhances GSIS and why variants in G6PC2 affect FBG in humans. PMID:25552595

  10. Global epigenomic analysis of primary human pancreatic islets provides insights into type 2 diabetes susceptibility loci

    PubMed Central

    Stitzel, Michael L.; Sethupathy, Praveen; Pearson, Daniel S.; Chines, Peter S.; Song, Lingyun; Erdos, Michael R.; Welch, Ryan; Parker, Stephen C. J.; Boyle, Alan P.; Scott, Laura J.; Margulies, Elliott H.; Boehnke, Michael; Furey, Terrence S.; Crawford, Gregory E.; Collins, Francis S.

    2010-01-01

    Summary Identifying cis-regulatory elements is important to understand how human pancreatic islets modulate gene expression in physiologic or pathophysiologic (e.g., diabetic) conditions. We conducted genome-wide analysis of DNase I hypersensitive sites, histone H3 lysine methylation modifications (K4me1, K4me3, K79me2), and CCCTC factor (CTCF) binding in human islets. This identified ~18,000 putative promoters (several hundred unannotated and islet-active). Surprisingly, active promoter modifications were absent at genes encoding islet-specific hormones, suggesting a distinct regulatory mechanism. Of 34,039 distal (non-promoter) regulatory elements, 47% are islet-unique and 22% are CTCF-bound. In the 18 type 2 diabetes (T2D)-associated loci, we identified 118 putative regulatory elements and confirmed enhancer activity for 12/33 tested. Among 6 regulatory elements harboring T2D-associated variants, 2 exhibit significant allele-specific differences in activity. These findings present a global snapshot of the human islet epigenome and should provide functional context for non-coding variants emerging from genetic studies of T2D and other islet disorders. PMID:21035756

  11. Application of Rotating Wall Vessel (RWV) Cell Culture for Pancreas Islet Cell Transplantation

    NASA Technical Reports Server (NTRS)

    Rutzky, Lynne P.

    1998-01-01

    Type I insulin-dependent diabetes mellitus (IDDM) remains a major cause of morbidity and mortality in both pediatric and adult populations, despite significant advances in medical management. While insulin therapy treats symptoms of acute diabetes, it fails to prevent chronic complications such as microvascular disease, blindness, neuropathy, and chronic renal failure. Strict control of blood glucose concentrations delays but does not prevent the onset and progression of secondary complications. Although, whole pancreas transplantation restores physiological blood glucose levels, a continuous process of allograft rejection causes vascular and exocrine-related complications. Recent advances in methods for isolation and purification of pancreatic islets make transplantation of islet allografts an attractive alternative to whole pancreas transplantation. However, immunosuppressive drugs are necessary to prevent rejection of islet allografts and many of these drugs are known to be toxic to the islets. Since auto-transplants of isolated islets following total pancreatectomy survive and function in vivo, it is apparent that a major obstacle to successful clinical islet transplantation is the immunogenicity of the islet allografts.

  12. Pig Pancreas Anatomy: Implications for Pancreas Procurement, Preservation, and Islet Isolation

    PubMed Central

    Ferrer, Joana; Scott, William E; Weegman, Bradley P; Suszynski, Thomas M; Sutherland, David E R; Hering, Bernhard J; Papas, Klearchos K

    2009-01-01

    Background Islet transplantation is emerging as a treatment option for selected patients with type 1 diabetes. The limited human islet supply from cadavers and poor islet yield and quality remain substantial impediments to progress in the field. Use of porcine islets holds great promise for large-scale application of islet transplantation. Consistent isolation of porcine islets is dependent on advances in pancreas procurement and preservation, and islet isolation requiring detailed knowledge of the porcine pancreatic anatomy. The primary aim of this study was to describe the vascular and ductal anatomy of the porcine pancreas in order to guide and improve organ preservation and enzyme perfusion. Methods Pancreata were removed by en bloc viscerectomy from 65 female Landrace pigs. Results 15% of organs exhibited inconsistent vascular branching from the celiac trunk. All organs had uniform patterns of branching at the superior mesenteric artery. The superior and inferior mesenteric veins (IMV) merged to become the portal vein in all but one case in which the IMV drained into the splenic vein. 97% of pancreata had three lobes: duodenal (DL), connecting (CL), and splenic (SL); 39% demonstrated ductal communication between the CL and the other two lobes; 50% had ductal communication only between the CL and DL; and 11% presented other types of ductal delineation. Conclusions Accounting for the variations in vascular and ductal anatomy, as detailed in this study, will facilitate development of protocols for preservation, optimal enzyme administration, and pancreas distention and digestion, and ultimately lead to substantial improvements in isolation outcomes. PMID:19077881

  13. Small islets are essential for successful intraportal transplantation in a diabetes mouse model.

    PubMed

    Su, Z; Xia, J; Shao, W; Cui, Y; Tai, S; Ekberg, H; Corbascio, M; Chen, J; Qi, Z

    2010-12-01

    Optimization of islet transplantation protocols is necessary for improved success of treatment for type 1 diabetes. Here, we investigated whether the size of islets transplanted into the portal vein (PV) of the liver can affect engraftment in the early post-transplantation in an experimental mouse model. Small (average diameter < 250 μm, group A) or large (average diameter > 250 μm, group B) islets (400 islet equivalents/recipient) purified from normal BALB/c mice were transplanted into syngenic recipients with diabetes induced by STZ. The percentage of mice returning to a non-diabetic status was higher in group A (100%) than that of group B (62.5%). Focal areas of liver necrosis associated with the islets emboli were observed in both groups, but the pathology in group B was significantly worse. Multiple proinflammatory cytokines were significantly higher in group B than that of A at 3 h post-transplantation. Our study determined that the size of islets plays a critical role in the success of intraportal islet transplantation (IPIT) and should be taken into account in future IPIT protocols for the treatment of diabetes.

  14. Preparation of suspensions of pancreatic islet cells: a comparison of methods.

    PubMed

    Kohnert, K D; Hehmke, B

    1986-01-01

    A comparative study for preparation of cell suspensions from pancreatic islets has been performed using mechanical or enzymatic dissociation with proteolytic enzymes such as trypsin, dispase, and pronase. Treatment of isolated pancreatic islets from neonatal rats with these enzymes proved to be superior to a mechanical dissociation method. The enzymatic dissociation was performed by fractionated treatment of pancreatic islets with low concentration of enzymes in Hanks' solution for 2-3 min at room temperature. With the exception of trypsin the percentage of single cells was consistently higher with dispase and pronase treatment, being 83-92%. Cell viability (dye exclusion) was more than 90%. Mechanical disintegration of pancreatic islets resulted in a low yield of single cells, and cell viability was considerably reduced in comparison with the enzymatic methods. Labeling of islet cells with Na2 51CrO4 and measurement of the basal 51Cr-release demonstrated superior membrane preservation after pronase or dispase treatment. Islet cells isolated either by fractionated dispase or pronase treatment were found to be well preserved and very suitable for the detection of circulating cell surface antibodies and their cytotoxic effects to islet cells.

  15. Genetic regulatory signatures underlying islet gene expression and type 2 diabetes

    PubMed Central

    Varshney, Arushi; Scott, Laura J.; Welch, Ryan P.; Erdos, Michael R.; Chines, Peter S.; Narisu, Narisu; Albanus, Ricardo D’O.; Orchard, Peter; Wolford, Brooke N.; Kursawe, Romy; Vadlamudi, Swarooparani; Cannon, Maren E.; Didion, John P.; Hensley, John; Kirilusha, Anthony; Bonnycastle, Lori L.; Taylor, D. Leland; Watanabe, Richard; Mohlke, Karen L.; Boehnke, Michael; Collins, Francis S.; Parker, Stephen C. J.; Stitzel, Michael L.

    2017-01-01

    Genome-wide association studies (GWAS) have identified >100 independent SNPs that modulate the risk of type 2 diabetes (T2D) and related traits. However, the pathogenic mechanisms of most of these SNPs remain elusive. Here, we examined genomic, epigenomic, and transcriptomic profiles in human pancreatic islets to understand the links between genetic variation, chromatin landscape, and gene expression in the context of T2D. We first integrated genome and transcriptome variation across 112 islet samples to produce dense cis-expression quantitative trait loci (cis-eQTL) maps. Additional integration with chromatin-state maps for islets and other diverse tissue types revealed that cis-eQTLs for islet-specific genes are specifically and significantly enriched in islet stretch enhancers. High-resolution chromatin accessibility profiling using assay for transposase-accessible chromatin sequencing (ATAC-seq) in two islet samples enabled us to identify specific transcription factor (TF) footprints embedded in active regulatory elements, which are highly enriched for islet cis-eQTL. Aggregate allelic bias signatures in TF footprints enabled us de novo to reconstruct TF binding affinities genetically, which support the high-quality nature of the TF footprint predictions. Interestingly, we found that T2D GWAS loci were strikingly and specifically enriched in islet Regulatory Factor X (RFX) footprints. Remarkably, within and across independent loci, T2D risk alleles that overlap with RFX footprints uniformly disrupt the RFX motifs at high-information content positions. Together, these results suggest that common regulatory variations have shaped islet TF footprints and the transcriptome and that a confluent RFX regulatory grammar plays a significant role in the genetic component of T2D predisposition. PMID:28193859

  16. β-cell metabolic alterations under chronic nutrient overload in rat and human islets.

    PubMed

    Vernier, Stephanie; Chiu, Angela; Schober, Joseph; Weber, Theresa; Nguyen, Phuong; Luer, Mark; McPherson, Timothy; Wanda, Paul E; Marshall, Connie A; Rohatgi, Nidhi; McDaniel, Michael L; Greenberg, Andrew S; Kwon, Guim

    2012-01-01

    The aim of this study was to assess multifactorial β-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 lipid droplets in β-cells in a time- and concentration-dependent manner. Glucose and FFAs synergistically stimulated the nutrient sensor mammalian target of rapamycin complex 1 (mTORC1). A potent mTORC1 inhibitor, rapamycin (25 nM), significantly reduced triglyceride accumulation in rat islets. Importantly, lipid droplets accumulated only in β-cells but not in α-cells in an mTORC1-dependent manner. Nutrient activation of mTORC1 upregulated the expression of adipose differentiation related protein (ADRP), known to stabilize lipid droplets. Rat islet size and new DNA synthesis also increased under nutrient overload. Insulin secretion into the culture medium increased steadily over a 4-day period without any significant difference between glucose (10 mM) alone and the combination of glucose (10 mM) and FFAs (240 μM). Insulin content and insulin biosynthesis, however, were significantly reduced under the combination of nutrients compared with glucose alone. Elevated nutrients also stimulated lipid droplet formation in human islets in an mTORC1-dependent manner. Unlike rat islets, however, human islets did not increase in size under nutrient overload despite a normal response to nutrients in releasing insulin. The different responses of islet cell growth under nutrient overload appear to impact insulin biosynthesis and storage differently in rat and human islets.

  17. Outcomes of Pancreatic Islet Allotransplantation Using the Edmonton Protocol at the University of Chicago

    PubMed Central

    Tekin, Zehra; Garfinkel, Marc R.; Chon, W. James; Schenck, Lindsay; Golab, Karolina; Savari, Omid; Thistlethwaite, J. Richard; Philipson, Louis H.; Majewski, Colleen; Pannain, Silvana; Ramachandran, Sabarinathan; Rezania, Kourosh; Hariprasad, Seenu M.; Millis, J. Michael; Witkowski, Piotr

    2016-01-01

    Objective The aim of this study was to assess short-term and long-term results of the pancreatic islet transplantation using the Edmonton protocol at the University of Chicago. Materials and Methods Nine patients underwent pancreatic islet cell transplantation using the Edmonton Protocol; they were followed up for 10 years after initial islet transplant with up to 3 separate islet infusions. They were given induction treatment using an IL-2R antibody and their maintenance immunosuppression regimen consisted of sirolimus and tacrolimus. Results Nine patients received a total of 18 islet infusions. Five patients dropped out in the early phase of the study. Greater than 50% drop-out and noncompliance rate resulted from both poor islet function and recurrent side effects of immunosuppression. The remaining 4 (44%) patients stayed insulin free with intervals for at least over 5 years (cumulative time) after the first transplant. Each of them received 3 infusions, on average 445 000 islet equivalent per transplant. Immunosuppression regimen required multiple adjustments in all patients due to recurrent side effects. In the long-term follow up, kidney function remained stable, and diabetic retinopathy and polyneuropathy did not progress in any of the patients. Patients' panel reactive antibodies remained zero and anti-glutamic acid decarboxylase 65 antibody did not rise after the transplant. Results of metabolic tests including hemoglobin A1c, arginine stimulation, and mixed meal tolerance test were correlated with clinical islet function. Conclusions Pancreatic islet transplantation initiated according to Edmonton protocol offered durable long-term insulin-free glycemic control in only highly selected brittle diabetics providing stable control of diabetic neuropathy and retinopathy and without increased sensitization or impaired renal function. Immunosuppression adjustments and close follow-up were critical for patient retention and ultimate success. PMID:27795987

  18. Characterization of the mouse pancreatic islet proteome and comparative analysis with other mouse tissues

    SciTech Connect

    Petyuk, Vladislav A.; Qian, Weijun; Hinault, Charlotte; Gritsenko, Marina A.; Singhal, Mudita; Monroe, Matthew E.; Camp, David G.; Kulkarni, Rohit N.; Smith, Richard D.

    2008-08-01

    The pancreatic islets of Langerhans and insulin-producing beta cells in particular play a central role in the maintenance of glucose homeostasis and the islet dysfunction is associated with the pathogenesis of both type 1 and type 2 diabetes mellitus. To contribute to the understanding of the biology of the pancreatic islets we applied proteomic techniques based on liquid chromatography coupled with mass spectrometry. Here as an initial step we present the first comprehensive proteomic characterization of pancreas islets of the mouse, the commonly used animal model for diabetes research. Two-dimensional SCX LC/RP LC-MS/MS has been applied to characterize of the mouse islet proteome, resulting in the confident identification of 17,350 different tryptic peptides covering 2,612 proteins with at least two unique peptide identifications per protein. The dataset also allowed identification of a number of post-translational modifications including several modifications relevant to oxidative stress and phosphorylation. While many of the identified phosphorylation sites corroborates with previous known sites, the oxidative modifications observed on cysteinyl residues potentially reveal novel information related to the role of oxidation stress in islet functions. Comparative analysis of the islet proteome database with 15 available proteomic datasets from other mouse tissues and cells revealed a set of 68 proteins uniquely detected only in the pancreatic islets. Besides proteins with known functions, like islet secreted peptide hormones, this unique set contains a number of proteins with yet unknown functions. The resulting peptide and protein database will be available at ncrr.pnl.gov web site of the NCRR proteomic center (ncrr.pnl.gov).

  19. Genetic regulatory signatures underlying islet gene expression and type 2 diabetes.

    PubMed

    Varshney, Arushi; Scott, Laura J; Welch, Ryan P; Erdos, Michael R; Chines, Peter S; Narisu, Narisu; Albanus, Ricardo D'O; Orchard, Peter; Wolford, Brooke N; Kursawe, Romy; Vadlamudi, Swarooparani; Cannon, Maren E; Didion, John P; Hensley, John; Kirilusha, Anthony; Bonnycastle, Lori L; Taylor, D Leland; Watanabe, Richard; Mohlke, Karen L; Boehnke, Michael; Collins, Francis S; Parker, Stephen C J; Stitzel, Michael L

    2017-02-28

    Genome-wide association studies (GWAS) have identified >100 independent SNPs that modulate the risk of type 2 diabetes (T2D) and related traits. However, the pathogenic mechanisms of most of these SNPs remain elusive. Here, we examined genomic, epigenomic, and transcriptomic profiles in human pancreatic islets to understand the links between genetic variation, chromatin landscape, and gene expression in the context of T2D. We first integrated genome and transcriptome variation across 112 islet samples to produce dense cis-expression quantitative trait loci (cis-eQTL) maps. Additional integration with chromatin-state maps for islets and other diverse tissue types revealed that cis-eQTLs for islet-specific genes are specifically and significantly enriched in islet stretch enhancers. High-resolution chromatin accessibility profiling using assay for transposase-accessible chromatin sequencing (ATAC-seq) in two islet samples enabled us to identify specific transcription factor (TF) footprints embedded in active regulatory elements, which are highly enriched for islet cis-eQTL. Aggregate allelic bias signatures in TF footprints enabled us de novo to reconstruct TF binding affinities genetically, which support the high-quality nature of the TF footprint predictions. Interestingly, we found that T2D GWAS loci were strikingly and specifically enriched in islet Regulatory Factor X (RFX) footprints. Remarkably, within and across independent loci, T2D risk alleles that overlap with RFX footprints uniformly disrupt the RFX motifs at high-information content positions. Together, these results suggest that common regulatory variations have shaped islet TF footprints and the transcriptome and that a confluent RFX regulatory grammar plays a significant role in the genetic component of T2D predisposition.

  20. Islet transplantation in type 1 diabetes: ongoing challenges, refined procedures, and long-term outcome.

    PubMed

    Shapiro, A M James

    2012-01-01

    Remarkable progress has been made in islet transplantation over a span of 40 years. Once just an experimental curiosity in mice, this therapy has moved forward, and can now provide robust therapy for highly selected patients with type 1 diabetes (T1D), refractory to stabilization by other means. This progress could not have occurred without extensive dynamic international collaboration. Currently, 1,085 patients have undergone islet transplantation at 40 international sites since the Edmonton Protocol was reported in 2000 (752 allografts, 333 autografts), according to the Collaborative Islet Transplant Registry. The long-term results of islet transplantation in selected centers now match registry data of pancreas-alone transplantation, with 6 sites reporting five-year insulin independence rates ≥50%. Islet transplantation has been criticized for the use of multiple donor pancreas organs, but progress has also occurred in single-donor success, with 10 sites reporting increased single-donor engraftment. The next wave of innovative clinical trial interventions will address instant blood-mediated inflammatory reaction (IBMIR), apoptosis, and inflammation, and will translate into further marked improvements in single-donor success. Effective control of auto- and alloimmunity is the key to long-term islet function, and high-resolution cellular and antibody-based assays will add considerable precision to this process. Advances in immunosuppression, with new antibody-based targeting of costimulatory blockade and other T-B cellular signaling, will have further profound impact on the safety record of immunotherapy. Clinical trials will move forward shortly to test out new human stem cell derived islets, and in parallel trials will move forward, testing pig islets for compatibility in patients. Induction of immunological tolerance to self-islet antigens and to allografts is a difficult challenge, but potentially within our grasp.

  1. Enterovirus infection of human islets of Langerhans affects β-cell function resulting in disintegrated islets, decreased glucose stimulated insulin secretion and loss of Golgi structure

    PubMed Central

    Hodik, M; Skog, O; Lukinius, A; Isaza-Correa, J M; Kuipers, J; Giepmans, B N G; Frisk, G

    2016-01-01

    Aims/hypothesis In type 1 diabetes (T1D), most insulin-producing β cells are destroyed, but the trigger is unknown. One of the possible triggers is a virus infection and the aim of this study was to test if enterovirus infection affects glucose stimulated insulin secretion and the effect of virus replication on cellular macromolecules and organelles involved in insulin secretion. Methods Isolated human islets were infected with different strains of coxsackievirus B (CVB) virus and the glucose-stimulated insulin release (GSIS) was measured in a dynamic perifusion system. Classical morphological electron microscopy, large-scale electron microscopy, so-called nanotomy, and immunohistochemistry were used to study to what extent virus-infected β cells contained insulin, and real-time PCR was used to analyze virus induced changes of islet specific genes. Results In islets infected with CVB, GSIS was reduced in correlation with the degree of virus-induced islet disintegration. The expression of the gene encoding insulin was decreased in infected islets, whereas the expression of glucagon was not affected. Also, in islets that were somewhat disintegrated, there were uninfected β cells. Ultrastructural analysis revealed that virus particles and virus replication complexes were only present in β cells. There was a significant number of insulin granules remaining in the virus-infected β cells, despite decreased expression of insulin mRNA. In addition, no typical Golgi apparatus was detected in these cells. Exposure of islets to synthetic dsRNA potentiated glucose-stimulated insulin secretion. Conclusions/interpretation Glucose-stimulated insulin secretion; organelles involved in insulin secretion and gene expression were all affected by CVB replication in β cells. PMID:27547409

  2. Anti-diabetic action of all-trans retinoic acid and the orphan G protein coupled receptor GPRC5C in pancreatic β-cells.

    PubMed

    Amisten, Stefan; Mohammad Al-Amily, Israa; Soni, Arvind; Hawkes, Ross; Atanes, Patricio; Persaud, Shanta Jean; Rorsman, Patrik; Salehi, Albert

    2017-03-31

    Pancreatic islets express high levels of the orphan G-protein coupled receptor C5C (GPRC5C), the function of which remains to be established. Here we have examined the role of GPRC5C in the regulation of insulin secretion and β-cell survival and proliferation using human and mouse pancreatic islets. The expression of GPRC5C was analysed by RNA-sequencing, qPCR, western blotting and confocal microscopy. Insulin secretion and cell viability were determined by RIA and MTS assays, respectively. GPRC5C mRNA expression and protein level were reduced in the islets from type-2 diabetic donors. RNA sequencing in human islets revealed GPRC5C expression correlated with the expression of genes controlling apoptosis, cell survival and proliferation. A reduction in Gprc5c mRNA and protein expression was observed in islets isolated from old mice (>46 weeks of age) compared to that in islets from newborn (<3 weeks) mice. Down-regulation of Gprc5c led to both moderately reduced glucose-stimulated insulin release and also reduced cAMP content in mouse islets. Potentiation of glucose-stimulated insulin secretion concomitant with enhanced islet cAMP level by all-trans retinoic acid (ATRA) was attenuated upon Gprc5c-KD. ATRA also increased [Ca(+2)]i in Huh7-cells. Gprc5c over expression in Huh7 cells was associated with increased ERK1/2 activity. Gprc5c-KD in clonal MIN6c4 cells reduced cell proliferation and in murine islets increased apoptosis and the sensitivity of primary islet cells to a cocktail of pro-apoptotic cytokines. Our results demonstrate that agents activating GPRC5C represent a novel modality for the treatment and/or prevention of diabetes by restoring and/or maintaining functional β-cell mass.

  3. That which does not kill us makes us stronger--does Nietzsche's quote apply to islets? A re-evaluation of the passenger leukocyte theory, free radicals, and glucose toxicity in islet cell transplantation.

    PubMed

    Wright, J R; Xu, B-Y

    2014-07-01

    In clinical islet transplantation, isolated islets are embolized into the liver via the portal vein (PV); however, up to 70% of the islets are lost in the first few days after transplantation (i.e., too quickly to be mediated by the adaptive immune system). Part of early loss is due to instant blood-mediated inflammatory reaction, an immune/thrombotic process caused by islets interacting with complement. We have shown that glucose toxicity (GT) also plays a critical role based upon the observation that islets embolized into the PVs of diabetic athymic mice are rapidly lost but, if recipients are not diabetic, the islet grafts persist. Using donor islets resistant to the β-cell toxin streptozotocin, we have shown that intraportal islets engrafted in non-diabetic athymic mice for as little as 3 days will maintain normoglycemia when streptozotocin is administered destroying the recipient's native pancreas β-cells. What is the mechanism of GT in β-cells? Chronic exposure to hyperglycemia over-exerts β-cells and their electron transport chains leak superoxide radicals during aerobic metabolism. Here we reinterpret old data and present some compelling new data supporting a new model of early intraportal islet graft loss. We hypothesize that diabetes stimulates overproduction of superoxide in both the β-cells of the islet grafts and the endothelial cells lining the intraportal microvasculature adjacent to where the embolized islets become lodged. This double dose of oxidant damage stresses both the islets, which are highly susceptible to free radicals because of inherent low levels of scavenging enzymes, and the adjacent hepatic endothelial cells. This, superimposed upon localized endothelial damage caused by embolization, precipitates inflammation and coagulation which further damages islet grafts. Based upon this model, we predict that pre-exposing islets to sub-lethal hyperoxia should up-regulate islet free radical scavenging enzyme levels and promote initial

  4. Phage library-screening: a powerful approach for generation of targeting-agents specific for normal pancreatic islet-cells and islet-cell carcinoma in vivo.

    PubMed

    Ueberberg, S; Schneider, S

    2010-02-25

    Phage display technology is a powerful approach for the generation of peptides and antibodies that target specific organ- or tumor-structures. By applying this approach to rats in vivo or freshly isolated rat islets in vitro, we have recently reported the successful isolation of internalizing single-chain antibodies (SCA-antibodies), which are highly specific for the endocrine-cells of a pancreatic islet (either beta- or alpha-cells) both in rodents and in humans. Moreover, others have reported peptides targeting specifically the vascular endothelium of normal or pre-malignant islets or advanced islet-cell tumors. The features of these antibodies and peptides are compatible with a potential use for in vivo delivery of molecular cargos (e.g. imaging agents and therapeutics). Therefore, this article reviews the principles of phage display, provides an overview about agents either specific for the endocrine-cells or the vascular endothelium of islets, discusses methododical key elements for the generation of these ligands and highlights remaining questions and potential future perspectives.

  5. Determination of Optimal Sample Size for Quantification of β-Cell Area, Amyloid Area and β-Cell Apoptosis in Isolated Islets

    PubMed Central

    Entrup, Leon; Templin, Andrew T.; Hogan, Meghan F.; Samarasekera, Thanya; Zraika, Sakeneh; Boyko, Edward J.; Kahn, Steven E.

    2015-01-01

    Culture of isolated rodent islets is widely used in diabetes research to assess different endpoints, including outcomes requiring histochemical staining. As islet yields during isolation are limited, we determined the number of islets required to obtain reliable data by histology. We found that mean values for insulin-positive β-cell area/islet area, thioflavin S-positive amyloid area/islet area and β-cell apoptosis do not vary markedly when more than 30 islets are examined. Measurement variability declines as more islets are quantified, so that the variability of the coefficient of variation (CV) in human islet amyloid polypeptide (hIAPP) transgenic islets for β-cell area/islet area, amyloid area/islet area and β-cell apoptosis are 13.20% ± 1.52%, 10.03% ± 1.76% and 6.78% ± 1.53%, respectively (non-transgenic: 7.65% ± 1.17% β-cell area/islet area and 8.93% ± 1.56% β-cell apoptosis). Increasing the number of islets beyond 30 had marginal effects on the CV. Using 30 islets, 6 hIAPP-transgenic preparations are required to detect treatment effects of 14% for β-cell area/islet area, 30% for amyloid area/islet area and 23% for β-cell apoptosis (non-transgenic: 9% for β-cell area/islet area and 45% for β-cell apoptosis). This information will be of value in the design of studies using isolated islets to examine β cells and islet amyloid. PMID:26216141

  6. A novel dual-glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide receptor agonist is neuroprotective in transient focal cerebral ischemia in the rat.

    PubMed

    Han, Ling; Hölscher, Christian; Xue, Guo-Fang; Li, Guanglai; Li, Dongfang

    2016-01-06

    Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) receptor agonists have been shown to be neuroprotective in previous studies in animal models of Alzheimer's or Parkinson's disease. Recently, novel dual-GLP-1/GIP receptor agonists that activate both receptors (DA) were developed to treat diabetes. We tested the protective effects of a novel potent DA against middle cerebral artery occlusion injury in rats and compared it with a potent GLP-1 analog, Val(8)-GLP-1(glu-PAL). Animals were evaluated for neurologic deficit score, infarct volume, and immunohistochemical analyses of the brain at several time points after ischemia. The Val(8)-GLP-1(glu-PAL)-treated and DA-treated groups showed significantly reduced scores of neurological dysfunction, cerebral infarction size, and percentage of TUNEL-positive apoptotic neurons. Furthermore, the expression of the apoptosis marker Bax, the inflammation marker iNOS, and the survival marker Bcl-2 was significantly increased. The DA-treated group was better protected against neurodegeneration than the Val(8)-GLP-1(glu-PAL) group, and the scores of neurological dysfunction, cerebral infarction size, and expression of Bcl-2 were higher, whereas the percentage of TUNEL-positive neurons and the levels of Bax and iNOS were lower in the DA group. DA treatment reduced the infarct volume and improved the functional deficit. It also suppressed the inflammatory response and cell apoptosis after reperfusion. In conclusion, the novel GIP and GLP-1 dual-receptor agonist is more neuroprotective than a GLP-1 receptor agonist in key biomarkers of neuronal degeneration.

  7. Three-dimensional optical method for integrated visualization of mouse islet microstructure and vascular network with subcellular-level resolution

    NASA Astrophysics Data System (ADS)

    Fu, Ya-Yuan; Lu, Chih-Hsuan; Lin, Chi-Wen; Juang, Jyuhn-Huarng; Enikolopov, Grigori; Sibley, Eric; Chiang, Ann-Shyn; Tang, Shiue-Cheng

    2010-07-01

    Microscopic visualization of islets of Langerhans under normal and diabetic conditions is essential for understanding the pathophysiology of the disease. The intrinsic opacity of pancreata, however, limits optical accessibility for high-resolution light microscopy of islets in situ. Because the standard microtome-based, 2-D tissue analysis confines visualization of the islet architecture at a specific cut plane, 3-D representation of image data is preferable for islet assessment. We applied optical clearing to minimize the random light scattering in the mouse pancreatic tissue. The optical-cleared pancreas allowed penetrative, 3-D microscopic imaging of the islet microstructure and vasculature. Specifically, the islet vasculature was revealed by vessel painting-lipophilic dye labeling of blood vessels-for confocal microscopy. The voxel-based confocal micrographs were digitally processed with projection algorithms for 3-D visualization. Unlike the microtome-based tissue imaging, this optical method for penetrative imaging of mouse islets yielded clear, continuous optical sections for an integrated visualization of the islet microstructure and vasculature with subcellular-level resolution. We thus provide a useful imaging approach to change our conventional planar view of the islet structure into a 3-D panorama for better understanding of the islet physiology.

  8. Distinctions between islet neogenesis and β-cell replication: implications for reversal of Type 1 and 2 diabetes.

    PubMed

    Levetan, Claresa

    2010-06-01

    The terms "islet" and "β-cell" are often used interchangeably, yet islets are highly complex multicellular organelles that contain the insulin-producing β-cells and four other cells types, all of which play a role in maintaining glucose homeostasis within a very narrow range. Although the formation of new islets in adults is rare, occurring primarily in response to pancreatic injury and major stress to the pancreas, β-cell replication from existing cells occurs throughout adulthood. An understanding of the regulatory factors controlling pancreatic development has more clearly defined the differences between new islet formation from progenitor cells located throughout the adult pancreas and β-cell replication occurring within existing islets. The present review sets forth to more clearly distinguish the differences between the postnatal pathways of islet neogenesis and β-cell replication with a discussion of the potential implications for reversal of Type 1 and 2 diabetic patients using islet neogenesis agents that are now in development. For Type 1 diabetic patients, an immune tolerance agent in conjunction with an islet neogenesis agent may allow achievement of adequate islet mass, perhaps with subsequent potential to withdraw medications. For Type 2 diabetic patients, lifestyle changes and/or medications may sustain the production of new islets and limit the accelerated β-cell apoptosis characteristic of the condition.

  9. Diabetes Is Reversed in a Murine Model by Marginal Mass Syngeneic Islet Transplantation Using a Subcutaneous Cell Pouch Device

    PubMed Central

    Pepper, Andrew R.; Pawlick, Rena; Gala-Lopez, Boris; MacGillivary, Amanda; Mazzuca, Delfina M.; White, David J. G.; Toleikis, Philip M.; Shapiro, A. M. James

    2015-01-01

    Background Islet transplantation is a successful β-cell replacement therapy for selected patients with type 1 diabetes mellitus. Although high rates of early insulin independence are achieved routinely, long-term function wanes over time. Intraportal transplantation is associated with procedural risks, requires multiple donors, and does not afford routine biopsy. Stem cell technologies may require potential for retrievability, and graft removal by hepatectomy is impractical. There is a clear clinical need for an alternative, optimized transplantation site. The subcutaneous space is a potential substitute, but transplantation of islets into this site has routinely failed to reverse diabetes. However, an implanted device, which becomes prevascularized before transplantation, may alter this equation. Methods Syngeneic mouse islets were transplanted subcutaneously within Sernova Corp's Cell Pouch (CP). All recipients were preimplanted with CPs 4 weeks before diabetes induction and transplantation. After transplantation, recipients were monitored for glycemic control and glucose tolerance. Results Mouse islets transplanted into the CP routinely restored glycemic control with modest delay and responded well to glucose challenge, comparable to renal subcapsular islet grafts, despite a marginal islet dose, and normoglycemia was maintained until graft explantation. In contrast, islets transplanted subcutaneously alone failed to engraft. Islets within CPs stained positively for insulin, glucagon, and microvessels. Conclusions The CP is biocompatible, forms an environment suitable for islet engraftment, and offers a potential alternative to the intraportal site for islet and future stem cell therapies. PMID:26308506

  10. Pancreastatin producing cell line from human pancreatic islet cell tumor.

    PubMed

    Funakoshi, A; Tateishi, K; Tsuru, M; Jimi, A; Wakasugi, H; Ikeda, Y; Kono, A

    1990-04-30

    It has been characterized that cell line QGP-1 derived from human non-functioning pancreatic islet cell tumor produces human pancreastatin. Exponentially growing cultures produced 5.7 fmol of pancreastatin/10(6) cells/hr. Human pancreastatin immunoreactivities in plasma and tumor after xenografting with QGP-1 into nude mouse were 92.7 fmol/ml and 160.2 pmol/g wet weight, respectively. Immunocytochemical study revealed both chromogranin A and pancreastatin immunoreactive cells in the tumor. Gel filtrations of culture medium and tumor extract identified heterogenous molecular forms of PST-LI which eluted as large and smaller molecular species. These results suggest that plasma pancreastatin levels may be useful as a tumor marker of endocrine tumor of the pancreas, and the pancreastatin producing cell line may be useful for studies of the mechanism of secretions and processing of chromogranin A and pancreastatin.

  11. Identification of in vitro parameters predictive of graft function: a study in an animal model of islet transplantation.

    PubMed

    Migliavacca, B; Nano, R; Antonioli, B; Marzorati, S; Davalli, A M; Di Carlo, V; Bertuzzi, F

    2004-04-01

    The quality of human islets is one of the factors decisive for the success of human islet transplantation. Several parameters have been proposed to characterize islet quality, but none of them has been able to predict the fate of a transplant. The aim of our study was to correlate a panel of in vitro parameters for islet viability with their in vivo function after transplantation in nude mice. Islets were obtained after enzymatic digestion of a human pancreas; they were purified from exocrine tissue using a continuous-density gradient. Two aliquots of islets (1000 and 2000 islets) were transplanted under the kidney capsule of diabetic nude mice. The animals were followed for 1 month with repeated measurements of blood glucose and body weight. One month after transplantation, mice were killed and their graft harvested for histologic analysis. In parallel we studied in vitro islet viability with propidium iodide and fura-2, their insulin content, their purity, and their insulin response to glucose upon static incubation. Ten islet preparations were transplanted: 3 out of 10 preparations did not restore normoglycemia; 4 out of 10 normalized glycemia only in mice receiving 2000 islets, and 3 out of 10 fully restore normoglycemia in all mice. The purity of preparations (R(2) = 0.63 and 0.85, respectively, with 1000 and 2000 islets) and the insulin content (R(2) = 0.75 with 2000 IE) correlated with transplant success. These data show that purity of islet preparations and their insulin content should be useful parameters for the selection of islet preparations for transplant purposes.

  12. Co-encapsulation of bioengineered IGF-II-producing cells and pancreatic islets: effect on beta-cell survival.

    PubMed

    Jourdan, G; Dusseault, J; Benhamou, P Y; Rosenberg, L; Hallé, J P

    2011-06-01

    Insulin-like growth factor-II (IGF-II) has been shown to promote pancreatic β-cell survival. We evaluated the effect of co-encapsulating islets and bioengineered IGF-II-producing cells on islet cell survival. IGF-II or green fast protein (GFP) genes were transferred into TM4 cells, and purified using a neomycin resistance gene, leading to pure cell cultures (TM4-IGF-II or TM4-GFP) with a stable overexpression of the transferred gene. Islets were co-encapsulated with TM4-IGF-II or TM4-GFP, or encapsulated alone in alginate microcapsules. Rat and mouse islet cell survival was studied in vitro and in vivo, respectively. After 12 days in culture, islet viability (dual staining, acridine orange/propidium iodide) was 83% with TM4-IGF-II, compared with 51% (P<0.05) and 41% (P<0.001) with TM4-GFP and islets alone, respectively. The study of islet necrotic centers and the evaluation of islet function, using the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) assay, yielded similar results. From 125 days after transplantation, more diabetic mice maintained normoglycemia when they were transplanted with islets co-encapsulated with TM4-IGF-II (4/7). A significant difference for the maintenance of normoglycemia was observed between recipients of islets co-encapsulated with TM4-IGF-II versus islets alone (P=0.023), or with TM4-GFP (P=0.048). In conclusion, the co-encapsulation of islets with bioengineered IGF-II-producing cells promotes islet cell survival.

  13. Microfabricated biocapsules for the immunoisolation of pancreatic islets of Langerhans

    NASA Astrophysics Data System (ADS)

    Desai, Tejal Ashwin

    1998-08-01

    A silicon-based microfabricated biocapsule was developed and evaluated for use in the immunoisolation of transplanted cells, specifically pancreatic islets of Langerhans for the treatment of Type I diabetes. The transplantation of cells with specific functions is a promising therapy for a wide variety of pathologies including diabetes, Parkinson's, and hemophilia. Such transplanted cells, however, are sensitive to both cellular and humoral immune rejection as well as damage by autoimmune activity, without chronic immunosuppression. The research presented in this dissertation investigated whether microfabricated silicon-based biocapsules, with uniform membrane pore sizes in the tens of nanometer range, could provide an immunoprotective environment for pancreatic islets and other insulin-secreting cell lines, while maintaining cell viability and functionality. By utilizing fabrication techniques commonly employed in the microelectronics industry (MEMS), membranes were fabricated with precisely controlled and uniform pore sizes, allowing the optimization of biocapsule membrane parameters for the encapsulation of specific hormone-secreting cell types. The biocapsule-forming process employed bulk micromachining to define cell-containing chambers within single crystalline silicon wafers. These chambers interface with the surrounding biological environment through polycrystalline silicon filter membranes, which were surface micromachined to present a high density of uniform pores to allow sufficient permeability to oxygen, glucose, and insulin. Both in vitro and in vivo experiments established the biocompatibility of the microfabricated biocapsule, and demonstrated that encapsulated cells could live and function normally in terms of insulin-secretion within microfabricated environments for extended periods of time. This novel research shows the potential of using microfabricated biocapsules for the encapsulation of several different cell xenografts. The semipermeability

  14. Total pancreatectomy and islet autotransplantation: A decade nationwide analysis

    PubMed Central

    Fazlalizadeh, Reza; Moghadamyeghaneh, Zhobin; Demirjian, Aram N; Imagawa, David K; Foster, Clarence E; Lakey, Jonathan R; Stamos, Michael J; Ichii, Hirohito

    2016-01-01

    AIM: To investigate outcomes and predictors of in-hospital morbidity and mortality after total pancreatectomy (TP) and islet autotransplantation. METHODS: The nationwide inpatient sample (NIS) database was used to identify patients who underwent TP and islet autotransplantation (IAT) between 2002-2012 in the United States. Variables of interest were inherent variables of NIS database which included demographic data (age, sex, and race), comorbidities (such as diabetes mellitus, hypertension, and deficiency anemia), and admission type (elective vs non-elective). The primary endpoints were mortality and postoperative complications according to the ICD-9 diagnosis codes which were reported as the second to 25th diagnosis of patients in the database. Risk adjusted analysis was performed to investigate morbidity predictors. Multivariate regression analysis was used to identify predictors of in-hospital morbidity. RESULTS: We evaluated a total of 923 patients who underwent IAT after pancreatectomy during 2002-2012. Among them, there were 754 patients who had TP + IAT. The most common indication of surgery was chronic pancreatitis (86%) followed by acute pancreatitis (12%). The number of patients undergoing TP + IAT annually significantly increased during the 11 years of study from 53 cases in 2002 to 155 cases in 2012. Overall mortality and morbidity of patients were 0% and 57.8 %, respectively. Post-surgical hypoinsulinemia was reported in 42.3% of patients, indicating that 57.7% of patients were insulin independent during hospitalization. Predictors of in-hospital morbidity were obesity [adjusted odds ratio (AOR): 3.02, P = 0.01], fluid and electrolyte disorders (AOR: 2.71, P < 0.01), alcohol abuse (AOR: 2.63, P < 0.01), and weight loss (AOR: 2.43, P < 0.01). CONCLUSION: TP + IAT is a safe procedure with no mortality, acceptable morbidity, and achieved high rate of early insulin independence. Obesity is the most significant predictor of in-hospital morbidity. PMID

  15. Metastatic pancreatic islet cell carcinoma to the orbit: a case report.

    PubMed

    Nasr, Amin M.; Teichmann, Klaus; Dabbagh, Najwa; Huaman, Antonio M.

    1998-03-01

    We report a rare case of pancreatic islet cell carcinoma metastatic to the orbit in a 29-year-old woman. The initial symptomatology, progression of the disease, and radiologic and histopathologic findings are presented and discussed.

  16. Engineered VEGF-releasing PEG-MAL hydrogel for pancreatic islet vascularization

    PubMed Central

    Phelps, Edward A.; Templeman, Kellie L.; Thulé, Peter M.; García, Andrés J.

    2013-01-01

    Biofunctionalized polyethylene glycol maleimide (PEG-MAL) hydrogels were engineered as a platform to deliver pancreatic islets to the small bowel mesentery and promote graft vascularization. VEGF, a potent stimulator of angiogenesis, was incorporated into the hydrogel to be released in an on-demand manner through enzymatic degradation. PEG-MAL hydrogel enabled extended in vivo release of VEGF. Isolated rat islets encapsulated in PEG-MAL hydrogels remained viable in culture and secreted insulin. Islets encapsulated in PEG-MAL matrix and transplanted to the small bowel mesentery of healthy rats grafted to the host tissue and revascularized by 4 weeks. Addition of VEGF release to the PEG-MAL matrix greatly augmented the vascularization response. These results establish PEG-MAL engineered matrices as a vascular-inductive cell delivery vehicle and warrant their further investigation as islet transplantation vehicles in diabetic animal models. PMID:25787738

  17. 'Giving and taking': endothelial and beta-cells in the islets of Langerhans.

    PubMed

    Eberhard, Daniel; Kragl, Martin; Lammert, Eckhard

    2010-08-01

    The beta-cells of the islets of Langerhans are embedded in a dense capillary network. The blood vessels supply the islet cells with nutrients and oxygen, and in turn take up the secreted islet hormones to deliver them to target tissues. In addition, vessels provide a basement membrane, which optimizes islet function. In this review we focus on the dynamic interactions between blood vessels and beta-cells, which are pivotal for enhancing insulin expression and beta-cell proliferation in response to increased insulin demand during body growth, pregnancy, and virtually all conditions associated with insulin resistance. Importantly, a failure in this adaptive response might contribute to the onset of type 2 diabetes mellitus.

  18. Use of Glucagon-Like Peptide-1 Agonists to Improve Islet Graft Performance

    PubMed Central

    Wang, Yong; Qi, Meirigeng; McGarrigle, James J.; Rady, Brian; Davis, Maureen; Vaca, Pilar

    2013-01-01

    Human islet transplantation is an effective and promising therapy for Type I diabetes. However, long-term insulin independence is both difficult to achieve and inconsistent. De novo or early administration of incretin-based drugs is being explored for improving islet engraftment. In addition to its glucose-dependent insulinotropic effects, incretins also lower postprandial glucose excursion by inhibiting glucagon secretion, delaying gastric emptying, and can protect beta-cell function. Incretin therapy has so far proven clinically safe and tolerable with little hypoglycemic risk. The present review aims at highlighting the new frontiers in research involving incretins from both in vitro and in vivo animal studies in the field of islet transplant. It also provides an overview of the current clinical status of incretin usage in islet transplantation in the management of Type I diabetes. PMID:23925432

  19. GPR54 peptide agonists stimulate insulin secretion from murine, porcine and human islets.

    PubMed

    Bowe, James E; Foot, Victoria L; Amiel, Stephanie A; Huang, Gao Cai; Lamb, Morgan W; Lakey, Jonathan; Jones, Peter M; Persaud, Shanta J

    2012-01-01

    This study was designed to determine the effects of 10 and 13 amino acid forms of kisspeptin on dynamic insulin secretion from mammalian islets since it is not clear from published data whether the shorter peptide is stimulatory while the longer peptide inhibits insulin release. Insulin secretion was measured by radioimmunoassay following perifusion of human, pig, rat and mouse isolated islets with kisspeptin-10 or kisspeptin-13 in the presence of 20 mM glucose. Both peptides stimulated rapid, reversible potentiation of glucose-stimulated insulin secretion from islets of all species tested. These data indicate that both kisspeptin-10 and kisspeptin-13, which is an extension of kisspeptin-10 by three amino acids, act directly at islet β-cells of various species to potentiate insulin secretion, and suggest that inhibitory effects reported in earlier studies may reflect differences in experimental protocols.

  20. Multiplex Sequential Immunoprecipitation of Insulin Secretory Granule Proteins from Radiolabeled Pancreatic Islets.

    PubMed

    Guest, Paul C

    2017-01-01

    Pulse radiolabeling of cells with radioactive amino acids is a common method for tracking the biosynthesis of proteins. Specific proteins can then be immunoprecipitated and analyzed by electrophoresis and imaging techniques. This chapter presents a protocol for the biosynthetic labeling of pancreatic islets with (35)S-methionine, followed by multiplex sequential immunoprecipitation of insulin and three other secretory granule accessory proteins. This provided a means of distinguishing those pancreatic islet proteins with different biosynthetic rates in response to the media glucose concentrations.