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Sample records for glucagon expressing cells

  1. Human adipose tissue-derived mesenchymal stem cells differentiate into insulin, somatostatin, and glucagon expressing cells

    SciTech Connect

    Timper, Katharina; Seboek, Dalma; Eberhardt, Michael; Linscheid, Philippe; Christ-Crain, Mirjam; Keller, Ulrich; Mueller, Beat; Zulewski, Henryk . E-mail: henryk.zulewski@unibas.ch

    2006-03-24

    Mesenchymal stem cells (MSC) from mouse bone marrow were shown to adopt a pancreatic endocrine phenotype in vitro and to reverse diabetes in an animal model. MSC from human bone marrow and adipose tissue represent very similar cell populations with comparable phenotypes. Adipose tissue is abundant and easily accessible and could thus also harbor cells with the potential to differentiate in insulin producing cells. We isolated human adipose tissue-derived MSC from four healthy donors. During the proliferation period, the cells expressed the stem cell markers nestin, ABCG2, SCF, Thy-1 as well as the pancreatic endocrine transcription factor Isl-1. The cells were induced to differentiate into a pancreatic endocrine phenotype by defined culture conditions within 3 days. Using quantitative PCR a down-regulation of ABCG2 and up-regulation of pancreatic developmental transcription factors Isl-1, Ipf-1, and Ngn3 were observed together with induction of the islet hormones insulin, glucagon, and somatostatin.

  2. Characterization of glucagon-expressing neurons in the chicken retina

    PubMed Central

    Fischer, Andy J.; Skorupa, Dana; Schonberg, David L.; Walton, Nathaniel A.

    2008-01-01

    We have recently identified large glucagon-expressing neurons that densely ramify neurites in the peripheral edge of the retina and regulate the proliferation of progenitors in the circumferential marginal zone (CMZ) of the postnatal chicken eye (Fischer et al., 2005). However, nothing is known about the transmitters and proteins that are expressed by the glucagon-expressing neurons in the avian retina. We used antibodies to cell-distinguishing markers to better characterize the different types of glucagon-expressing neurons. We found that the large glucagon-expressing neurons were immunoreactive for substance P, neurofilament, Pax6, AP2α, HuD, calretinin, trkB and trkC. Colocalization of glucagon and substance P in the large glucagon-expressing neurons indicates that these cells are the “bullwhip cells” that have been briefly described by Ehrlich, Keyser and Karten (1987). Similar to the bullwhip cells, the conventional glucagon-expressing amacrine cells were immunoreactive for calretinin, HuD, Pax6, and AP2α. Unlike bullwhip cells, the conventional glucagon-expressing amacrine cells were immunoreactive for GABA. While glucagon-immunoreactive amacrine cells were negative for substance P in central regions of the retina, a subset of this type of amacrine cell was immunoreactive for substance P in far peripheral regions of the retina. An additional type of glucagon/substance P-expressing neuron, resembling the bullwhip cells, was found in far peripheral and dorsal regions of the retina. Based on morphology, distribution within the retina, and histological markers, we conclude that there may be 4 different types of glucagon-expressing neurons in the avian retina. PMID:16572462

  3. Glucagon

    MedlinePlus

    Glucagon is a hormone produced in the pancreas. Glucagon is used to raise very low blood sugar. Glucagon is also used in diagnostic ... sweating, confusion, nervousness or irritability, sudden changes in behavior or mood, headache, numbness or tingling around the ...

  4. Glucagon secretion from pancreatic α-cells

    PubMed Central

    Briant, Linford; Salehi, Albert; Vergari, Elisa; Zhang, Quan; Rorsman, Patrik

    2016-01-01

    Type 2 diabetes involves a ménage à trois of impaired glucose regulation of pancreatic hormone release: in addition to impaired glucose-induced insulin secretion, the release of the hyperglycaemic hormone glucagon becomes dysregulated; these last-mentioned defects exacerbate the metabolic consequences of hypoinsulinaemia and are compounded further by hypersecretion of somatostatin (which inhibits both insulin and glucagon secretion). Glucagon secretion has been proposed to be regulated by either intrinsic or paracrine mechanisms, but their relative significance and the conditions under which they operate are debated. Importantly, the paracrine and intrinsic modes of regulation are not mutually exclusive; they could operate in parallel to control glucagon secretion. Here we have applied mathematical modelling of α-cell electrical activity as a novel means of dissecting the processes that underlie metabolic regulation of glucagon secretion. Our analyses indicate that basal hypersecretion of somatostatin and/or increased activity of somatostatin receptors may explain the loss of adequate counter-regulation under hypoglycaemic conditions, as well as the physiologically inappropriate stimulation of glucagon secretion during hyperglycaemia seen in diabetic patients. We therefore advocate studying the interaction of the paracrine and intrinsic mechanisms; unifying these processes may give a more complete picture of the regulation of glucagon secretion from α-cells than studying the individual parts. PMID:27044683

  5. Glucagon

    MedlinePlus

    ... pancreas. Glucagon is used to raise very low blood sugar. Glucagon is also used in diagnostic testing of ... discovering that the patient is unconscious from low blood sugar. After the injection, the patient should be turned ...

  6. Oleic acid and glucose regulate glucagon-like peptide 1 receptor expression in a rat pancreatic ductal cell line

    SciTech Connect

    Zhang, Leshuai W.; McMahon Tobin, Grainne A.; Rouse, Rodney L.

    2012-10-15

    The glucagon-like peptide 1 receptor (GLP1R) plays a critical role in glucose metabolism and has become an important target for a growing class of drugs designed to treat type 2 diabetes. In vitro studies were designed to investigate the effect of the GLP1R agonist, exenatide (Ex4), in “on-target” RIN-5mF (islet) cells as well as in “off-target” AR42J (acinar) and DSL-6A/C1 (ductal) cells in a diabetic environment. Ex4 increased islet cell proliferation but did not affect acinar cells or ductal cells at relevant concentrations. A high caloric, high fat diet is a risk factor for impaired glucose tolerance and type-2 diabetes. An in vitro Oleic acid (OA) model was used to investigate the effect of Ex4 in a high calorie, high fat environment. At 0.1 and 0.4 mM, OA mildly decreased the proliferation of all pancreatic cell types. Ex4 did not potentiate the inhibitory effect of OA on cell proliferation. Akt phosphorylation in response to Ex4 was diminished in OA-treated ductal cells. GLP1R protein detected by western blot was time and concentration dependently decreased after glucose stimulation in OA-treated ductal cells. In ductal cells, OA treatment altered the intracellular localization of GLP1R and its co-localization with early endosome and recycling endosomes. Chloroquine (lysosomal inhibitor), N-acetyl-L-cysteine (reactive oxygen species scavenger) and wortmannin (a phosphatidylinositol-3-kinase inhibitor), fully or partially, rescued GLP1R protein in OA-pretreated, glucose-stimulated ductal cells. The impact of altered regulation on phenotype/function is presently unknown. However, these data suggest that GLP1R regulation in ductal cells can be altered by a high fat, high calorie environment. -- Highlights: ► Exenatide did not inhibit islet, acinar or ductal cell proliferation. ► GLP1R protein decreased after glucose stimulation in oleic acid-treated ductal cells. ► Oleic acid treatment altered localization of GLP1R with early and recycling

  7. Autophagy deficiency in β cells blunts incretin-induced suppression of glucagon release from α cells.

    PubMed

    Kim, Min Joo; Choi, Ok Kyong; Chae, Kyung Sil; Lee, Hakmo; Chung, Sung Soo; Ham, Dong-Sik; Kim, Ji-Won; Yoon, Kun-Ho; Park, Kyong Soo; Jung, Hye Seung

    2015-09-01

    Incretin-based therapy such as GLP-1 receptor agonists and DPP-4 inhibitors for type 2 diabetes mellitus is characterized by glucose-dependent insulin secretion and glucose-inhibited glucagon secretion. Recently, autophagy deficiency in islet β cells has been shown to contribute to the pathogenesis of type 2 diabetes mellitus however, with the role of incretin has not been established. To evaluate the role of autophagy in incretin effects, 8-week-old male β cell-specific Atg7 knockout (Atg7(Δβ cell)) mice and wild-type mice were administered vildagliptin for 12 weeks. Vildagliptin treatment improved glucose intolerance and hypoinsulinemia; however, it failed to suppress serum glucagon levels after glucose loading in the Atg7(Δβ cell) mice. Ex vivo glucose-induced glucagon suppression was also blunted in the islets from vildagliptin-treated Atg7(Δβ cell) mice. The α cell mass was not affected by β cell autophagy deficiency or vildagliptin. However, glucagon mRNA expression was significantly increased by vildagliptin in the autophagy-deficient islets, and was significantly reduced by vildagliptin in wild-type islets. Pancreatic glucagon contents were not in agreement with the changes in mRNA expression, suggesting a dysregulation in glucagon translation and secretion. In vitro studies revealed that glucose-stimulated cAMP production was impaired in the autophagy-deficient islets exposed to exendin-4. Taken together, the results suggest that the constitutive autophagy in β cells could regulate incretin-induced glucagon expression and release in α cells, and that cAMP may play a role in this process. PMID:26744903

  8. Autophagy deficiency in β cells blunts incretin-induced suppression of glucagon release from α cells

    PubMed Central

    Kim, Min Joo; Choi, Ok Kyong; Chae, Kyung Sil; Lee, Hakmo; Chung, Sung Soo; Ham, Dong-Sik; Kim, Ji-Won; Yoon, Kun-Ho; Park, Kyong Soo; Jung, Hye Seung

    2015-01-01

    Incretin-based therapy such as GLP-1 receptor agonists and DPP-4 inhibitors for type 2 diabetes mellitus is characterized by glucose-dependent insulin secretion and glucose-inhibited glucagon secretion. Recently, autophagy deficiency in islet β cells has been shown to contribute to the pathogenesis of type 2 diabetes mellitus however, with the role of incretin has not been established. To evaluate the role of autophagy in incretin effects, 8-week-old male β cell-specific Atg7 knockout (Atg7Δβ cell) mice and wild-type mice were administered vildagliptin for 12 weeks. Vildagliptin treatment improved glucose intolerance and hypoinsulinemia; however, it failed to suppress serum glucagon levels after glucose loading in the Atg7Δβ cell mice. Ex vivo glucose-induced glucagon suppression was also blunted in the islets from vildagliptin-treated Atg7Δβ cell mice. The α cell mass was not affected by β cell autophagy deficiency or vildagliptin. However, glucagon mRNA expression was significantly increased by vildagliptin in the autophagy-deficient islets, and was significantly reduced by vildagliptin in wild-type islets. Pancreatic glucagon contents were not in agreement with the changes in mRNA expression, suggesting a dysregulation in glucagon translation and secretion. In vitro studies revealed that glucose-stimulated cAMP production was impaired in the autophagy-deficient islets exposed to exendin-4. Taken together, the results suggest that the constitutive autophagy in β cells could regulate incretin-induced glucagon expression and release in α cells, and that cAMP may play a role in this process. PMID:26744903

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

    PubMed Central

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

    2015-01-01

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

  10. Heterodimeric Pbx-Prep1 homeodomain protein binding to the glucagon gene restricting transcription in a cell type-dependent manner.

    PubMed

    Herzig, S; Fuzesi, L; Knepel, W

    2000-09-01

    Homeodomain proteins specify developmental pathways and cell-specific gene transcription whereby proteins of the PBC subclass can direct target gene specificity of Hox proteins. Proteins encoded by nonclustered homeobox genes have been shown to be essential for cell lineage differentiation and gene expression in pancreatic islets. Using specific antiserum in an electrophoretic mobility shift assay and in vitro transcribed/translated proteins, the nuclear proteins binding domain B of the G3 enhancer-like element of the glucagon gene were identified in the present study as heterodimers consisting of the ubiquitously expressed homeodomain protein Prep1 and the also widely expressed PBC homeoprotein Pbx (isoform 1a, 1b, or 2). These heterodimeric complexes were found to bind also to the glucagon cAMP response element and to a newly identified element termed G5 (from -169 to -140). Whereas the expression of Prep1 or Pbx forms alone had no effect, coexpression of Pbx1a/1b-Prep1 inhibited the glucagon promoter when activated by cotransfected Pax6 or another transcription factor in non-glucagon-producing cells. In contrast, in glucagon-producing pancreatic islet cells, Pbx-Prep1 had no effect on GAL4-Pax6-induced mutant glucagon promoter activity or on Pax6-dependent wild-type glucagon promoter activity. Furthermore, 5'-deletion of G5 enhanced glucagon promoter activity in a non-glucagon-producing cell line but not in glucagon-producing islet cells. This study thus identifies a novel target and Hox-independent function of Pbx-Prep1 heterodimers that, through repression of glucagon gene transcription in non-glucagon-producing cells, may help to establish islet cell-specific expression of the glucagon gene. PMID:10869353

  11. Model for Glucagon Secretion by Pancreatic α-Cells

    PubMed Central

    González-Vélez, Virginia; Dupont, Geneviève; Gil, Amparo; González, Alejandro; Quesada, Iván

    2012-01-01

    Glucagon hormone is synthesized and released by pancreatic α-cells, one of the islet-cell types. This hormone, along with insulin, maintains blood glucose levels within the physiological range. Glucose stimulates glucagon release at low concentrations (hypoglycemia). However, the mechanisms involved in this secretion are still not completely clear. Here, using experimental calcium time series obtained in mouse pancreatic islets at low and high glucose conditions, we propose a glucagon secretion model for α-cells. Our model takes into account that the resupply of releasable granules is not only controlled by cytoplasmic , as in other neuroendocrine and endocrine cells, but also by the level of extracellular glucose. We found that, although calcium oscillations are highly variable, the average secretion rates predicted by the model fall into the range of values reported in the literature, for both stimulated and non-stimulated conditions. For low glucose levels, the model predicts that there would be a well-controlled number of releasable granules refilled slowly from a large reserve pool, probably to ensure a secretion rate that could last for several minutes. Studying the α-cell response to the addition of insulin at low glucose, we observe that the presence of insulin reduces glucagon release by decreasing the islet level. This observation is in line with previous work reporting that dynamics, mainly frequency, is altered by insulin [1]. Thus, the present results emphasize the main role played by and glucose in the control of glucagon secretion by α-cells. Our modeling approach also shows that calcium oscillations potentiate glucagon secretion as compared to constant levels of this cellular messenger. Altogether, the model sheds new light on the subcellular mechanisms involved in α-cell exocytosis, and provides a quantitative predictive tool for studying glucagon secretion modulators in physiological and pathological conditions. PMID:22412861

  12. The β-cell specific transcription factor Nkx6.1 inhibits glucagon gene transcription by interfering with Pax6

    PubMed Central

    Gauthier, Benoit R.; Gosmain, Yvan; Mamin, Aline; Philippe, Jacques

    2007-01-01

    The transcription factor Nkx6.1 is required for the establishment of functional insulin-producing β-cells in the endocrine pancreas. Overexpression of Nkx6.1 has been shown to inhibit glucagon gene expression while favouring insulin gene activation. Down-regulation resulted in the opposite effect, suggesting that absence of Nkx6.1 favours glucagon gene expression. To understand the mechanism by which Nkx6.1 suppresses glucagon gene expression, we studied its effect on the glucagon gene promoter activity in non-islet cells using transient transfections and gel-shift analyses. In glucagonoma cells transfected with an Nkx6.1-encoding vector, the glucagon promoter activity was reduced by 65%. In BHK21 cells, Nkx6.1 inhibited by 93% Pax6-mediated activation of the glucagon promoter, whereas Cdx2/3 and Maf stimulations were unaltered. Although Nkx6.1 could interact with both the G1 and G3 element, only the former displayed specificity for Nkx6.1. Mutagenesis of the three potential AT-rich motifs within the G1 revealed that only the Pax6-binding site preferentially interacted with Nkx6.1. Chromatin immunoprecipitation confirmed interaction of Nkx6.1 with the glucagon promoter and revealed a direct competition for binding between Pax6 and Nkx6.1. A weak physical interaction between Pax6 and Nkx6.1 was detected in vitro and in vivo suggesting that Nkx6.1 predominantly inhibits glucagon gene transcription through G1-binding competition. We suggest that cell-specific expression of the glucagon gene may only proceed when Nkx6.1, in combination with Pdx1 and Pax4, are silenced in early α-cell precursors. PMID:17263687

  13. Angptl4 links α-cell proliferation following glucagon receptor inhibition with adipose tissue triglyceride metabolism

    PubMed Central

    Ben-Zvi, Danny; Barrandon, Ornella; Hadley, Stephanie; Blum, Barak; Peterson, Quinn P.; Melton, Douglas A.

    2015-01-01

    Type 2 diabetes is characterized by a reduction in insulin function and an increase in glucagon activity that together result in hyperglycemia. Glucagon receptor antagonists have been developed as drugs for diabetes; however, they often increase glucagon plasma levels and induce the proliferation of glucagon-secreting α-cells. We find that the secreted protein Angiopoietin-like 4 (Angptl4) is up-regulated via Pparγ activation in white adipose tissue and plasma following an acute treatment with a glucagon receptor antagonist. Induction of adipose angptl4 and Angptl4 supplementation promote α-cell proliferation specifically. Finally, glucagon receptor antagonist improves glycemia in diet-induced obese angptl4 knockout mice without increasing glucagon levels or α-cell proliferation, underscoring the importance of this protein. Overall, we demonstrate that triglyceride metabolism in adipose tissue regulates α-cells in the endocrine pancreas. PMID:26621734

  14. Glucagon gene 5'-flanking sequences direct expression of simian virus 40 large T antigen to the intestine, producing carcinoma of the large bowel in transgenic mice.

    PubMed

    Lee, Y C; Asa, S L; Drucker, D J

    1992-05-25

    Glucagon and the glucagon-like peptides play important roles in the regulation of glucose homeostasis. Previous studies have demonstrated that approximately 1300 base pairs of rat glucagon gene 5'-flanking sequences direct transgene expression to the pancreas and brain, but not to the intestine, of transgenic mice. These observations suggested that different tissue-specific enhancer elements mediate activation of glucagon gene transcription in the pancreas and intestine. We have now generated mice that express SV40 large T antigen under the control of approximately 2000 base pairs of glucagon gene 5'-flanking sequences. Transgene expression was observed in the brain and pancreas in association with the development of pancreatic endocrine tumors. In contrast to the mice described previously, we also detected transgene expression throughout the gastrointestinal tract in endocrine cells of the stomach and small and large intestine. Focal areas of enteroendocrine cell hyperplasia in the large bowel invariably progressed to invasive and metastasizing plurihormonal endocrine carcinoma, which was clinically and pathologically evident by 4 weeks of age. In contrast, transgene expression in the small bowel and stomach was not associated with progression to either hyperplasia or carcinoma. The results of these studies provide functional evidence for the existence of an upstream cis-acting regulatory domain that directs glucagon gene transcription to the endocrine cells of the intestine in transgenic mice. PMID:1587847

  15. Glucagon-like-peptide-1 receptor expression in normal and diseased human thyroid and pancreas.

    PubMed

    Waser, Beatrice; Blank, Annika; Karamitopoulou, Eva; Perren, Aurel; Reubi, Jean C

    2015-03-01

    Glucagon-like-peptide-1 (GLP1) analogs may induce thyroid or pancreatic diseases in animals, raising questions about their use in diabetic patients. There is, however, controversy regarding expression of GLP1 receptors (GLP1R) in human normal and diseased thyroid and pancreas. Here, 221 human thyroid and pancreas samples were analyzed for GLP1R immunohistochemistry and compared with quantitative in vitro GLP1R autoradiography. Neither normal nor hyperplastic human thyroids containing parafollicular C cells express GLP1R with either method. Papillary thyroid cancer do not, and medullary thyroid carcinomas rarely express GLP1R. Insulin- and somatostatin-producing cells in the normal pancreas express a high density of GLP1R, whereas acinar cells express them in low amounts. Ductal epithelial cells do not express GLP1R. All benign insulinomas express high densities of GLP1R, whereas malignant insulinomas rarely express them. All ductal pancreatic carcinomas are GLP1R negative, whereas 6/20 PanIN 1/2 and 0/12 PanIN 3 express GLP1R. Therefore, normal thyroid, including normal and hyperplastic C cells, or papillary thyroid cancer are not targets for GLP1 analogs in humans. Conversely, all pancreatic insulin- and somatostatin-producing cells are physiological GLP1 targets, as well as most acini. As normal ductal epithelial cells or PanIN 3 or ductal pancreatic carcinomas do not express GLP1R, it seems unlikely that GLP1R is related to neoplastic transformation in pancreas. GLP1R-positive medullary thyroid carcinomas and all benign insulinomas are candidates for in vivo GLP1R targeting. PMID:25216224

  16. In Situ Electrophysiological Examination of Pancreatic α Cells in the Streptozotocin-Induced Diabetes Model, Revealing the Cellular Basis of Glucagon Hypersecretion

    PubMed Central

    Huang, Ya-Chi; Rupnik, Marjan S.; Karimian, Negar; Herrera, Pedro L.; Gilon, Patrick; Feng, Zhong-Ping; Gaisano, Herbert Y.

    2013-01-01

    Early-stage type 1 diabetes (T1D) exhibits hyperglucagonemia by undefined cellular mechanisms. Here we characterized α-cell voltage-gated ion channels in a streptozotocin (STZ)-induced diabetes model that lead to increased glucagon secretion mimicking T1D. GYY mice expressing enhanced yellow fluorescence protein in α cells were used to identify α cells within pancreas slices. Mice treated with low-dose STZ exhibited hyperglucagonemia, hyperglycemia, and glucose intolerance, with 71% reduction of β-cell mass. Although α-cell mass of STZ-treated mice remained unchanged, total pancreatic glucagon content was elevated, coinciding with increase in size of glucagon granules. Pancreas tissue slices enabled in situ examination of α-cell electrophysiology. α cells of STZ-treated mice exhibited the following: 1) increased exocytosis (serial depolarization-induced capacitance), 2) enhanced voltage-gated Na+ current density, 3) reduced voltage-gated K+ current density, and 4) increased action potential (AP) amplitude and firing frequency. Hyperglucagonemia in STZ-induced diabetes is thus likely due to increased glucagon content arising from enlarged glucagon granules and increased AP firing frequency and amplitude coinciding with enhanced Na+ and reduced K+ currents. These alterations may prime α cells in STZ-treated mice for more glucagon release per cell in response to low glucose stimulation. Thus, our study provides the first insight that STZ treatment sensitizes release mechanisms of α cells. PMID:23043159

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

    PubMed

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

    1985-04-01

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

  18. Dapagliflozin stimulates glucagon secretion at high glucose: experiments and mathematical simulations of human A-cells.

    PubMed

    Pedersen, Morten Gram; Ahlstedt, Ingela; El Hachmane, Mickaël F; Göpel, Sven O

    2016-01-01

    Glucagon is one of the main regulators of blood glucose levels and dysfunctional stimulus secretion coupling in pancreatic A-cells is believed to be an important factor during development of diabetes. However, regulation of glucagon secretion is poorly understood. Recently it has been shown that Na(+)/glucose co-transporter (SGLT) inhibitors used for the treatment of diabetes increase glucagon levels in man. Here, we show experimentally that the SGLT2 inhibitor dapagliflozin increases glucagon secretion at high glucose levels both in human and mouse islets, but has little effect at low glucose concentrations. Because glucagon secretion is regulated by electrical activity we developed a mathematical model of A-cell electrical activity based on published data from human A-cells. With operating SGLT2, simulated glucose application leads to cell depolarization and inactivation of the voltage-gated ion channels carrying the action potential, and hence to reduce action potential height. According to our model, inhibition of SGLT2 reduces glucose-induced depolarization via electrical mechanisms. We suggest that blocking SGLTs partly relieves glucose suppression of glucagon secretion by allowing full-scale action potentials to develop. Based on our simulations we propose that SGLT2 is a glucose sensor and actively contributes to regulation of glucagon levels in humans which has clinical implications. PMID:27535321

  19. Dapagliflozin stimulates glucagon secretion at high glucose: experiments and mathematical simulations of human A-cells

    PubMed Central

    Pedersen, Morten Gram; Ahlstedt, Ingela; El Hachmane, Mickaël F.; Göpel, Sven O.

    2016-01-01

    Glucagon is one of the main regulators of blood glucose levels and dysfunctional stimulus secretion coupling in pancreatic A-cells is believed to be an important factor during development of diabetes. However, regulation of glucagon secretion is poorly understood. Recently it has been shown that Na+/glucose co-transporter (SGLT) inhibitors used for the treatment of diabetes increase glucagon levels in man. Here, we show experimentally that the SGLT2 inhibitor dapagliflozin increases glucagon secretion at high glucose levels both in human and mouse islets, but has little effect at low glucose concentrations. Because glucagon secretion is regulated by electrical activity we developed a mathematical model of A-cell electrical activity based on published data from human A-cells. With operating SGLT2, simulated glucose application leads to cell depolarization and inactivation of the voltage-gated ion channels carrying the action potential, and hence to reduce action potential height. According to our model, inhibition of SGLT2 reduces glucose-induced depolarization via electrical mechanisms. We suggest that blocking SGLTs partly relieves glucose suppression of glucagon secretion by allowing full-scale action potentials to develop. Based on our simulations we propose that SGLT2 is a glucose sensor and actively contributes to regulation of glucagon levels in humans which has clinical implications. PMID:27535321

  20. β-Cell Uncoupling Protein 2 Regulates Reactive Oxygen Species Production, Which Influences Both Insulin and Glucagon Secretion

    PubMed Central

    Robson-Doucette, Christine A.; Sultan, Sobia; Allister, Emma M.; Wikstrom, Jakob D.; Koshkin, Vasilij; Bhatacharjee, Alpana; Prentice, Kacey J.; Sereda, Samuel B.; Shirihai, Orian S.; Wheeler, Michael B.

    2011-01-01

    OBJECTIVE The role of uncoupling protein 2 (UCP2) in pancreatic β-cells is highly debated, partly because of the broad tissue distribution of UCP2 and thus limitations of whole-body UCP2 knockout mouse models. To investigate the function of UCP2 in the β-cell, β-cell–specific UCP2 knockout mice (UCP2BKO) were generated and characterized. RESEARCH DESIGN AND METHODS UCP2BKO mice were generated by crossing loxUCP2 mice with mice expressing rat insulin promoter-driven Cre recombinase. Several in vitro and in vivo parameters were measured, including respiration rate, mitochondrial membrane potential, islet ATP content, reactive oxygen species (ROS) levels, glucose-stimulated insulin secretion (GSIS), glucagon secretion, glucose and insulin tolerance, and plasma hormone levels. RESULTS UCP2BKO β-cells displayed mildly increased glucose-induced mitochondrial membrane hyperpolarization but unchanged rates of uncoupled respiration and islet ATP content. UCP2BKO islets had elevated intracellular ROS levels that associated with enhanced GSIS. Surprisingly, UCP2BKO mice were glucose-intolerant, showing greater α-cell area, higher islet glucagon content, and aberrant ROS-dependent glucagon secretion under high glucose conditions. CONCLUSIONS Using a novel β-cell–specific UCP2KO mouse model, we have shed light on UCP2 function in primary β-cells. UCP2 does not behave as a classical metabolic uncoupler in the β-cell, but has a more prominent role in the regulation of intracellular ROS levels that contribute to GSIS amplification. In addition, β-cell UCP2 contributes to the regulation of intraislet ROS signals that mediate changes in α-cell morphology and glucagon secretion. PMID:21984579

  1. Farnesoid X receptor inhibits glucagon-like peptide-1 production by enteroendocrine L cells.

    PubMed

    Trabelsi, Mohamed-Sami; Daoudi, Mehdi; Prawitt, Janne; Ducastel, Sarah; Touche, Véronique; Sayin, Sama I; Perino, Alessia; Brighton, Cheryl A; Sebti, Yasmine; Kluza, Jérôme; Briand, Olivier; Dehondt, Hélène; Vallez, Emmanuelle; Dorchies, Emilie; Baud, Grégory; Spinelli, Valeria; Hennuyer, Nathalie; Caron, Sandrine; Bantubungi, Kadiombo; Caiazzo, Robert; Reimann, Frank; Marchetti, Philippe; Lefebvre, Philippe; Bäckhed, Fredrik; Gribble, Fiona M; Schoonjans, Kristina; Pattou, François; Tailleux, Anne; Staels, Bart; Lestavel, Sophie

    2015-01-01

    Bile acids are signalling molecules, which activate the transmembrane receptor TGR5 and the nuclear receptor FXR. BA sequestrants (BAS) complex bile acids in the intestinal lumen and decrease intestinal FXR activity. The BAS-BA complex also induces glucagon-like peptide-1 (GLP-1) production by L cells which potentiates β-cell glucose-induced insulin secretion. Whether FXR is expressed in L cells and controls GLP-1 production is unknown. Here, we show that FXR activation in L cells decreases proglucagon expression by interfering with the glucose-responsive factor Carbohydrate-Responsive Element Binding Protein (ChREBP) and GLP-1 secretion by inhibiting glycolysis. In vivo, FXR deficiency increases GLP-1 gene expression and secretion in response to glucose hence improving glucose metabolism. Moreover, treatment of ob/ob mice with the BAS colesevelam increases intestinal proglucagon gene expression and improves glycaemia in a FXR-dependent manner. These findings identify the FXR/GLP-1 pathway as a new mechanism of BA control of glucose metabolism and a pharmacological target for type 2 diabetes. PMID:26134028

  2. Farnesoid X Receptor Inhibits Glucagon-Like Peptide-1 Production by Enteroendocrine L-cells

    PubMed Central

    TRABELSI, Mohamed-Sami; DAOUDI, Mehdi; PRAWITT, Janne; DUCASTEL, Sarah; TOUCHE, Véronique; SAYIN, Sama I.; PERINO, Alessia; BRIGHTON, Cheryl A.; SEBTI, Yasmine; KLUZA, Jérôme; BRIAND, Olivier; DEHONDT, Hélène; VALLEZ, Emmanuelle; DORCHIES, Emilie; BAUD, Grégory; SPINELLI, Valeria; HENNUYER, Nathalie; CARON, Sandrine; BANTUBUNGI, Kadiombo; CAIAZZO, Robert; REIMANN, Frank; MARCHETTI, Philippe; LEFEBVRE, Philippe; BÄCKHED, Fredrik; GRIBBLE, Fiona M.; SCHOONJANS, Kristina; PATTOU, François; TAILLEUX, Anne; STAELS, Bart; LESTAVEL, Sophie

    2015-01-01

    Bile acids (BA) are signalling molecules which activate the transmembrane receptor TGR5 and the nuclear receptor FXR. BA sequestrants (BAS) complex BA in the intestinal lumen and decrease intestinal FXR activity. The BAS-BA complex also induces Glucagon-Like Peptide-1 (GLP-1) production by L-cells which potentiates β-cell glucose-induced insulin secretion. Whether FXR is expressed in L-cells and controls GLP-1 production is unknown. Here we show that FXR activation in L-cells decreases proglucagon expression by interfering with the glucose-responsive factor Carbohydrate-Responsive Element Binding Protein (ChREBP) and GLP-1 secretion by inhibiting glycolysis. In vivo, FXR-deficiency increases GLP-1 gene expression and secretion in response to glucose hence improving glucose metabolism. Moreover, treatment of ob/ob mice with the BAS colesevelam increases intestinal proglucagon gene expression and improves glycemia in a FXR-dependent manner. These findings identify the FXR/GLP-1 pathway as a new mechanism of BA control of glucose metabolism and a pharmacological target for type 2 diabetes. PMID:26134028

  3. High fat diet impairs the function of glucagon-like peptide-1 producing L-cells

    PubMed Central

    Richards, Paul; Pais, Ramona; Habib, Abdella M.; Brighton, Cheryl A.; Yeo, Giles S.H.; Reimann, Frank; Gribble, Fiona M.

    2016-01-01

    Glucagon-like peptide-1 (GLP-1) acts as a satiety signal and enhances insulin release. This study examined how GLP-1 production from intestinal L-cells is modified by dietary changes. Methods Transgenic mouse models were utilized in which L-cells could be purified by cell specific expression of a yellow fluorescent protein, Venus. Mice were fed on chow or 60% high fat diet (HFD) for 2 or 16 weeks. L-cells were purified by flow cytometry and analysed by microarray and quantitative RT-PCR. Enteroendocrine cell populations were examined by FACS analysis, and GLP-1 secretion was assessed in primary intestinal cultures. Results Two weeks HFD reduced the numbers of GLP-1 positive cells in the colon, and of GIP positive cells in the small intestine. Purified small intestinal L-cells showed major shifts in their gene expression profiles. In mice on HFD for 16 weeks, significant reductions were observed in the expression of L-cell specific genes, including those encoding gut hormones (Gip, Cck, Sct, Nts), prohormone processing enzymes (Pcsk1, Cpe), granins (Chgb, Scg2), nutrient sensing machinery (Slc5a1, Slc15a1, Abcc8, Gpr120) and enteroendocrine-specific transcription factors (Etv1, Isl1, Mlxipl, Nkx2.2 and Rfx6). A corresponding reduction in the GLP-1 secretory responsiveness to nutrient stimuli was observed in primary small intestinal cultures. Conclusion Mice fed on HFD exhibited reduced expression in L-cells of many L-cell specific genes, suggesting an impairment of enteroendocrine cell function. Our results suggest that a western style diet may detrimentally affect the secretion of gut hormones and normal post-prandial signaling, which could impact on insulin secretion and satiety. PMID:26145551

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

    PubMed

    van Raalte, Daniël H; Verchere, C Bruce

    2016-07-01

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

  5. Effect of glucagon on digestive enzyme synthesis, transport and secretion in mouse pancreatic acinar cells.

    PubMed Central

    Singh, M

    1980-01-01

    1. Effect of glucagon on amylase secretion and lactic dehydrogenase (LDH) release from functionally intact dissociated pancreatic acinar cells and acini was studied. 2. In dissociated rat pancreatic acinar cells, the rate of amylase secretion was increased by 70% with bethanechol (maximally effective concentration, 10(-4) M) and 125% with A23187 (10(-5) M), but the response to cholecystokinin-pancreozymin (CCK-PZ) was inconsistent. In dissociated cells from mouse pancreas, the increases amounted to 78% with bethanechol (10(-4) M), 134% with A23187 (10(-5) M) and 82% with CCK-PZ (maximally effective concentration, 0 . 01 u. ml.-1). Glucagon in concentrations ranging from 10(-7) to 10(-4) M increased amylase secretion by 3, 26, 67 and 80%, whereas secretin (10(-8)--10(-5) M) increased amylase secretion by 8, 39, 88 and 138%. LDH release was increased with A23187 in concentrations greater than 10(-6) M. 3. CCK-PZ, bethanechol and A23187 used in maximal concentrations potentiated the effect of a submaximal dose of glucagon whereas secretin did not have an additive or a potentiating effect. 4. Pancreatic acini were approximately 3 times more responsive to secretagogues than cells. The dose--response curves to bethanechol, glucagon and CCK-PZ for increase in amylase secretion were similar. LDH release was not increased by these agents. Cytochalasin B (5 microgram ml.-1) which is known to disrupt the integrity of luminal membrane inhibited the amylase secretion stimulated by glucagon, bethanechol and CCK-PZ. 5. Glucagon inhibited incorporation of a mixture of fifteen 14C-labelled amino acids (algal profile, Schwarz Mann) into perchloric acid precipitable proteins in dissociated mouse pancreatic acini within 30 min. 6. In 'pulse-chase' experiments, glucagon decreased the specific activity of zymogen granules isolated by differential centrifugation, from pancreatic lobules (120 min) and increased the specific activity of radiolabelled proteins in the medium (60 and 120 min

  6. Blockade of glucagon signaling prevents or reverses diabetes onset only if residual β-cells persist

    PubMed Central

    Damond, Nicolas; Thorel, Fabrizio; Moyers, Julie S; Charron, Maureen J; Vuguin, Patricia M; Powers, Alvin C; Herrera, Pedro L

    2016-01-01

    Glucagon secretion dysregulation in diabetes fosters hyperglycemia. Recent studies report that mice lacking glucagon receptor (Gcgr-/-) do not develop diabetes following streptozotocin (STZ)-mediated ablation of insulin-producing β-cells. Here, we show that diabetes prevention in STZ-treated Gcgr-/- animals requires remnant insulin action originating from spared residual β-cells: these mice indeed became hyperglycemic after insulin receptor blockade. Accordingly, Gcgr-/- mice developed hyperglycemia after induction of a more complete, diphtheria toxin (DT)-induced β-cell loss, a situation of near-absolute insulin deficiency similar to type 1 diabetes. In addition, glucagon deficiency did not impair the natural capacity of α-cells to reprogram into insulin production after extreme β-cell loss. α-to-β-cell conversion was improved in Gcgr-/- mice as a consequence of α-cell hyperplasia. Collectively, these results indicate that glucagon antagonism could i) be a useful adjuvant therapy in diabetes only when residual insulin action persists, and ii) help devising future β-cell regeneration therapies relying upon α-cell reprogramming. DOI: http://dx.doi.org/10.7554/eLife.13828.001 PMID:27092792

  7. Blockade of glucagon signaling prevents or reverses diabetes onset only if residual β-cells persist.

    PubMed

    Damond, Nicolas; Thorel, Fabrizio; Moyers, Julie S; Charron, Maureen J; Vuguin, Patricia M; Powers, Alvin C; Herrera, Pedro L

    2016-01-01

    Glucagon secretion dysregulation in diabetes fosters hyperglycemia. Recent studies report that mice lacking glucagon receptor (Gcgr(-/-)) do not develop diabetes following streptozotocin (STZ)-mediated ablation of insulin-producing β-cells. Here, we show that diabetes prevention in STZ-treated Gcgr(-/-) animals requires remnant insulin action originating from spared residual β-cells: these mice indeed became hyperglycemic after insulin receptor blockade. Accordingly, Gcgr(-/-) mice developed hyperglycemia after induction of a more complete, diphtheria toxin (DT)-induced β-cell loss, a situation of near-absolute insulin deficiency similar to type 1 diabetes. In addition, glucagon deficiency did not impair the natural capacity of α-cells to reprogram into insulin production after extreme β-cell loss. α-to-β-cell conversion was improved in Gcgr(-/-) mice as a consequence of α-cell hyperplasia. Collectively, these results indicate that glucagon antagonism could i) be a useful adjuvant therapy in diabetes only when residual insulin action persists, and ii) help devising future β-cell regeneration therapies relying upon α-cell reprogramming. PMID:27092792

  8. Amylase release from dissociated mouse pancreatic acinar cells stimulated by glucagon: effect of membrane stabilizers.

    PubMed Central

    Singh, M

    1980-01-01

    1. The effect of membrane stabilizers and cytochalasin-B on amylase secretion, basal and induced by ionophore A23187, CCK-PZ, bethanechol and glucagon, was studied in dissociated mouse pancreatic acinar cells. 2. Cytochalasin-B did not affect basal or secretagogue-stimulated amylase secretion. 3. Membrane stabilizers [thymol (10(-7)-10(-4) M), chlorpromazine (10(-7)-10(-4) M) and propranolol (10(-7)-10(-5) M) did not alter basal release of amylase. At higher concentrations of thymol (10(-3) M), chlorpromazine (10(-3) M) and propranolol (10(-4) M), dissociated acinar cells were lysed as indicated by an increase in release of lactic dehydrogenase (LDH). 4. Ionophore A23187, CCK-PZ (maximal effective concentrations, 0.01 u. ml.-1), bethanechol (maximal effective concentrations, 10(-4) M) and glucagon increased amylase secretion in a dose-dependent fashion. Concentrations of CCK-PZ and bethanechol beyond optimal levels decreased amylase secretion. Concentrations of ionophore A23187 and glucagon when tested beyond 10(-6) M and 10(-4) M respectively increased the release of LDH. In concentrations that were non-toxic, membrane stabilizers blocked the stimulating effect of cholecystokinin-pancreozymin and bethanechol on amylase secretion but did not alter the response to A23187 and glucagon. 5. Unlike bethanechol, glucagon neither increased the uptake of 45Ca nor did it alter the release of 45Ca from cells previously loaded with 45CaCl2. 6. These data provide evidence that stimulus-secretion coupling in dissociated pancreatic acinar cells is basically similar to cells in situ. The effect of glucagon is consistent with the model in which hormone-dependent mobilization of Ca2+ from intra- or extracellular sources is bypassed leading to digestive enzyme secretion. PMID:6166745

  9. Electrical activity-triggered glucagon-like peptide-1 secretion from primary murine L-cells

    PubMed Central

    Rogers, G J; Tolhurst, G; Ramzan, A; Habib, A M; Parker, H E; Gribble, F M; Reimann, F

    2011-01-01

    Glucagon like peptide 1 (GLP-1) based therapies are now widely used for the treatment of type 2 diabetes. Developing our understanding of intestinal GLP-1 release may facilitate the development of new therapeutics aimed at targeting the GLP-1 producing L-cells. This study was undertaken to characterise the electrical activity of primary L-cells and the importance of voltage gated sodium and calcium channels for GLP-1 secretion. Primary murine L-cells were identified and purified using transgenic mice expressing a fluorescent protein driven by the proglucagon promoter. Fluorescent L-cells were identified within primary colonic cultures for patch clamp recordings. GLP-1 secretion was measured from primary colonic cultures. L-cells purified by flow cytometry were used to measure gene expression by microarray and quantitative RT-PCR. Electrical activity in L-cells was due to large voltage gated sodium currents, inhibition of which by tetrodotoxin reduced both basal and glutamine-stimulated GLP-1 secretion. Voltage gated calcium channels were predominantly of the L-type, Q-type and T-type, by expression analysis, consistent with the finding that GLP-1 release was blocked both by nifedipine and ω-conotoxin MVIIC. We observed large voltage-dependent potassium currents, but only a small chromanol sensitive current that might be attributable to KCNQ1. GLP-1 release from primary L-cells is linked to electrical activity and activation of L-type and Q-type calcium currents. The concept of an electrically excitable L-cell provides a basis for understanding how GLP-1 release may be modulated by nutrient, hormonal and pharmaceutical stimuli. PMID:21224236

  10. Quantitative ultrastructural analysis of the human parietal cell during acid inhibition and increase of gastric potential difference by glucagon.

    PubMed Central

    Ivey, K J; Tarnawski, A; Sherman, D; Krause, W J; Ackman, K; Burks, M; Hewett, J

    1980-01-01

    Glucagon inhibits gastric acid secretion and increases the negativity of gastric mucosal potential difference (PD) in man. To test the hypothesis that the increased negativity of PD after glucagon in man could be due to decreased parietal cell canalicular membrane area, a quantitative ultrastructural analysis was carried out. Four healthy volunteers with normal gastric mucosa were submitted to biopsy before and 20 minutes after intravenous injection of 2 mg glucagon (G). This time corresponded with the maximal change in PD and a decrease in gastric acid secretion. Canalicular and tubulovesicular membrane area of 80 parietal cells (40 cells before glucagon and 40 cells after glucagon) were quantified by the Loud morphometric method. After glucagon, the oxyntic cell canalicular membrane area was reduced by one-fourth (P less than 0.05), while tubulovesicular membrane area showed an increase (P less than 0.05) at the same time. The decrease in the area of parietal cell canalicular membrane caused by glucagon may in part be responsible for increased negativity of the gastric PD caused by this hormone. Images Fig. 2 PMID:7364316

  11. Autoreactive T-cell receptor (Vbeta/D/Jbeta) sequences in diabetes are homologous to insulin, glucagon, the insulin receptor, and the glucagon receptor.

    PubMed

    Root-Bernstein, Robert

    2009-01-01

    The hypervariable (Vbeta/D/Jbeta) regions of T-cell receptors (TCR) have been sequenced in a variety of autoimmune diseases by various investigators. An analysis of some of these sequences shows that TCR from both human diabetics and NOD mice mimic insulin, glucagon, the insulin receptor, and the glucagon receptor. Such similarities are not found in the TCR produced in other human autoimmune diseases. These data may explain how insulin, glucagon, and their receptors are targets of autoimmunity in diabetes and also suggest that TCR mimicking insulin and its receptor may be targets of anti-insulin autoantibodies. Such intra-systemic mimicry of self-proteins also raises complex questions about how "self" and "nonself" are regulated during TCR production, especially in light of the complementarity of insulin for its receptor and glucagon for its receptor. The data presented here suggest that some TCR may be complementary to other TCR in autoimmune diseases, a possibility that is experimentally testable. Such complementarity, if it exists, could either serve to down-regulate the clones bearing such TCR or, alternatively, trigger an intra-immune system civil war between them. PMID:19051206

  12. Intranasal Glucagon

    PubMed Central

    2014-01-01

    Prevention of diabetic complications is mainly obtained through optimal control of blood glucose levels. With hypoglycemic drugs like beta-cell stimulating drugs and especially insulin, the limit to treatment is represented by hypoglycemia, a life-threatening occurrence that is dangerous itself and can induce fear of other episodes. Glucagon, injected subcutaneously (SC) or intramuscularly (IM), is the treatment of choice for severe hypoglycemia outside of the hospital setting. However, due to practical aspects such as preparation of solutions for administration and injection by untrained persons, there are obstacles to its routine use. This review focuses on the current status of alternative routes of administration of peptide hormones, and in particular the intranasal (IN) route of glucagon, as a promising approach for the treatment of severe hypoglycemia. PMID:25385946

  13. Glucagon-Like Peptide-1 Regulates Cholecystokinin Production in β-Cells to Protect From Apoptosis

    PubMed Central

    Linnemann, Amelia K.; Neuman, Joshua C.; Battiola, Therese J.; Wisinski, Jaclyn A.; Kimple, Michelle E.

    2015-01-01

    Cholecystokinin (CCK) is a classic gut hormone that is also expressed in the pancreatic islet, where it is highly up-regulated with obesity. Loss of CCK results in increased β-cell apoptosis in obese mice. Similarly, islet α-cells produce increased amounts of another gut peptide, glucagon-like peptide 1 (GLP-1), in response to cytokine and nutrient stimulation. GLP-1 also protects β-cells from apoptosis via cAMP-mediated mechanisms. Therefore, we hypothesized that the activation of islet-derived CCK and GLP-1 may be linked. We show here that both human and mouse islets secrete active GLP-1 as a function of body mass index/obesity. Furthermore, GLP-1 can rapidly stimulate β-cell CCK production and secretion through direct targeting by the cAMP-modulated transcription factor, cAMP response element binding protein (CREB). We find that cAMP-mediated signaling is required for Cck expression, but CCK regulation by cAMP does not require stimulatory levels of glucose or insulin secretion. We also show that CREB directly targets the Cck promoter in islets from obese (Leptinob/ob) mice. Finally, we demonstrate that the ability of GLP-1 to protect β-cells from cytokine-induced apoptosis is partially dependent on CCK receptor signaling. Taken together, our work suggests that in obesity, active GLP-1 produced in the islet stimulates CCK production and secretion in a paracrine manner via cAMP and CREB. This intraislet incretin loop may be one mechanism whereby GLP-1 protects β-cells from apoptosis. PMID:25984632

  14. Glucagon-Like Peptide-1 Regulates Cholecystokinin Production in β-Cells to Protect From Apoptosis.

    PubMed

    Linnemann, Amelia K; Neuman, Joshua C; Battiola, Therese J; Wisinski, Jaclyn A; Kimple, Michelle E; Davis, Dawn Belt

    2015-07-01

    Cholecystokinin (CCK) is a classic gut hormone that is also expressed in the pancreatic islet, where it is highly up-regulated with obesity. Loss of CCK results in increased β-cell apoptosis in obese mice. Similarly, islet α-cells produce increased amounts of another gut peptide, glucagon-like peptide 1 (GLP-1), in response to cytokine and nutrient stimulation. GLP-1 also protects β-cells from apoptosis via cAMP-mediated mechanisms. Therefore, we hypothesized that the activation of islet-derived CCK and GLP-1 may be linked. We show here that both human and mouse islets secrete active GLP-1 as a function of body mass index/obesity. Furthermore, GLP-1 can rapidly stimulate β-cell CCK production and secretion through direct targeting by the cAMP-modulated transcription factor, cAMP response element binding protein (CREB). We find that cAMP-mediated signaling is required for Cck expression, but CCK regulation by cAMP does not require stimulatory levels of glucose or insulin secretion. We also show that CREB directly targets the Cck promoter in islets from obese (Leptin(ob/ob)) mice. Finally, we demonstrate that the ability of GLP-1 to protect β-cells from cytokine-induced apoptosis is partially dependent on CCK receptor signaling. Taken together, our work suggests that in obesity, active GLP-1 produced in the islet stimulates CCK production and secretion in a paracrine manner via cAMP and CREB. This intraislet incretin loop may be one mechanism whereby GLP-1 protects β-cells from apoptosis. PMID:25984632

  15. The Zinc Transporter Slc30a8/ZnT8 Is Required in a Subpopulation of Pancreatic α-Cells for Hypoglycemia-induced Glucagon Secretion*

    PubMed Central

    Solomou, Antonia; Meur, Gargi; Bellomo, Elisa; Hodson, David J.; Tomas, Alejandra; Migrenne Li, Stéphanie; Philippe, Erwann; Herrera, Pedro L.; Magnan, Christophe; Rutter, Guy A.

    2015-01-01

    SLC30A8 encodes a zinc transporter ZnT8 largely restricted to pancreatic islet β- and α-cells, and responsible for zinc accumulation into secretory granules. Although common SLC30A8 variants, believed to reduce ZnT8 activity, increase type 2 diabetes risk in humans, rare inactivating mutations are protective. To investigate the role of Slc30a8 in the control of glucagon secretion, Slc30a8 was inactivated selectively in α-cells by crossing mice with alleles floxed at exon 1 to animals expressing Cre recombinase under the pre-proglucagon promoter. Further crossing to Rosa26:tdRFP mice, and sorting of RFP+: glucagon+ cells from KO mice, revealed recombination in ∼30% of α-cells, of which ∼50% were ZnT8-negative (14 ± 1.8% of all α-cells). Although glucose and insulin tolerance were normal, female αZnT8KO mice required lower glucose infusion rates during hypoglycemic clamps and displayed enhanced glucagon release (p < 0.001) versus WT mice. Correspondingly, islets isolated from αZnT8KO mice secreted more glucagon at 1 mm glucose, but not 17 mm glucose, than WT controls (n = 5; p = 0.008). Although the expression of other ZnT family members was unchanged, cytoplasmic (n = 4 mice per genotype; p < 0.0001) and granular (n = 3, p < 0.01) free Zn2+ levels were significantly lower in KO α-cells versus control cells. In response to low glucose, the amplitude and frequency of intracellular Ca2+ increases were unchanged in α-cells of αZnT8KO KO mice. ZnT8 is thus important in a subset of α-cells for normal responses to hypoglycemia and acts via Ca2+-independent mechanisms. PMID:26178371

  16. Normal Glucagon Signaling and β-Cell Function After Near-Total α-Cell Ablation in Adult Mice

    PubMed Central

    Thorel, Fabrizio; Damond, Nicolas; Chera, Simona; Wiederkehr, Andreas; Thorens, Bernard; Meda, Paolo; Wollheim, Claes B.; Herrera, Pedro L.

    2011-01-01

    OBJECTIVE To evaluate whether healthy or diabetic adult mice can tolerate an extreme loss of pancreatic α-cells and how this sudden massive depletion affects β-cell function and blood glucose homeostasis. RESEARCH DESIGN AND METHODS We generated a new transgenic model allowing near-total α-cell removal specifically in adult mice. Massive α-cell ablation was triggered in normally grown and healthy adult animals upon diphtheria toxin (DT) administration. The metabolic status of these mice was assessed in 1) physiologic conditions, 2) a situation requiring glucagon action, and 3) after β-cell loss. RESULTS Adult transgenic mice enduring extreme (98%) α-cell removal remained healthy and did not display major defects in insulin counter-regulatory response. We observed that 2% of the normal α-cell mass produced enough glucagon to ensure near-normal glucagonemia. β-Cell function and blood glucose homeostasis remained unaltered after α-cell loss, indicating that direct local intraislet signaling between α- and β-cells is dispensable. Escaping α-cells increased their glucagon content during subsequent months, but there was no significant α-cell regeneration. Near-total α-cell ablation did not prevent hyperglycemia in mice having also undergone massive β-cell loss, indicating that a minimal amount of α-cells can still guarantee normal glucagon signaling in diabetic conditions. CONCLUSIONS An extremely low amount of α-cells is sufficient to prevent a major counter-regulatory deregulation, both under physiologic and diabetic conditions. We previously reported that α-cells reprogram to insulin production after extreme β-cell loss and now conjecture that the low α-cell requirement could be exploited in future diabetic therapies aimed at regenerating β-cells by reprogramming adult α-cells. PMID:21926270

  17. Characterization of a novel functional protein in the pancreatic islet: IHoP regulation of glucagon synthesis in alpha-cells

    PubMed Central

    Oh, Seh-Hoon; Darwiche, Houda; Cho, Jae-Hyoung; Shupe, Thomas; Petersen, Bryon E.

    2011-01-01

    Objective We have identified a novel protein in bone marrow (BM)-derived insulin-producing cells (IPCs). Here we characterize this protein, hereby named Islet Homeostasis Protein (IHoP), in the pancreatic islet. Methods Detection of IHoP mRNA and protein were performed using RT-PCR, immunocytochemistry and in-situ hybridization. IHoP functions were utilizing proliferation, insulin secretion by in vitro assays, and as well as following siRNA protocols for suppression of IHoP. Results We found that IHoP did not homologue with known pancreatic hormones. IHoP expression was seen in both BM-derived IPCs and isolated pancreatic islets. Immunohistochemistry on pancreatic islet revealed that IHoP localized to the glucagon synthesizing α (alpha)-cells. Inhibition of IHoP by siRNA resulted in the loss of glucagon expression, which induced low blood glucose levels (63–85 mg/dL). Subsequently, cellular apoptosis was observed throughout the islet, including the insulin-producing β (beta)-cells. Islets of pre-onset diabetic patients showed normal expression of IHoP and glucagon; however IHoP was lost upon onset of the disease. Conclusion These data suggest that IHoP could be a new functional protein in the islet, and may play a role in islet homeostasis. PMID:22143342

  18. UCP2 Regulates the Glucagon Response to Fasting and Starvation

    PubMed Central

    Allister, Emma M.; Robson-Doucette, Christine A.; Prentice, Kacey J.; Hardy, Alexandre B.; Sultan, Sobia; Gaisano, Herbert Y.; Kong, Dong; Gilon, Patrick; Herrera, Pedro L.; Lowell, Bradford B.; Wheeler, Michael B.

    2013-01-01

    Glucagon is important for maintaining euglycemia during fasting/starvation, and abnormal glucagon secretion is associated with type 1 and type 2 diabetes; however, the mechanisms of hypoglycemia-induced glucagon secretion are poorly understood. We previously demonstrated that global deletion of mitochondrial uncoupling protein 2 (UCP2−/−) in mice impaired glucagon secretion from isolated islets. Therefore, UCP2 may contribute to the regulation of hypoglycemia-induced glucagon secretion, which is supported by our current finding that UCP2 expression is increased in nutrient-deprived murine and human islets. Further to this, we created α-cell–specific UCP2 knockout (UCP2AKO) mice, which we used to demonstrate that blood glucose recovery in response to hypoglycemia is impaired owing to attenuated glucagon secretion. UCP2-deleted α-cells have higher levels of intracellular reactive oxygen species (ROS) due to enhanced mitochondrial coupling, which translated into defective stimulus/secretion coupling. The effects of UCP2 deletion were mimicked by the UCP2 inhibitor genipin on both murine and human islets and also by application of exogenous ROS, confirming that changes in oxidative status and electrical activity directly reduce glucagon secretion. Therefore, α-cell UCP2 deletion perturbs the fasting/hypoglycemic glucagon response and shows that UCP2 is necessary for normal α-cell glucose sensing and the maintenance of euglycemia. PMID:23434936

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

    PubMed Central

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

    2013-01-01

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

  20. Interleukin-6 enhances insulin secretion by increasing glucagon-like peptide-1 secretion from L cells and alpha cells

    PubMed Central

    Ellingsgaard, Helga; Hauselmann, Irina; Schuler, Beat; Habib, Abdella M; Baggio, Laurie L; Meier, Daniel T; Eppler, Elisabeth; Bouzakri, Karim; Wueest, Stephan; Muller, Yannick D; Hansen, Ann Maria Kruse; Reinecke, Manfred; Konrad, Daniel; Gassmann, Max; Reimann, Frank; Halban, Philippe A; Gromada, Jesper; Drucker, Daniel J; Gribble, Fiona M; Ehses, Jan A; Donath, Marc Y

    2014-01-01

    Exercise, obesity and type 2 diabetes are associated with elevated plasma concentrations of interleukin-6 (IL-6). Glucagon-like peptide-1 (GLP-1) is a hormone that induces insulin secretion. Here we show that administration of IL-6 or elevated IL-6 concentrations in response to exercise stimulate GLP-1 secretion from intestinal L cells and pancreatic alpha cells, improving insulin secretion and glycemia. IL-6 increased GLP-1 production from alpha cells through increased proglucagon (which is encoded by GCG) and prohormone convertase 1/3 expression. In models of type 2 diabetes, the beneficial effects of IL-6 were maintained, and IL-6 neutralization resulted in further elevation of glycemia and reduced pancreatic GLP-1. Hence, IL-6 mediates crosstalk between insulin-sensitive tissues, intestinal L cells and pancreatic islets to adapt to changes in insulin demand. This previously unidentified endocrine loop implicates IL-6 in the regulation of insulin secretion and suggests that drugs modulating this loop may be useful in type 2 diabetes. PMID:22037645

  1. Interleukin-6 enhances insulin secretion by increasing glucagon-like peptide-1 secretion from L cells and alpha cells.

    PubMed

    Ellingsgaard, Helga; Hauselmann, Irina; Schuler, Beat; Habib, Abdella M; Baggio, Laurie L; Meier, Daniel T; Eppler, Elisabeth; Bouzakri, Karim; Wueest, Stephan; Muller, Yannick D; Hansen, Ann Maria Kruse; Reinecke, Manfred; Konrad, Daniel; Gassmann, Max; Reimann, Frank; Halban, Philippe A; Gromada, Jesper; Drucker, Daniel J; Gribble, Fiona M; Ehses, Jan A; Donath, Marc Y

    2011-01-01

    Exercise, obesity and type 2 diabetes are associated with elevated plasma concentrations of interleukin-6 (IL-6). Glucagon-like peptide-1 (GLP-1) is a hormone that induces insulin secretion. Here we show that administration of IL-6 or elevated IL-6 concentrations in response to exercise stimulate GLP-1 secretion from intestinal L cells and pancreatic alpha cells, improving insulin secretion and glycemia. IL-6 increased GLP-1 production from alpha cells through increased proglucagon (which is encoded by GCG) and prohormone convertase 1/3 expression. In models of type 2 diabetes, the beneficial effects of IL-6 were maintained, and IL-6 neutralization resulted in further elevation of glycemia and reduced pancreatic GLP-1. Hence, IL-6 mediates crosstalk between insulin-sensitive tissues, intestinal L cells and pancreatic islets to adapt to changes in insulin demand. This previously unidentified endocrine loop implicates IL-6 in the regulation of insulin secretion and suggests that drugs modulating this loop may be useful in type 2 diabetes. PMID:22037645

  2. The Reaction of Glucagon with Its Receptor: Evidence for Discrete Regions of Activity and Binding in the Glucagon Molecule

    PubMed Central

    Rodbell, Martin; Birnbaumer, Lutz; Pohl, Stephen L.; Sundby, F.

    1971-01-01

    Des-histidine-glucagon (DH-glucagon, glucagon2-29) does not activate the glucagon-sensitive adenylate cyclase system present in either liver plasma membranes or in fat-cell “ghosts”, but inhibits the response of these systems to submaximal concentrations of glucagon. DH-glucagon also inhibits, competitively, the binding of [125I]glucagon to its receptor in liver plasma membranes. Amino-terminal fragments of glucagon (glucagon1-21, glucagon1-23) and carboxy-terminal fragments (glucagon20-29, glucagon22-29) failed to activate adenylate cyclase, to inhibit the response of the enzyme to glucagon, or to compete with labeled glucagon at its receptor. It is concluded that the amino-terminal histidine residue of glucagon is essential for biological activity and that a hydrophobic near-carboxy-terminal region (residues 22-27) is essential for binding of glucagon to its receptor. Amino-terminal histidine may also contribute to the binding of glucagon, since the apparent affinity of DH-glucagon for the receptor is only about one-sixth that of glucagon. Thus, essentially the entire molecule of glucagon must be considered to be the biologically active species. Because, as shown elsewhere, the binding of glucagon to its receptor shows characteristics of hydrophobic bonding, and because certain detergents induce conformational changes in the carboxy-terminal binding region of glucagon, the binding is probably of a lipophilic type. PMID:5280527

  3. Small Mouse Islets Are Deficient in Glucagon-Producing Alpha Cells but Rich in Somatostatin-Secreting Delta Cells

    PubMed Central

    Grapengiesser, Eva; Hellman, Bo

    2016-01-01

    Small and big mouse islets were compared with special reference to their content of glucagon-producing α-cells and somatostatin-producing δ-cells. Areas stained for glucagon and somatostatin were measured in the largest cross section of small (diameter < 60 μm) and big (diameter > 100 μm) islets. Comparison of the areas indicated proportionally more δ- than α-cells in the small islets. After isolation with collagenase these islets were practically devoid of α-cells. We evaluated the functional importance of the islet size by measuring the Ca2+ signal for insulin release. A majority of the small islets responded to the hyperpolarization action of somatostatin with periodic decrease of cytoplasmic Ca2+ when glucose was elevated after tolbutamide blockade of the KATP channels. PMID:27504459

  4. Expression, purification, and C-terminal amidation of recombinant human glucagon-like peptide-1.

    PubMed

    Zhang, Zhi-Zhen; Yang, Sheng-Sheng; Dou, Hong; Mao, Ji-Fang; Li, Kang-Sheng

    2004-08-01

    Human glucagon-like peptide-1 (hGLP-1) (7-36) amide, a gastrointestinal hormone with a pharmaceutical potential in treating type 2 diabetes mellitus, is composed of 30 amino acid residues as a mature protein. We report here the development of a method for high-level expression and purification of recombinant hGLP-1 (7-36) amide (rhGLP-1) through glutathione S-transferase (GST) fusion expression system. The cDNA of hGLP-1-Leu, the 31st-residue leucine-extended precursor peptide, was prepared by annealing and ligating of artificially synthetic oligonucleotide fragments, inserted into pBluescript SK (+/-) plasmid, and then cloned into pGEX-4T-3 GST fusion vector. The fusion protein GST-hGLP-1-Leu, expressed in Escherichia coli strain BL21 (DE3), was purified by affinity chromatography after high-level culture and sonication of bacteria. Following cleavage of GST-hGLP-1-Leu by cyanogen bromide, the recombinant hGLP-1-Leu was released from fusion protein, and purified using QAE Sepharose ion exchange and RP C(18) chromatography. After purification, the precursor hGLP-1-Leu was transacylated by carboxypeptidase Y, Arg-NH(2) as a nucleophile, to produce rhGLP-1. Electrospray ionization mass spectrometry showed the molecular weight was as expected. The biological activity of rhGLP-1 in a rat model demonstrated that plasma glucose concentrations were significantly lower and insulin concentrations higher after intraperitoneal injection of rhGLP-1 together with glucose compared with glucose alone (P < 0.001). PMID:15249052

  5. Glucagon actions on the kidney revisited: possible role in potassium homeostasis.

    PubMed

    Bankir, Lise; Bouby, Nadine; Blondeau, Bertrand; Crambert, Gilles

    2016-08-01

    It is now recognized that the metabolic disorders observed in diabetes are not, or not only due to the lack of insulin or insulin resistance, but also to elevated glucagon secretion. Accordingly, selective glucagon receptor antagonists are now proposed as a novel strategy for the treatment of diabetes. However, besides its metabolic actions, glucagon also influences kidney function. The glucagon receptor is expressed in the thick ascending limb, distal tubule, and collecting duct, and glucagon regulates the transepithelial transport of several solutes in these nephron segments. Moreover, it also influences solute transport in the proximal tubule, possibly by an indirect mechanism. This review summarizes the knowledge accumulated over the last 30 years about the influence of glucagon on the renal handling of electrolytes and urea. It also describes a possible novel role of glucagon in the short-term regulation of potassium homeostasis. Several original findings suggest that pancreatic α-cells may express a "potassium sensor" sensitive to changes in plasma K concentration and could respond by adapting glucagon secretion that, in turn, would regulate urinary K excretion. By their combined actions, glucagon and insulin, working in a combinatory mode, could ensure an independent regulation of both plasma glucose and plasma K concentrations. The results and hypotheses reviewed here suggest that the use of glucagon receptor antagonists for the treatment of diabetes should take into account their potential consequences on electrolyte handling by the kidney. PMID:27194722

  6. Impairment by interleukin 1 beta and tumour necrosis factor alpha of the glucagon-induced increase in phosphoenolpyruvate carboxykinase gene expression and gluconeogenesis in cultured rat hepatocytes.

    PubMed Central

    Christ, B; Nath, A

    1996-01-01

    The influence of the inflammatory mediators interleukin 1 beta (IL1 beta) and tumour necrosis factor alpha (TNF alpha) on the glucagon-induced expression of phosphoenolpyruvate carboxykinase (PCK) and on glucose formation via gluconeogenesis was investigated in cultured rat hepatocytes. Gene expression was monitored by determination of mRNA levels and of enzyme activity. Glucose formation was estimated with newly synthesized radioactive glucose derived from a radiolabelled lactate precursor. Glucagon (0.1 or 1 nM) induced PCK mRNA transiently to a maximum 2 h after its application. In the presence of recombinant human (rh) IL1 beta or rhTNF alpha the increase in PCK mRNA levels was totally inhibited at 0.1 nM glucagon, whereas at 1 nM glucagon the maximal increase was inhibited by only 25%. Glucagon (0.1 or 1 nM) induced PCK activity to a maximum after 4 h (4-fold and 6-fold over prestimulatory activity respectively). In the presence of rhIL1 beta or rhTNF alpha the maximal increase was inhibited by approx. 50%. Addition of rhIL1 beta or rhTNF alpha 2 h after glucagon, at the maximal glucagon-induced PCK mRNA levels, accelerated the decay of PCK mRNA. Glucagon (1 or 10 nM) [corrected] increased glucose formation from lactate by 1.3-fold and 1.7-fold respectively over unstimulated rates. In the presence of rhIL1 beta or rhTNF alpha this increase in glucose formation was inhibited by 60-90%. At 0.1 nM, glucagon doubled the intracellular cAMP concentration. This increase was prevented by rhIL1 beta or rhTNF alpha. At 1 nM, glucagon increased cAMP concentrations by 10-fold. In the presence of rhIL1 beta or rhTNF alpha this increase was inhibited by 70%. From the results it is suggested that rhIL1 beta and rhTNF alpha prevented glucagon-stimulated PCK gene expression and gluconeogenesis at least in part by inhibition of the glucagon-stimulated increase in cAMP concentrations. PMID:8947481

  7. Glucagon-like peptide-1 receptor is present in pancreatic acinar cells and regulates amylase secretion through cAMP.

    PubMed

    Hou, Yanan; Ernst, Stephen A; Heidenreich, Kaeli; Williams, John A

    2016-01-01

    Glucagon-like peptide-1 (GLP-1) is a glucoincretin hormone that can act through its receptor (GLP-1R) on pancreatic β-cells and increase insulin secretion and production. GLP-1R agonists are used clinically to treat type 2 diabetes. GLP-1 may also regulate the exocrine pancreas at multiple levels, including inhibition through the central nervous system, stimulation indirectly through insulin, and stimulation directly on acinar cells. However, it has been unclear whether GLP-1R is present in pancreatic acini and what physiological functions these receptors regulate. In the current study we utilized GLP-1R knockout (KO) mice to study the role of GLP-1R in acinar cells. RNA expression of GLP-1R was detected in acutely isolated pancreatic acini. Acinar cell morphology and expression of digestive enzymes were not affected by loss of GLP-1R. GLP-1 induced amylase secretion in wild-type (WT) acini. In GLP-1R KO mice, this effect was abolished, whereas vasoactive intestinal peptide-induced amylase release in KO acini showed a pattern similar to that in WT acini. GLP-1 stimulated cAMP production and increased protein kinase A-mediated protein phosphorylation in WT acini, and these effects were absent in KO acini. These data show that GLP-1R is present in pancreatic acinar cells and that GLP-1 can regulate secretion through its receptor and cAMP signaling pathway. PMID:26542397

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

    PubMed

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

    2016-06-01

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

  9. Glucagon-like peptide-1 inhibits vascular smooth muscle cell dedifferentiation through mitochondrial dynamics regulation.

    PubMed

    Torres, Gloria; Morales, Pablo E; García-Miguel, Marina; Norambuena-Soto, Ignacio; Cartes-Saavedra, Benjamín; Vidal-Peña, Gonzalo; Moncada-Ruff, David; Sanhueza-Olivares, Fernanda; San Martín, Alejandra; Chiong, Mario

    2016-03-15

    Glucagon-like peptide-1 (GLP-1) is a neuroendocrine hormone produced by gastrointestinal tract in response to food ingestion. GLP-1 plays a very important role in the glucose homeostasis by stimulating glucose-dependent insulin secretion, inhibiting glucagon secretion, inhibiting gastric emptying, reducing appetite and food intake. Because of these actions, the GLP-1 peptide-mimetic exenatide is one of the most promising new medicines for the treatment of type 2 diabetes. In vivo treatments with GLP-1 or exenatide prevent neo-intima layer formation in response to endothelial damage and atherosclerotic lesion formation in aortic tissue. Whether GLP-1 modulates vascular smooth muscle cell (VSMC) migration and proliferation by controlling mitochondrial dynamics is unknown. In this report, we showed that GLP-1 increased mitochondrial fusion and activity in a PKA-dependent manner in the VSMC cell line A7r5. GLP-1 induced a Ser-637 phosphorylation in the mitochondrial fission protein Drp1, and decreased Drp1 mitochondrial localization. GLP-1 inhibited PDGF-BB-induced VSMC migration and proliferation, actions inhibited by overexpressing wild type Drp1 and mimicked by the Drp1 inhibitor Mdivi-1 and by overexpressing dominant negative Drp1. These results show that GLP-1 stimulates mitochondrial fusion, increases mitochondrial activity and decreases PDGF-BB-induced VSMC dedifferentiation by a PKA/Drp1 signaling pathway. Our data suggest that GLP-1 inhibits vascular remodeling through a mitochondrial dynamics-dependent mechanism. PMID:26807480

  10. Glucose regulation of glucagon secretion.

    PubMed

    Gylfe, Erik; Gilon, Patrick

    2014-01-01

    Glucagon secreted by pancreatic α-cells is the major hyperglycemic hormone correcting acute hypoglycaemia (glucose counterregulation). In diabetes the glucagon response to hypoglycaemia becomes compromised and chronic hyperglucagonemia appears. There is increasing awareness that glucagon excess may underlie important manifestations of diabetes. However opinions differ widely how glucose controls glucagon secretion. The autonomous nervous system plays an important role in the glucagon response to hypoglycaemia. But it is clear that glucose controls glucagon secretion also by mechanisms involving direct effects on α-cells or indirect effects via paracrine factors released from non-α-cells within the pancreatic islets. The present review discusses these mechanisms and argues that different regulatory processes are involved in a glucose concentration-dependent manner. Direct glucose effects on the α-cell and autocrine mechanisms are probably most significant for the glucagon response to hypoglycaemia. During hyperglycaemia, when secretion from β- and δ-cells is stimulated, paracrine inhibitory factors generate pulsatile glucagon release in opposite phase to pulsatile release of insulin and somatostatin. High concentrations of glucose have also stimulatory effects on glucagon secretion that tend to balance and even exceed the inhibitory influence. The latter actions might underlie the paradoxical hyperglucagonemia that aggravates hyperglycaemia in persons with diabetes. PMID:24367972

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

    PubMed

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

    2016-06-01

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

  12. Recombinant expression, in vitro refolding, and biophysical characterization of the human glucagon-like peptide-1 receptor.

    PubMed

    Schröder-Tittmann, Kathrin; Bosse-Doenecke, Eva; Reedtz-Runge, Steffen; Ihling, Christian; Sinz, Andrea; Tittmann, Kai; Rudolph, Rainer

    2010-09-14

    Activation of the glucagon-like peptide-1 receptor (GLP-1R) upon ligand binding leads to the release of insulin from pancreatic cells. This strictly glucose-dependent process renders the receptor and its ligands useful in the treatment of type II diabetes mellitus. To enable a biophysical characterization in vitro, we expressed the human full-length GLP-1R in the cytosol of Escherichia coli as inclusion bodies. After purification, refolding of the SDS-solubilized receptor was achieved by the exchange of SDS against the detergent Brij78 using an artificial chaperone system. Far-UV circular dichroism spectroscopic studies revealed that the receptor adopts a characteristic alpha-helical structure in Brij78 micelles. Ligand binding of the renatured protein was quantified by fluorescence quenching and surface plasmon resonance spectroscopy. In the presence of Brij micelles, the refolded receptor binds the agonist exendin-4 with an apparent dissociation constant of approximately 100 nM in a reversible one-step mechanism. To demonstrate that the detected ligand binding activity is not only due to an autonomously functional N-terminal domain (nGLP-1R) but also due to additional contacts with the juxtamembrane part, we separately expressed and refolded the extracellular domain relying on identical protocols established for the full-length GLP-1R. In support of the suggested multidomain binding mode, the nGLP-1R binds exendin-4 with a lower affinity (K(app) in the micromolar range) and a different kinetic mechanism. The lower ligand affinity of the nGLP-1R results entirely from a decreased kinetic stability of the receptor-ligand complex, dissociation of which is approximately 40-fold faster in the case of the nGLP-1R compared to the full-length GLP-1R. In summary, a framework was developed to produce functional human full-length GLP-1R by recombinant expression in E. coli as a prerequisite for eventual structure determination and a rigorous biophysical characterization

  13. Effect of Glucagon-like Peptide-1 on the Differentiation of Adipose-derived Stem Cells into Osteoblasts and Adipocytes

    PubMed Central

    Lee, Hye Min; Joo, Bo Sun; Lee, Chang Hoon; Kim, Heung Yeol

    2015-01-01

    Objectives Glucagon-like peptide-1 (GLP-1) is an intestinally secreted hormone and it plays an important role in the regulation of glucose homeostasis. However, the possible role of GLP-1 in the differentiation of adipose-derived stem cells (ADSCs) remains unknown. Therefore this study investigated the effect of GLP-1 on the differentiation of ADSCs into osteoblasts and adipocytes. Methods ADSCs were isolated from human adipose tissues of the abdomens, cultured and characterized by flow cytometry and multi-lineage potential assay. ADSCs were induced in osteogenic and adipogenic media treated with two different doses (10 and 100 nM) of GLP-1, and then the effect of GLP-1 on differentiation of ADSCs into osteoblast and adipocyte was examined. The signaling pathway involved in these processes was also examined. Results Isolated human ADSCs expressed mesenchymal stem cell (MSC) specific markers as well as GLP-1 receptor (GLP-1R) proteins. They also showed multiple-lineage potential of MSC. GLP-1 was upregulated the activity and mRNA expression of osteoblast-specific marker, alkaline phosphatase and the mineralization of calcium. In contrast, GLP-1 significantly suppressed the expression of adipocyte-specific markers, peroxisome proliferator-activated receptor gamma (PPAR-γ), lipoprotein lipase (LPL) and adipocyte protein 2 (AP2). This decreased expression of adipocyte specific markers caused by GLP-1 was significantly reversed by the treatment of extracellular signal-regulated kinase (ERK) inhibitor, PD98059 (P < 0.05). Conclusion This result demonstrates that GLP-1 stimulates osteoblast differentiation in ADSCs, whereas it inhibits adipocyte differentiation. The ERK signaling pathway seems to be involved in these differentiation processes mediated by GLP-1. PMID:26357647

  14. Molecular physiology of glucagon-like peptide-1 insulin secretagogue action in pancreatic β cells.

    PubMed

    Leech, Colin A; Dzhura, Igor; Chepurny, Oleg G; Kang, Guoxin; Schwede, Frank; Genieser, Hans-G; Holz, George G

    2011-11-01

    Insulin secretion from pancreatic β cells is stimulated by glucagon-like peptide-1 (GLP-1), a blood glucose-lowering hormone that is released from enteroendocrine L cells of the distal intestine after the ingestion of a meal. GLP-1 mimetics (e.g., Byetta) and GLP-1 analogs (e.g., Victoza) activate the β cell GLP-1 receptor (GLP-1R), and these compounds stimulate insulin secretion while also lowering levels of blood glucose in patients diagnosed with type 2 diabetes mellitus (T2DM). An additional option for the treatment of T2DM involves the administration of dipeptidyl peptidase-IV (DPP-IV) inhibitors (e.g., Januvia, Galvus). These compounds slow metabolic degradation of intestinally released GLP-1, thereby raising post-prandial levels of circulating GLP-1 substantially. Investigational compounds that stimulate GLP-1 secretion also exist, and in this regard a noteworthy advance is the demonstration that small molecule GPR119 agonists (e.g., AR231453) stimulate L cell GLP-1 secretion while also directly stimulating β cell insulin release. In this review, we summarize what is currently known concerning the signal transduction properties of the β cell GLP-1R as they relate to insulin secretion. Emphasized are the cyclic AMP, protein kinase A, and Epac2-mediated actions of GLP-1 to regulate ATP-sensitive K⁺ channels, voltage-dependent K⁺ channels, TRPM2 cation channels, intracellular Ca⁺ release channels, and Ca⁺-dependent exocytosis. We also discuss new evidence that provides a conceptual framework with which to understand why GLP-1R agonists are less likely to induce hypoglycemia when they are administered for the treatment of T2DM. PMID:21782840

  15. Molecular Physiology of Glucagon-Like Peptide-1 Insulin Secretagogue Action in Pancreatic β Cells

    PubMed Central

    Leech, Colin A.; Dzhura, Igor; Chepurny, Oleg G.; Kang, Guoxin; Schwede, Frank; Genieser, Hans-G.; Holz, George G.

    2011-01-01

    Insulin secretion from pancreatic β cells is stimulated by glucagon-like peptide-1 (GLP-1), a blood glucose-lowering hormone that is released from enteroendocrine L cells of the distal intestine after the ingestion of a meal. GLP-1 mimetics (e.g., Byetta) and GLP-1 analogs (e.g., Victoza) activate the β cell GLP-1 receptor (GLP-1R), and these compounds stimulate insulin secretion while also lowering levels of blood glucose in patients diagnosed with type 2 diabetes mellitus (T2DM). An additional therapeutic option for the treatment of T2DM involves the administration of dipeptidyl peptidase-IV (DPP-IV) inhibitors (e.g., Januvia, Galvus). These compounds slow metabolic degradation of intestinally released GLP-1, thereby raising post-prandial levels of circulating GLP-1 substantially. Investigational compounds that stimulate GLP-1 secretion also exist, and in this regard a noteworthy advance is the demonstration that small molecule GPR119 agonists (e.g., AR231453) stimulate L cell GLP-1 secretion while also directly stimulating β cell insulin release. In this review, we summarize what is currently known concerning the signal transduction properties of the β cell GLP-1R as they relate to insulin secretion. Emphasized are the cyclic AMP, protein kinase A, and Epac2 mediated actions of GLP-1 to regulate ATP-sensitive K+ channels, voltage-dependent K+ channels, TRPM2 cation channels, intracellular Ca2+ release channels, and Ca2+-dependent exocytosis. We also discuss new evidence that provides a conceptual framework with which to understand why GLP-1R agonists are less likely to induce hypoglycemia when they are administered for the treatment of T2DM. PMID:21782840

  16. Functional studies of a glucagon receptor isolated from frog Rana tigrina rugulosa: implications on the molecular evolution of glucagon receptors in vertebrates.

    PubMed

    Ngan, E S; Chow, L S; Tse, D L; Du, X; Wei, Y; Mojsov, S; Chow, B K

    1999-09-01

    In this report, the first amphibian glucagon receptor (GluR) cDNA was characterized from the liver of the frog Rana tigrina rugulosa. Functional expression of the frog GluR in CHO and COS-7 cells showed a high specificity of the receptor towards human glucagon with an EC(50) value of 0.8+/-0.5 nM. The binding of radioiodinated human glucagon to GluR was displaced in a dose-dependent manner only with human glucagon and its antagonist (des-His(1)-[Nle(9)-Ala(11)-Ala(16)]) with IC(50) values of 12.0+/-3. 0 and 7.8+/-1.0 nM, respectively. The frog GluR did not display any affinity towards fish and human GLP-1s, and towards glucagon peptides derived from two species of teleost fishes (goldfish, zebrafish). These fish glucagons contain substitutions in several key residues that were previously shown to be critical for the binding of human glucagon to its receptor. By RT-PCR, mRNA transcripts of frog GluR were located in the liver, brain, small intestine and colon. These results demonstrate a conservation of the functional characteristics of the GluRs in frog and mammalian species and provide a framework for a better understanding of the molecular evolution of the GluR and its physiological function in vertebrates. PMID:10471837

  17. Nesfatin-1 stimulates glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide secretion from STC-1 cells in vitro.

    PubMed

    Ramesh, Naresh; Mortazavi, Sima; Unniappan, Suraj

    2015-06-26

    Nesfatin-1 is an 82 amino acid peptide encoded in a secreted precursor, nucleobindin 2. It is an anorexigenic and insulinotropic peptide found abundantly in the hypothalamus, pancreas and gastric oxyntic mucosa. NUCB2 mRNA expression is 10 fold higher in the gastric mucosa than in brain, suggesting gastrointestinal tract as a main source of nesfatin-1. Meal responsive insulin secretion is regulated by incretins glucagon-like peptide-1 (GLP-1) and glucose dependent insulinotropic polypeptide (GIP). Since both nesfatin-1 and incretins modulate insulin secretion, we hypothesized that nesfatin-1 is present in the enteroendocrine cells, and that it regulates incretin secretion. RT-PCR analysis found NUCB2 mRNA expression, and immunofluorescence microscopy determined nesfatin-1 immunoreactivity in STC-1, an enteroendocrine cell line. NUCB2/nesfatin-1 is co-localized with GLP-1 and GIP in mouse small intestinal cells. Static incubation of STC-1 cells with nesfatin-1 upregulated preproglucagon (GLP-1 precursor) mRNA (0.01, 0.1, 1 and 10 nM) and GLP-1 secretion (0.1, 1 and 10 nM). Nesfatin-1 also enhanced GIP mRNA (0.1, 1 and 10 nM) and GIP secretion (1 and 10 nM). Together, our data support the hypothesis that nesfatin-1 is present in enteroendocrine cells and that it stimulates incretin secretion. Future studies should aim for nesfatin-1 and incretin interactions in vivo. PMID:25930999

  18. Increased Glucose-induced Secretion of Glucagon-like Peptide-1 in Mice Lacking the Carcinoembryonic Antigen-related Cell Adhesion Molecule 2 (CEACAM2).

    PubMed

    Ghanem, Simona S; Heinrich, Garrett; Lester, Sumona G; Pfeiffer, Verena; Bhattacharya, Sumit; Patel, Payal R; DeAngelis, Anthony M; Dai, Tong; Ramakrishnan, Sadeesh K; Smiley, Zachary N; Jung, Dae Y; Lee, Yongjin; Kitamura, Tadahiro; Ergun, Suleyman; Kulkarni, Rohit N; Kim, Jason K; Giovannucci, David R; Najjar, Sonia M

    2016-01-01

    Carcinoembryonic antigen-related cell adhesion molecule 2 (CEACAM2) regulates food intake as demonstrated by hyperphagia in mice with the Ceacam2 null mutation (Cc2(-/-)). This study investigated whether CEACAM2 also regulates insulin secretion. Ceacam2 deletion caused an increase in β-cell secretory function, as assessed by hyperglycemic clamp analysis, without affecting insulin response. Although CEACAM2 is expressed in pancreatic islets predominantly in non-β-cells, basal plasma levels of insulin, glucagon and somatostatin, islet areas, and glucose-induced insulin secretion in pooled Cc2(-/-) islets were all normal. Consistent with immunofluorescence analysis showing CEACAM2 expression in distal intestinal villi, Cc2(-/-) mice exhibited a higher release of oral glucose-mediated GLP-1, an incretin that potentiates insulin secretion in response to glucose. Compared with wild type, Cc2(-/-) mice also showed a higher insulin excursion during the oral glucose tolerance test. Pretreating with exendin(9-39), a GLP-1 receptor antagonist, suppressed the effect of Ceacam2 deletion on glucose-induced insulin secretion. Moreover, GLP-1 release into the medium of GLUTag enteroendocrine cells was increased with siRNA-mediated Ceacam2 down-regulation in parallel to an increase in Ca(2+) entry through L-type voltage-dependent Ca(2+) channels. Thus, CEACAM2 regulates insulin secretion, at least in part, by a GLP-1-mediated mechanism, independent of confounding metabolic factors. PMID:26586918

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

    SciTech Connect

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

    2010-07-30

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

  20. Effect of Cell Adhesion Molecule 1 Expression on Intracellular Granule Movement in Pancreatic α Cells.

    PubMed

    Yokawa, Satoru; Furuno, Tadahide; Suzuki, Takahiro; Inoh, Yoshikazu; Suzuki, Ryo; Hirashima, Naohide

    2016-09-01

    Although glucagon secreted from pancreatic α cells plays a role in increasing glucose concentrations in serum, the mechanism regulating glucagon secretion from α cells remains unclear. Cell adhesion molecule 1 (CADM1), identified as an adhesion molecule in α cells, has been reported not only to communicate among α cells and between nerve fibers, but also to prevent excessive glucagon secretion from α cells. Here, we investigated the effect of CADM1 expression on the movement of intracellular secretory granules in α cells because the granule transport is an important step in secretion. Spinning disk microscopic analysis showed that granules moved at a mean velocity of 0.236 ± 0.010 μm/s in the mouse α cell line αTC6 that expressed CADM1 endogenously. The mean velocity was significantly decreased in CADM1-knockdown (KD) cells (mean velocity: 0.190 ± 0.016 μm/s). The velocity of granule movement decreased greatly in αTC6 cells treated with the microtubule-depolymerizing reagent nocodazole, but not in αTC6 cells treated with the actin-depolymerizing reagent cytochalasin D. No difference in the mean velocity was observed between αTC6 and CADM1-KD cells treated with nocodazole. These results suggest that intracellular granules in pancreatic α cells move along the microtubule network, and that CADM1 influences their velocity. PMID:27262873

  1. Insulin and Glucagon Secretion In Vitro

    NASA Technical Reports Server (NTRS)

    Rajan, Arun S.

    1998-01-01

    Long-duration space flight is associated with many physiological abnormalities in astronauts. In particular, altered regulation of the hormones insulin and glucagon may contribute to metabolic disturbances such as increased blood sugar levels, which if persistently elevated result in toxic effects. These changes are also observed in the highly prevalent disease diabetes, which affects 16 million Americans and consumes over $100 billion in annual healthcare costs. By mimicking the microgravity environment of space in the research laboratory using a NASA-developed bioreactor, one can study the physiology of insulin and glucagon secretion and determine if there are alterations in these cellular processes. The original specific objectives of the project included: (1) growing ('cell culture') of pancreatic islet beta and alpha cells that secrete insulin and glucagon respectively, in the NASA bioreactor; (2) examination of the effects of microgravity on insulin and glucagon secretion; and (3) study of molecular mechanisms of insulin and glucagon secretion if altered by microgravity.

  2. Progesterone Receptor Membrane Component 1 Is a Functional Part of the Glucagon-like Peptide-1 (GLP-1) Receptor Complex in Pancreatic β Cells*

    PubMed Central

    Zhang, Ming; Robitaille, Mélanie; Showalter, Aaron D.; Huang, Xinyi; Liu, Ying; Bhattacharjee, Alpana; Willard, Francis S.; Han, Junfeng; Froese, Sean; Wei, Li; Gaisano, Herbert Y.; Angers, Stéphane; Sloop, Kyle W.; Dai, Feihan F.; Wheeler, Michael B.

    2014-01-01

    Glucagon-like peptide-1 (GLP-1) is an incretin hormone that regulates glucose homeostasis. Because of their direct stimulation of insulin secretion from pancreatic β cells, GLP-1 receptor (GLP-1R) agonists are now important therapeutic options for the treatment of type 2 diabetes. To better understand the mechanisms that control the insulinotropic actions of GLP-1, affinity purification and mass spectrometry (AP-MS) were employed to uncover potential proteins that functionally interact with the GLP-1R. AP-MS performed on Chinese hamster ovary cells or MIN6 β cells, both expressing the human GLP-1R, revealed 99 proteins potentially associated with the GLP-1R. Three novel GLP-1R interactors (PGRMC1, Rab5b, and Rab5c) were further validated through co-immunoprecipitation/immunoblotting, fluorescence resonance energy transfer, and immunofluorescence. Functional studies revealed that overexpression of PGRMC1, a novel cell surface receptor that associated with liganded GLP-1R, enhanced GLP-1-induced insulin secretion (GIIS) with the most robust effect. Knockdown of PGRMC1 in β cells decreased GIIS, indicative of positive interaction with GLP-1R. To gain insight mechanistically, we demonstrated that the cell surface PGRMC1 ligand P4-BSA increased GIIS, whereas its antagonist AG-205 decreased GIIS. It was then found that PGRMC1 increased GLP-1-induced cAMP accumulation. PGRMC1 activation and GIIS induced by P4-BSA could be blocked by inhibition of adenylyl cyclase/EPAC signaling or the EGF receptor–PI3K signal transduction pathway. These data reveal a dual mechanism for PGRMC1-increased GIIS mediated through cAMP and EGF receptor signaling. In conclusion, we identified several novel GLP-1R interacting proteins. PGRMC1 expressed on the cell surface of β cells was shown to interact with the activated GLP-1R to enhance the insulinotropic actions of GLP-1. PMID:25044020

  3. N-Glycan Remodeling on Glucagon Receptor Is an Effector of Nutrient Sensing by the Hexosamine Biosynthesis Pathway*

    PubMed Central

    Johswich, Anita; Longuet, Christine; Pawling, Judy; Rahman, Anas Abdel; Ryczko, Michael; Drucker, Daniel J.; Dennis, James W.

    2014-01-01

    Glucose homeostasis in mammals is dependent on the opposing actions of insulin and glucagon. The Golgi N-acetylglucosaminyltransferases encoded by Mgat1, Mgat2, Mgat4a/b/c, and Mgat5 modify the N-glycans on receptors and solute transporter, possibly adapting activities in response to the metabolic environment. Herein we report that Mgat5−/− mice display diminished glycemic response to exogenous glucagon, together with increased insulin sensitivity. Glucagon receptor signaling and gluconeogenesis in Mgat5−/− cultured hepatocytes was impaired. In HEK293 cells, signaling by ectopically expressed glucagon receptor was increased by Mgat5 expression and GlcNAc supplementation to UDP-GlcNAc, the donor substrate shared by Mgat branching enzymes. The mobility of glucagon receptor in primary hepatocytes was reduced by galectin-9 binding, and the strength of the interaction was dependent on Mgat5 and UDP-GlcNAc levels. Finally, oral GlcNAc supplementation rescued the glucagon response in Mgat5−/− hepatocytes and mice, as well as glycolytic metabolites and UDP-GlcNAc levels in liver. Our results reveal that the hexosamine biosynthesis pathway and GlcNAc salvage contribute to glucose homeostasis through N-glycan branching on glucagon receptor. PMID:24742675

  4. Expression of Secretogranin III in Chicken Endocrine Cells

    PubMed Central

    Morikawa, Satomi; Shinmura, Naoki; Moki, Hiroaki; Yasui, Tadashi; Tsukise, Azuma; Torii, Seiji; Watanabe, Tsuyoshi; Maeda, Yoshinori; Hosaka, Masahiro

    2015-01-01

    The expression of secretogranin III (SgIII) in chicken endocrine cells has not been investigated. There is limited data available for the immunohistochemical localization of SgIII in the brain, pituitary, and pancreatic islets of humans and rodents. In the present study, we used immunoblotting to reveal the similarities between the expression patterns of SgIII in the common endocrine glands of chickens and rats. The protein–protein interactions between SgIII and chromogranin A (CgA) mediate the sorting of CgA/prohormone core aggregates to the secretory granule membrane. We examined these interactions using co-immunoprecipitation in chicken endocrine tissues. Using immunohistochemistry, we also examined the expression of SgIII in a wide range of chicken endocrine glands and gastrointestinal endocrine cells (GECs). SgIII was expressed in the pituitary, pineal, adrenal (medullary parts), parathyroid, and ultimobranchial glands, but not in the thyroid gland. It was also expressed in GECs of the stomach (proventriculus and gizzard), small and large intestines, and pancreatic islet cells. These SgIII-expressing cells co-expressed serotonin, somatostatin, gastric inhibitory polypeptide, glucagon-like peptide-1, glucagon, or insulin. These results suggest that SgIII is expressed in the endocrine cells that secrete peptide hormones, which mature via the intragranular enzymatic processing of prohormones and physiologically active amines in chickens. PMID:25673289

  5. The glucagon-like peptide 1 receptor agonist enhances intrinsic peroxisome proliferator-activated receptor γ activity in endothelial cells

    SciTech Connect

    Onuma, Hirohisa; Inukai, Kouichi Kitahara, Atsuko; Moriya, Rie; Nishida, Susumu; Tanaka, Toshiaki; Katsuta, Hidenori; Takahashi, Kazuto; Sumitani, Yoshikazu; Hosaka, Toshio; Ishida, Hitoshi

    2014-08-22

    Highlights: • PPARγ activation was involved in the GLP-1-mediated anti-inflammatory action. • Exendin-4 enhanced endogenous PPARγ transcriptional activity in HUVECs. • H89, a PKA inhibitor, abolished GLP-1-induced PPARγ enhancement. • The anti-inflammatory effects of GLP-1 may be explained by PPARγ activation. - Abstract: Recent studies have suggested glucagon-like peptide-1 (GLP-1) signaling to exert anti-inflammatory effects on endothelial cells, although the precise underlying mechanism remains to be elucidated. In the present study, we investigated whether PPARγ activation is involved in the GLP-1-mediated anti-inflammatory action on endothelial cells. When we treated HUVEC cells with 0.2 ng/ml exendin-4, a GLP-1 receptor agonist, endogenous PPARγ transcriptional activity was significantly elevated, by approximately 20%, as compared with control cells. The maximum PPARγ activity enhancing effect of exendin-4 was observed 12 h after the initiation of incubation with exendin-4. As H89, a PKA inhibitor, abolished GLP-1-induced PPARγ enhancement, the signaling downstream from GLP-1 cross-talk must have been involved in PPARγ activation. In conclusion, our results suggest that GLP-1 has the potential to induce PPARγ activity, partially explaining the anti-inflammatory effects of GLP-1 on endothelial cells. Cross-talk between GLP-1 signaling and PPARγ activation would have major impacts on treatments for patients at high risk for cardiovascular disease.

  6. Spergularia marina Induces Glucagon-Like Peptide-1 Secretion in NCI-H716 Cells Through Bile Acid Receptor Activation

    PubMed Central

    Kim, Kyong; Lee, Yu Mi; Rhyu, Mee-Ra

    2014-01-01

    Abstract Spergularia marina Griseb. (SM) is a halophyte that grows in mud flats. The aerial portions of SM have been eaten as vegetables and traditionally used to prevent chronic diseases in Korea. However, there has been no scientific report that demonstrates the pharmacological effects of SM. Glucagon-like peptide-1 (GLP-1) is important for the maintenance of glucose and energy homeostasis through acting as a signal in peripheral and neural systems. To discover a functional food for regulating glucose and energy homeostasis, we evaluated the effect of an aqueous ethanolic extract (AEE) of SM on GLP-1 release from enteroendocrine NCI-H716 cells. In addition, we explored the Takeda G-protein-coupled receptor 5 (TGR5) agonist activity of AEE-SM in Chinese hamster ovary (CHO)-K1 cells transiently transfected with human TGR5. As a result, treatment of NCI-H716 cells with AEE-SM increased GLP-1 secretion and intracellular Ca2+ and cyclic AMP (cAMP) levels in a dose-dependent manner. Transfection of NCI-H716 cells with TGR5-specific small interference RNA inhibited AEE-SM-induced GLP-1 secretion and the increase in Ca2+ and cAMP levels. Moreover, AEE-SM showed that the TGR5 agonist activity in CHO-K1 cells transiently transfected with TGR5. The results suggest that AEE-SM might be a candidate for a functional food to regulate glucose and energy homeostasis. PMID:25260089

  7. Glucagon is the key factor in the development of diabetes.

    PubMed

    Lee, Young H; Wang, May-Yun; Yu, Xin-Xin; Unger, Roger H

    2016-07-01

    Glucagon plays important roles in normal glucose homeostasis and in metabolic abnormalities, particularly diabetes. Glucagon excess, rather than insulin deficiency, is essential for the development of diabetes for several reasons. Glucagon increases hepatic glucose and ketone production, the catabolic features of insulin deficiency. Hyperglucagonaemia is present in every form of diabetes. Beta cell destruction in glucagon receptor null mice does not cause diabetes unless mice are administered adenovirus encoding the glucagon receptor. In rodent studies the glucagon suppressors leptin and glucagon receptor antibody suppressed all catabolic manifestations of diabetes during insulin deficiency. Insulin prevents hyperglycaemia; however, insulin monotherapy cannot cure diabetes such that non-diabetic glucose homeostasis is achieved. Glucose-responsive beta cells normally regulate alpha cells, and diminished insulin action on alpha cells will favour hypersecretion of glucagon by the alpha cells, thus altering the insulin:glucagon ratio. Treating diabetes by suppression of glucagon, with leptin or antibody against the glucagon receptor, normalised glucose level (without glycaemic volatility) and HbA1c. Glucagon suppression also improved insulin sensitivity and glucose tolerance. If these results can be translated to humans, suppression of glucagon action will represent a step forward in the treatment of diabetes. This review summarises a presentation given at the 'Novel data on glucagon' symposium at the 2015 annual meeting of the EASD. It is accompanied by two other reviews on topics from this symposium (by Mona Abraham and Tony Lam, DOI: 10.1007/s00125-016-3950-3 , and by Russell Miller and Morris Birnbaum, DOI: 10.1007/s00125-016-3955-y ) and an overview by the Session Chair, Isabel Valverde (DOI: 10.1007/s00125-016-3946-z ). PMID:27115412

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

    PubMed

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

    2016-01-01

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

  9. Thyroid calcitonin cells in response to glucagon-induced hypocalcaemia in the Indian jackal, Canis aureus (Linnaeus--lex).

    PubMed

    Swarup, K; Tewari, N P

    1980-01-01

    Jackal (Canis aureus) puppies (10) were subjected to hypocalcaemia by a single intravenous injection of crystalline porcine glucagon (Eli Lilly and Co.) in a dosage of 0.2 mg/kg body weight. Fasting blood samples from each specimen were collected 30 minutes before injection. Then again after an interval of 15, 30, 60, 90, 120, 150, 180, 210, 240, 270 and 300 minutes blood samples were taken. For histological study animals were killed after 30, 90, 120, 240 and 300 minutes of the injection. The mean serum calcium level records a fall upto 90 minutes but it tends to return to normal and at 300 minutes it returns to the preinjection level. The mean serum inorganic phosphate level records a fall upto 180 minutes and therafter the value increases approaching the preinjection levles after 300 minutes. Specific stains were used for staining the calcitonin cells. Animals killed 30 minutes after the injection exhibit beginning of degranulation of secretory granules in their C cells, while those killed after 300 minutes show marked degranulation. A progressive degranulation of calcitonin cells at the various stages of experimentation displays correspondingly poorer response to the staining reaction. There is no change in the histological picture of the parathyroid. PMID:7424080

  10. New insight into the mechanisms underlying the function of the incretin hormone glucagon-like peptide-1 in pancreatic β-cells

    PubMed Central

    Xiong, Xiaoquan; Shao, Weijuan; Jin, Tianru

    2012-01-01

    During the past two decades, the exploration of function of two incretin hormones, namely glucagon-like peptide-1 (GLP-1) and gastric inhibitory peptide (GIP), has led to the development of two categories of novel therapeutic agents for diabetes and its complications, known as GLP-1 receptor (GLP-1R) agonists and DPP-IV inhibitors. Mechanisms underlying the function of GLP-1, however, still need to be further explored. GLP-1 not only functions as an incretin hormone in stimulating insulin secretion in response to nutritional, hormonal and neuronal stimulations, but also acts as an “insulin-like” factor in β-cell and extra-pancreatic organs. In addition to these insulinotropic and insulinomimetic effects, GLP-1 was shown to exert its protective effect in β-cell by repressing the expression of TxNIP, a mediator of glucolipotoxicity. A number of recent studies have shown that the Wnt signaling pathway effector, the bipartite transcription factor β-catenin/TCF, controls not only the production of GLP-1, but also the function of GLP-1. Furthermore, previously assumed “degradation” products of GLP-1(7–36)amide, including GLP-1(9–36)amide and GLP-1(28–36)amide, have been shown to exert beneficial effect in pancreas and extra-pancreatic tissues or cell lineages. Here we summarized our current knowledge on the metabolic, proliferative and protective effects of GLP-1(7–36)amide and its cleavage fragments, mainly focusing on pancreatic β-cells and the involvement of the Wnt signaling pathway effector β-catenin. PMID:23314611

  11. Simultaneous quantification of intracellular and secreted active and inactive glucagon-like peptide-1 from cultured cells.

    PubMed

    Amao, Michiko; Kitahara, Yoshiro; Tokunaga, Ayaka; Shimbo, Kazutaka; Eto, Yuzuru; Yamada, Naoyuki

    2015-03-01

    Glucagon-like peptide-1 (GLP-1) is an incretin peptide that regulates islet hormone secretion. During recent years, incretin-based therapies have been widely used for patients with type 2 diabetes. GLP-1 peptides undergo N- and C-terminal processing for gain or loss of functions. We developed a method to quantify picomolar quantities of intact GLP-1 peptides using liquid chromatography-tandem mass spectrometry (LC-MS/MS). By employing this label-free selected reaction monitoring (SRM) method, we were able to analyze secreted GLP-1(1-37), GLP-1(7-37), and GLP-1(7-36 amid from human enteroendocrine NCI-H716 cells after stimulation with nateglinide, glucose, and sucralose. The absolute total concentrations of secreted GLP-1 peptides at baseline and after stimulation with nateglinide, glucose, and sucralose were 167.3, 498.9, 238.3, and 143.1 pM, respectively. Meanwhile, the ratios of GLP-1(1-37), GLP-1(7-37), and GLP-1(7-36 amide) to total GLP-1 peptides were similar (6 ± 3, 26 ± 3, and 78 ± 5%, respectively). The SRM assay can analyze the concentrations of individual GLP-1 peptides and, therefore, is a tool to investigate the physiological roles of GLP-1 peptides. Furthermore, the molecular species secreted from NCI-H716 cells were unknown. Therefore, we performed a secretopeptidome analysis of supernatants collected from cultured NCI-H716 cells. Together with GLP-1 peptides, we detected neuroendocrine convertase 1, which regulates peptide hormones released from intestinal endocrine L-cells. PMID:25461479

  12. Lamprey proglucagon and the origin of glucagon-like peptides.

    PubMed

    Irwin, D M; Huner, O; Youson, J H

    1999-11-01

    We characterized two proglucagon cDNAs from the intestine of the sea lamprey Petromyzon marinus. As in other vertebrates, sea lamprey proglucagon genes encode three glucagon-like sequences, glucagon, and glucagon-like peptides 1 and 2 (GLP-1 and GLP-2). This observation indicates that all three glucagon-like sequences encoded by the proglucagon gene originated prior to the divergence of jawed and jawless vertebrates. Estimates of the rates of evolution for the glucagon-like sequences suggest that glucagon originated first, about 1 billion years ago, while GLP-1 and GLP-2 diverged from each other about 700 MYA. The two sea lamprey intestinal proglucagon cDNAs have differing coding potential. Proglucagon I cDNA encodes the previously characterized glucagon and the glucagon-like peptide GLP-1, while proglucagon II cDNA encodes a predicted GLP-2 and, possibly, a glucagon. The existence of two proglucagon cDNAs which differ with regard to their potential to encode glucagon-like peptides suggests that the lamprey may use differential gene expression as a third mechanism, in addition to alternative proteolytic processing and mRNA splicing, to regulate the production of proglucagon-derived peptides. PMID:10555286

  13. Glucagon and gastroesophageal reflux.

    PubMed

    Drane, W E; Haggar, A M; Engel, M A

    1984-04-01

    Using radionuclide gastroesophageal reflux techniques, the effect of glucagon on the occurrence of spontaneous gastroesophageal reflux was tested in 24 normal, asymptomatic volunteers, who served as their own controls. Before glucagon administration, spontaneous gastroesophageal reflux did not occur in any of the volunteers. After 1 mg of glucagon was given, gastroesophageal reflux occurred in two (8%) of the 24 volunteers. Gastroesophageal reflux did not occur after the administration of high-density barium sulfate and an effervescent agent to simulate the circumstances of a routine double-contrast upper gastrointestinal examination. Although the effect of glucagon may facilitate gastroesophageal reflux in a small percentage of normal individuals, most do not exhibit spontaneous gastroesophageal reflux, either before or after glucagon administration. PMID:6608226

  14. Evidence of Extrapancreatic Glucagon Secretion in Man.

    PubMed

    Lund, Asger; Bagger, Jonatan I; Wewer Albrechtsen, Nicolai J; Christensen, Mikkel; Grøndahl, Magnus; Hartmann, Bolette; Mathiesen, Elisabeth R; Hansen, Carsten P; Storkholm, Jan H; van Hall, Gerrit; Rehfeld, Jens F; Hornburg, Daniel; Meissner, Felix; Mann, Matthias; Larsen, Steen; Holst, Jens J; Vilsbøll, Tina; Knop, Filip K

    2016-03-01

    Glucagon is believed to be a pancreas-specific hormone, and hyperglucagonemia has been shown to contribute significantly to the hyperglycemic state of patients with diabetes. This hyperglucagonemia has been thought to arise from α-cell insensitivity to suppressive effects of glucose and insulin combined with reduced insulin secretion. We hypothesized that postabsorptive hyperglucagonemia represents a gut-dependent phenomenon and subjected 10 totally pancreatectomized patients and 10 healthy control subjects to a 75-g oral glucose tolerance test and a corresponding isoglycemic intravenous glucose infusion. We applied novel analytical methods of plasma glucagon (sandwich ELISA and mass spectrometry-based proteomics) and show that 29-amino acid glucagon circulates in patients without a pancreas and that glucose stimulation of the gastrointestinal tract elicits significant hyperglucagonemia in these patients. These findings emphasize the existence of extrapancreatic glucagon (perhaps originating from the gut) in man and suggest that it may play a role in diabetes secondary to total pancreatectomy. PMID:26672094

  15. Role of fatty acid transport protein 4 in oleic acid-induced glucagon-like peptide-1 secretion from murine intestinal L cells

    PubMed Central

    Poreba, M. A.; Dong, C. X.; Li, S. K.; Stahl, A.; Miner, J. H.

    2012-01-01

    The antidiabetic intestinal L cell hormone glucagon-like peptide-1 (GLP-1) enhances glucose-dependent insulin secretion and inhibits gastric emptying. GLP-1 secretion is stimulated by luminal oleic acid (OA), which crosses the cell membrane by an unknown mechanism. We hypothesized that L cell fatty acid transport proteins (FATPs) are essential for OA-induced GLP-1 release. Therefore, the murine GLUTag L cell model was used for immunoblotting, [3H]OA uptake assay, and GLP-1 secretion assay as determined by radioimmunoassay following treatment with OA ± phloretin, sulfo-N-succinimidyl oleate, or siRNA against FATP4. FATP4−/− and cluster-of-differentiation 36 (CD36)−/− mice received intraileal OA, and plasma GLP-1 was measured by sandwich immunoassay. GLUTag cells were found to express CD36, FATP1, FATP3, and FATP4. The cells demonstrated specific 3H[OA] uptake that was dose-dependently inhibited by 500 and 1,000 μM unlabeled OA (P < 0.001). Cell viability was not altered by treatment with OA. Phloretin and sulfo-N-succinimidyl oleate, inhibitors of protein-mediated transport and CD36, respectively, also decreased [3H]OA uptake, as did knockdown of FATP4 by siRNA transfection (P < 0.05–0.001). OA dose-dependently increased GLP-1 secretion at 500 and 1,000 μM (P < 0.001), whereas phloretin, sulfo-N-succinimidyl oleate, and FATP4 knockdown decreased this response (P < 0.05–0.01). FATP4−/− mice displayed lower plasma GLP-1 at 60 min in response to intraileal OA (P < 0.05), whereas, unexpectedly, CD36−/− mice displayed higher basal GLP-1 levels (P < 0.01) but a normal response to intraileal OA. Together, these findings demonstrate a key role for FATP4 in OA-induced GLP-1 secretion from the murine L cell in vitro and in vivo, whereas the precise role of CD36 remains unclear. PMID:22871340

  16. Biosynthesis of glucagon in isolated pancreatic islets of guinea pigs

    PubMed Central

    Hellerström, Claes; Howell, Simon L.; Edwards, John C.; Andersson, Arne; Östenson, Claes-Göran

    1974-01-01

    1. The biosynthesis of glucagon in guinea-pig A2 cells was investigated by incubation of isolated islets of Langerhans in the presence of [3H]tryptophan for periods of up to 14 days. Proteins were extracted from islets and incubation media and analysed by gel filtration. 2. In addition to very-high-molecular-weight (100000) proteins, the principal tryptophan-containing biosynthetic product after incubation for up to 17h was a protein of minimum mol.wt. 9000, which co-eluted on gel filtration with a peak of glucagon-like immunoreactivity, but was apparently devoid of biological activity in a fat-cell assay. A discrete peak of labelled glucagon was only recovered after incubation for at least 6 days. Losses of glucagon during the extraction and rapid secretion of newly synthesized glucagon into incubation media were excluded as reasons for the lack of recovery of labelled hormone from islets after shorter incubations. 3. The 9000-mol.wt. protein was localized to A2 cells in experiments using B-cell-depleted islets, and to A2-cell granules by subcellular fractionation and electron-microscopic radioautography. Only glucagon was secreted into the incubation medium. 4. Possible relationships between the 9000-mol.wt. protein and glucagon are discussed in the light of postulated mechanisms of glucagon biosynthesis. PMID:4615708

  17. Cardiomyocyte glucagon receptor signaling modulates outcomes in mice with experimental myocardial infarction

    PubMed Central

    Ali, Safina; Ussher, John R.; Baggio, Laurie L.; Kabir, M. Golam; Charron, Maureen J.; Ilkayeva, Olga; Newgard, Christopher B.; Drucker, Daniel J.

    2014-01-01

    Objective Glucagon is a hormone with metabolic actions that maintains normoglycemia during the fasting state. Strategies enabling either inhibition or activation of glucagon receptor (Gcgr) signaling are being explored for the treatment of diabetes or obesity. However, the cardiovascular consequences of manipulating glucagon action are poorly understood. Methods We assessed infarct size and the following outcomes following left anterior descending (LAD) coronary artery ligation; cardiac gene and protein expression, acylcarnitine profiles, and cardiomyocyte survival in normoglycemic non-obese wildtype mice, and in newly generated mice with selective inactivation of the cardiomyocyte Gcgr. Complementary experiments analyzed Gcgr signaling and cell survival in cardiomyocyte cultures and cell lines, in the presence or absence of exogenous glucagon. Results Exogenous glucagon administration directly impaired recovery of ventricular pressure in ischemic mouse hearts ex vivo, and increased mortality from myocardial infarction after LAD coronary artery ligation in mice in a p38 MAPK-dependent manner. In contrast, cardiomyocyte-specific reduction of glucagon action in adult GcgrCM−/− mice significantly improved survival, and reduced hypertrophy and infarct size following myocardial infarction. Metabolic profiling of hearts from GcgrCM−/− mice revealed a marked reduction in long chain acylcarnitines in both aerobic and ischemic hearts, and following high fat feeding, consistent with an essential role for Gcgr signaling in the control of cardiac fatty acid utilization. Conclusions Activation or reduction of cardiac Gcgr signaling in the ischemic heart produces substantial cardiac phenotypes, findings with implications for therapeutic strategies designed to augment or inhibit Gcgr signaling for the treatment of metabolic disorders. PMID:25685700

  18. Adrenergic Modulation of Pancreatic Glucagon Secretion in Man

    PubMed Central

    Gerich, John E.; Langlois, Maurice; Noacco, Claudio; Schneider, Victor; Forsham, Peter H.

    1974-01-01

    In order to characterize the influence of the adrenergic system on pancreatic glucagon secretion in man, changes in basal glucagon secretion during infusions of pure alpha and beta adrenergic agonists and their specific antagonists were studied. During infusion of isoproterenol (3 μg/min), a beta adrenergic agonist, plasma glucagon rose from a mean (±SE) basal level of 104±10 to 171±15 pg/ml, P < 0.0002. Concomitant infusion of propranolol (80 μg/min), a beta adrenergic antagonist, prevented the effects of isoproterenol, although propranolol itself had no effect on basal glucagon secretion. During infusion of methoxamine (0.5 mg/min), an alpha adrenergic agonist, plasma glucagon declined from a mean basal level of 122±15 to 75±17 pg/ml, P < 0.001. Infusion of phentolamine (0.5 mg/min), an alpha adrenergic antagonist, caused a rise in plasma glucagon from a mean basal level of 118±16 to 175±21 pg/ml, P < 0.0001. Concomitant infusion of methoxamine with phentolamine caused a reversal of the effects of phentolamine. The present studies thus confirm that catecholamines affect glucagon secretion in man and demonstrate that the pancreatic alpha cell possesses both alpha and beta adrenergic receptors. Beta adrenergic stimulation augments basal glucagon secretion, while alpha adrenergic stimulation diminishes basal glucagon secretion. Furthermore, since infusion of phentolamine, an alpha adrenergic antagonist, resulted in an elevation of basal plasma glucagon levels, there appears to be an inhibitory alpha adrenergic tone governing basal glucagon secretion. The above findings suggest that catecholamines may influence glucose homeostasis in man through their effects on both pancreatic alpha and beta cell function. Images PMID:4825234

  19. Imaging exocytosis of single glucagon-like peptide-1 containing granules in a murine enteroendocrine cell line with total internal reflection fluorescent microscopy

    SciTech Connect

    Ohara-Imaizumi, Mica; Aoyagi, Kyota; Akimoto, Yoshihiro; Nakamichi, Yoko; Nishiwaki, Chiyono; Kawakami, Hayato; Nagamatsu, Shinya

    2009-12-04

    To analyze the exocytosis of glucagon-like peptide-1 (GLP-1) granules, we imaged the motion of GLP-1 granules labeled with enhanced yellow fluorescent protein (Venus) fused to human growth hormone (hGH-Venus) in an enteroendocrine cell line, STC-1 cells, by total internal reflection fluorescent (TIRF) microscopy. We found glucose stimulation caused biphasic GLP-1 granule exocytosis: during the first phase, fusion events occurred from two types of granules (previously docked granules and newcomers), and thereafter continuous fusion was observed mostly from newcomers during the second phase. Closely similar to the insulin granule fusion from pancreatic {beta} cells, the regulated biphasic exocytosis from two types of granules may be a common mechanism in glucose-evoked hormone release from endocrine cells.

  20. The Effects of Glucagon-like Peptide-2 on the Tight Junction and Barrier Function in IPEC-J2 Cells through Phosphatidylinositol 3-kinase–Protein Kinase B–Mammalian Target of Rapamycin Signaling Pathway

    PubMed Central

    Yu, Changsong; Jia, Gang; Deng, Qiuhong; Zhao, Hua; Chen, Xiaoling; Liu, Guangmang; Wang, Kangning

    2016-01-01

    Glucagon-like peptide-2 (GLP-2) is important for intestinal barrier function and regulation of tight junction (TJ) proteins, but the intracellular mechanisms of action remain undefined. The purpose of this research was to determine the protective effect of GLP-2 mediated TJ and transepithelial electrical resistance (TER) in lipopolysaccharide (LPS) stressed IPEC-J2 cells and to test the hypothesis that GLP-2 regulate TJ and TER through the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt)-mammalian target of rapamycin (mTOR) signaling pathway in IPEC-J2 cells. Wortmannin and LY294002 are specific inhibitors of PI3K. The results showed that 100 μg/mL LPS stress decreased TER and TJ proteins occludin, claudin-1 and zonula occludens protein 1 (ZO-1) mRNA, proteins expressions (p<0.01) respectively. GLP-2 (100 nmol/L) promote TER and TJ proteins occludin, claudin-1, and zo-1 mRNA, proteins expressions in LPS stressed and normal IPEC-J2 cells (p<0.01) respectively. In normal cells, both wortmannin and LY294002, PI3K inhibitors, prevented the mRNA and protein expressions of Akt and mTOR increase induced by GLP-2 (p<0.01) following with the significant decreasing of occludin, claudin-1, ZO-1 mRNA and proteins expressions and TER (p<0.01). In conclusion, these results indicated that GLP-2 can promote TJ’s expression and TER in LPS stressed and normal IPEC-J2 cells and GLP-2 could regulate TJ and TER through the PI3K/Akt/mTOR pathway. PMID:26954146

  1. Hypoglycemia unawareness prevention: Targeting glucagon production.

    PubMed

    Samson, Willis K; Stein, Lauren M; Elrick, Mollisa; Salvatori, Alison; Kolar, Grant; Corbett, John A; Yosten, Gina L C

    2016-08-01

    Insulin-dependent individuals with diabetes are at risk for a severe hypoglycemic event that may predispose them to several repeat episodes during which the normal counter regulatory mechanisms that protect against hypoglycemia fail to be activated. This state of hypoglycemia unawareness is characterized by a failure of glucagon release, preventing mobilization of endogenous glucose stores from the liver. We describe the discovery of a novel hormone, produced in pancreatic delta cells, which stimulates glucagon production and release, particularly under low glucose conditions. We hypothesize that this hormone, called neuronostatin, may be effective as a co-therapy with insulin to prevent repeated, potentially fatal episodes of recurrent hypoglycemia. PMID:27080082

  2. Hypothalamic CaMKKβ mediates glucagon anorectic effect and its diet-induced resistance

    PubMed Central

    Quiñones, Mar; Al-Massadi, Omar; Gallego, Rosalía; Fernø, Johan; Diéguez, Carlos; López, Miguel; Nogueiras, Ruben

    2015-01-01

    Objective Glucagon receptor antagonists and humanized glucagon antibodies are currently studied as promising therapies for obesity and type II diabetes. Among its variety of actions, glucagon reduces food intake, but the molecular mechanisms mediating this effect as well as glucagon resistance are totally unknown. Methods Glucagon and adenoviral vectors were administered in specific hypothalamic nuclei of lean and diet-induced obese rats. The expression of neuropeptides controlling food intake was performed by in situ hybridization. The regulation of factors of the glucagon signaling pathway was assessed by western blot. Results The central injection of glucagon decreased feeding through a hypothalamic pathway involving protein kinase A (PKA)/Ca2+-calmodulin-dependent protein kinase kinase β (CaMKKβ)/AMP-activated protein kinase (AMPK)-dependent mechanism. More specifically, the central injection of glucagon increases PKA activity and reduces protein levels of CaMKKβ and its downstream target phosphorylated AMPK in the hypothalamic arcuate nucleus (ARC). Consistently, central glucagon significantly decreased AgRP expression. Inhibition of PKA and genetic activation of AMPK in the ARC blocked glucagon-induced anorexia in lean rats. Genetic down-regulation of glucagon receptors in the ARC stimulates fasting-induced hyperphagia. Although glucagon was unable to decrease food intake in DIO rats, glucagon sensitivity was restored after inactivation of CaMKKβ, specifically in the ARC. Thus, glucagon decreases food intake acutely via PKA/CaMKKβ/AMPK dependent pathways in the ARC, and CaMKKβ mediates its obesity-induced hypothalamic resistance. Conclusions This work reveals the molecular underpinnings by which glucagon controls feeding that may lead to a better understanding of disease states linked to anorexia and cachexia. PMID:26909312

  3. Copeptin under glucagon stimulation.

    PubMed

    Lewandowski, Krzysztof C; Lewiński, Andrzej; Skowrońska-Jóźwiak, Elżbieta; Stasiak, Magdalena; Horzelski, Wojciech; Brabant, Georg

    2016-05-01

    Stimulation of growth hormone (GH) and adrenocorticotropic hormone (ACTH) secretion by glucagon is a standard procedure to assess pituitary dysfunction but the pathomechanism of glucagon action remains unclear. As arginine vasopressin (AVP) may act on the release of both, GH and ACTH, we tested here the role of AVP in GST by measuring a stable precursor fragment, copeptin, which is stoichiometrically secreted with AVP in a 1:1 ratio. ACTH, cortisol, GH, and copeptin were measured at 0, 60, 90, 120, 150, and 180 min during GST in 79 subjects: healthy controls (Group 1, n = 32), subjects with pituitary disease, but with adequate cortisol and GH responses during GST (Group 2, n = 29), and those with overt hypopituitarism (Group 3, n = 18). Copeptin concentrations significantly increased over baseline 150 and 180 min following glucagon stimulation in controls and patients with intact pituitary function but not in hypopituitarism. Copeptin concentrations were stimulated over time and the maximal increment correlated with ACTH, while correlations between copeptin and GH were weaker. Interestingly, copeptin as well as GH secretion was significantly attenuated when comparing subjects within the highest to those in the lowest BMI quartile (p < 0.05). Copeptin is significantly released following glucagon stimulation. As this release is BMI-dependent, the time-dependent relation between copeptin and GH may be obscured, whereas the close relation to ACTH suggests that AVP/copeptin release might be linked to the activation of the adrenal axis. PMID:26578365

  4. Altered enteroendocrine cell expression in T cell receptor alpha chain knock-out mice.

    PubMed

    Rubin, D C; Zhang, H; Qian, P; Lorenz, R G; Hutton, K; Peters, M G

    2000-10-15

    Mice lacking T cell receptor alpha chain (TCRalpha(-/-)) develop inflammation of the colon. We have examined the effect of this inflammation on the colonic epithelium by studying markers of epithelial cuff, enteroendocrine, and immune cell differentiation. Using immunohistochemical techniques, colons were compared in normal C57/BL6 and murine TCR alpha(-/-) mice aged 2 and 3 weeks and 3-11 months. TCR alpha(-/-) mice aged 3-11 months had histologic evidence of inflammation with increased expression of CD45, CD4+, CD8+, and B220+ cells and a decrease in expression of IgA+ cells. There was a decrease in the number of cholecystokinin, serotonin, and neurotensin enteroendocrine expressing cells in the colon of TCR alpha(-/-) mice. These changes were not present in 2-3-week-old suckling/weaning mice. In contrast, peptide tyrosine tyrosine (PYY), glucagon-like peptide-1, and gastrin expression did not change and small intestinal enteroendocrine cells remained unaltered. The change in colonic enteroendocrine cell expression appears to be a specific response, since only a subset of these cells was altered, and the epithelium was intact by histologic analysis. The absence of functional T cells in TCR alpha(-/-) colon has a marked effect on differentiation of a specific subpopulation of enteroendocrine cells, prior to loss of integrity of the epithelium. PMID:11054861

  5. Multiple Factors Related to the Secretion of Glucagon-Like Peptide-1

    PubMed Central

    Wang, XingChun; Liu, Huan; Chen, Jiaqi; Li, Yan; Qu, Shen

    2015-01-01

    The glucagon-like peptide-1 is secreted by intestinal L cells in response to nutrient ingestion. It regulates the secretion and sensitivity of insulin while suppressing glucagon secretion and decreasing postprandial glucose levels. It also improves beta-cell proliferation and prevents beta-cell apoptosis induced by cytotoxic agents. Additionally, glucagon-like peptide-1 delays gastric emptying and suppresses appetite. The impaired secretion of glucagon-like peptide-1 has negative influence on diabetes, hyperlipidemia, and insulin resistance related diseases. Thus, glucagon-like peptide-1-based therapies (glucagon-like peptide-1 receptor agonists and dipeptidyl peptidase-4 inhibitors) are now well accepted in the management of type 2 diabetes. The levels of glucagon-like peptide-1 are influenced by multiple factors including a variety of nutrients. The component of a meal acts as potent stimulants of glucagon-like peptide-1 secretion. The levels of its secretion change with the intake of different nutrients. Some drugs also have influence on GLP-1 secretion. Bariatric surgery may improve metabolism through the action on GLP-1 levels. In recent years, there has been a great interest in developing effective methods to regulate glucagon-like peptide-1 secretion. This review summarizes the literature on glucagon-like peptide-1 and related factors affecting its levels. PMID:26366173

  6. The aglycone of ginsenoside Rg3 enables glucagon-like peptide-1 secretion in enteroendocrine cells and alleviates hyperglycemia in type 2 diabetic mice

    PubMed Central

    Kim, Ki-Suk; Jung Yang, Hea; Lee, In-Seung; Kim, Kang-Hoon; Park, Jiyoung; Jeong, Hyeon-Soo; Kim, Yoomi; Seok Ahn, Kwang; Na, Yun-Cheol; Jang, Hyeung-Jin

    2015-01-01

    Ginsenosides can be classified on the basis of the skeleton of their aglycones. Here, we hypothesized that the sugar moieties attached to the dammarane backbone enable binding of the ginsenosides to the sweet taste receptor, eliciting glucagon-like peptide-1 (GLP-1) secretion in the enteroendocrine L cells. Using the human enteroendocrine NCI-H716 cells, we demonstrated that 15 ginsenosides stimulate GLP-1 secretion according to the position of their sugar moieties. Through a pharmacological approach and RNA interference technique to inhibit the cellular signal cascade and using the Gαgust−/− mice, we elucidated that GLP-1 secreting effect of Rg3 mediated by the sweet taste receptor mediated the signaling pathway. Rg3, a ginsenoside metabolite that transformed the structure through a steaming process, showed the strongest GLP-1 secreting effects in NCI-H716 cells and also showed an anti-hyperglycemic effect on a type 2 diabetic mouse model through increased plasma GLP-1 and plasma insulin levels during an oral glucose tolerance test. Our study reveals a novel mechanism where the sugar moieties of ginsenosides Rg3 stimulates GLP-1 secretion in enteroendocrine L cells through a sweet taste receptor-mediated signal transduction pathway and thus has an anti-hyperglycemic effect on the type 2 diabetic mouse model. PMID:26675132

  7. Self-Assembling Glucagon-Like Peptide 1-Mimetic Peptide Amphiphiles for Enhanced Activity and Proliferation of Insulin-Secreting Cells

    PubMed Central

    Khan, Saahir; Sur, Shantanu; Newcomb, Christina J.; Appelt, Elizabeth A.

    2012-01-01

    Current treatment for type 1 diabetes mellitus requires daily insulin injections that fail to produce physiological glycemic control. Islet cell transplantation has been proposed as a permanent cure but is limited by loss of β-cell viability and function. These limitations could potentially be overcome by relying on the activity of glucagon-like peptide 1 (GLP-1), which acts on β-cells to promote insulin release, proliferation, and survival. We have developed a peptide amphiphile (PA) molecule incorporating a peptide mimetic for GLP-1. This GLP-1-mimetic PA self-assembles into one-dimensional nanofibers that stabilize the active secondary structure of GLP-1 and can be cross-linked by calcium ions to form a macroscopic gel capable of cell encapsulation and 3-dimensional culture. The GLP-1-mimetic PA nanofibers were found to stimulate insulin secretion from rat insulinoma (RINm5f) cells to a significantly greater extent than the mimetic peptide alone and to a level equivalent to that of the clinically used agonist exendin-4. The activity of the GLP-1-mimetic PA is glucose-dependent, lipid-raft dependent, and partially PKA-dependent consistent with native GLP-1. The GLP-1-mimetic PA also completely abrogates inflammatory cytokine-induced cell death to the level of untreated controls. When used as a PA gel to encapsulate RINm5f cells, the GLP-1-mimetic PA stimulates insulin secretion and proliferation in a cytokine-resistant manner that is significantly greater than a non-bioactive PA gel containing exendin-4. Due to its self-assembling property and bioactivity, the GLP-1-mimetic PA can be incorporated into previously developed islet cell transplantation protocols with the potential for significant enhancement of β-cell viability and function. PMID:22342354

  8. The Glucagon-Like Peptide 1 Analogue Exendin-4 Attenuates the Nicotine-Induced Locomotor Stimulation, Accumbal Dopamine Release, Conditioned Place Preference as well as the Expression of Locomotor Sensitization in Mice

    PubMed Central

    Egecioglu, Emil; Engel, Jörgen A.; Jerlhag, Elisabet

    2013-01-01

    The gastrointestinal peptide glucagon-like peptide 1 (GLP-1) is known to regulate consummatory behavior and is released in response to nutrient ingestion. Analogues of this peptide recently emerged as novel pharmacotherapies for treatment of type II diabetes since they reduce gastric emptying, glucagon secretion as well as enhance glucose-dependent insulin secretion. The findings that GLP-1 targets reward related areas including mesolimbic dopamine areas indicate that the physiological role of GLP-1 extends beyond food intake and glucose homeostasis control to include reward regulation. The present series of experiments was therefore designed to investigate the effects of the GLP-1 receptor agonist, Exendin-4 (Ex4), on established nicotine-induced effects on the mesolimbic dopamine system in mice. Specifically, we show that treatment with Ex4, at a dose with no effect per se, attenuate nicotine-induced locomotor stimulation, accumbal dopamine release as well as the expression of conditioned place preference in mice. In accordance, Ex4 also blocks nicotine-induced expression of locomotor sensitization in mice. Given that development of nicotine addiction largely depends on the effects of nicotine on the mesolimbic dopamine system these findings indicate that the GLP-1 receptor may be a potential target for the development of novel treatment strategies for nicotine cessations in humans. PMID:24204788

  9. Glucagon-Like Peptide-1 Induced Signaling and Insulin Secretion Do Not Drive Fuel and Energy Metabolism in Primary Rodent Pancreatic β-Cells

    PubMed Central

    Peyot, Marie-Line; Gray, Joshua P.; Lamontagne, Julien; Smith, Peter J. S.; Holz, George G.; Madiraju, S. R. Murthy

    2009-01-01

    Background Glucagon like peptide-1 (GLP-1) and its analogue exendin-4 (Ex-4) enhance glucose stimulated insulin secretion (GSIS) and activate various signaling pathways in pancreatic β-cells, in particular cAMP, Ca2+ and protein kinase-B (PKB/Akt). In many cells these signals activate intermediary metabolism. However, it is not clear whether the acute amplification of GSIS by GLP-1 involves in part metabolic alterations and the production of metabolic coupling factors. Methodology/Prinicipal Findings GLP-1 or Ex-4 at high glucose caused release (∼20%) of the total rat islet insulin content over 1 h. While both GLP-1 and Ex-4 markedly potentiated GSIS in isolated rat and mouse islets, neither had an effect on β-cell fuel and energy metabolism over a 5 min to 3 h time period. GLP-1 activated PKB without changing glucose usage and oxidation, fatty acid oxidation, lipolysis or esterification into various lipids in rat islets. Ex-4 caused a rise in [Ca2+]i and cAMP but did not enhance energy utilization, as neither oxygen consumption nor mitochondrial ATP levels were altered. Conclusions/Significance The results indicate that GLP-1 barely affects β-cell intermediary metabolism and that metabolic signaling does not significantly contribute to GLP-1 potentiation of GSIS. The data also indicate that insulin secretion is a minor energy consuming process in the β-cell, and that the β-cell is different from most cell types in that its metabolic activation appears to be primarily governed by a “push” (fuel substrate driven) process, rather than a “pull” mechanism secondary to enhanced insulin release as well as to Ca2+, cAMP and PKB signaling. PMID:19593440

  10. The Anthocyanin Delphinidin 3-Rutinoside Stimulates Glucagon-Like Peptide-1 Secretion in Murine GLUTag Cell Line via the Ca2+/Calmodulin-Dependent Kinase II Pathway

    PubMed Central

    Kato, Masaki; Tani, Tsubasa; Terahara, Norihiko; Tsuda, Takanori

    2015-01-01

    Glucagon-like peptide-1 (GLP-1) is an incretin hormone secreted from enteroendocrine L-cells. Although several nutrients induce GLP-1 secretion, there is little evidence to suggest that non-nutritive compounds directly increase GLP-1 secretion. Here, we hypothesized that anthocyanins induce GLP-1 secretion and thereby significantly contribute to the prevention and treatment of diabetes. Delphinidin 3-rutinoside (D3R) was shown to increase GLP-1 secretion in GLUTag L cells. The results suggested that three hydroxyl or two methoxyl moieties on the aromatic ring are essential for the stimulation of GLP-1 secretion. Notably, the rutinose moiety was shown to be a potent enhancer of GLP-1 secretion, but only in conjunction with three hydroxyl moieties on the aromatic ring (D3R). Receptor antagonist studies revealed that D3R-stimulates GLP-1 secretion involving inositol 1,4,5-trisphosphate receptor-mediated intracellular Ca2+ mobilization. Treatment of GLUTag cells with a Ca2+/calmodulin-dependent kinaseII (CaMKII) inhibitor (KN-93) abolished D3R-stimulated GLP-1 secretion. In addition, treatment of GLUTag cells with D3R resulted in activation of CaMKII. Pre-treatment of cells with a G protein-coupled receptor (GPR) 40/120 antagonist (GW1100) also significantly decreased D3R-stimulated GLP-1 secretion. These observations suggest that D3R stimulates GLP-1 secretion in GLUTag cells, and that stimulation of GLP-1 secretion by D3R is mediated via Ca2+-CaMKII pathway, which may possibly be mediated by GPR40/120. These findings provide a possible molecular mechanism of GLP-1 secretion in intestinal L-cells mediated by foods or drugs and demonstrate a novel biological function of anthocyanins in regards to GLP-1 secretion. PMID:25962102

  11. Hepatic glucagon metabolism. Correlation of hormone processing by isolated canine hepatocytes with glucagon metabolism in man and in the dog.

    PubMed Central

    Hagopian, W A; Tager, H S

    1987-01-01

    We have found that canine and rat hepatocytes convert (125I)iodoTyr10-glucagon to a peptide metabolite lacking the NH2-terminal three residues of the hormone. The peptide is released into the cell incubation medium and its formation is unaffected by a variety of lysosomotropic or other agents. Use of specific radioimmunoassays and gel filtration demonstrated in both normal subjects and in chronic renal failure patients a plasma peptide having the properties of the hormone fragment identified by cell studies. Studies of the dog revealed a positive gradient of the fragment across the liver and no differential gradient of the fragment and glucagon across the kidney. We conclude that the glucagon fragment arises from the cell-mediated processing of the hormone on a superficial aspect of the hepatocyte, the glucagon fragment identified during experiments in vitro represents the cognate of a peptide formed during the hepatic metabolism of glucagon in vivo, and measurement of the fragment by COOH-terminal radioimmunoassays could lead to an understimulation of hepatic glucagon extraction. PMID:2879853

  12. Differentiation of embryonic stem cells conditionally expressing neurogenin 3.

    PubMed

    Treff, Nathan R; Vincent, Robert K; Budde, Melisa L; Browning, Victoria L; Magliocca, Joseph F; Kapur, Vivek; Odorico, Jon S

    2006-11-01

    Expression of the proendocrine gene neurogenin 3 (Ngn3) is required for the development of pancreatic islets. To better characterize the molecular events regulated by Ngn3 during development, we have determined the expression profiles of murine embryonic stem cells (mESCs) uniformly induced to overexpress Ngn3. An mESC line was created in order to induce Ngn3 by adding doxycycline to the culture medium. Genome-wide microarray analysis was performed to identify genes regulated by Ngn3 in a variety of contexts, including undifferentiated ESCs and differentiating embryoid bodies (EBs). Genes regulated by Ngn3 in a context-independent manner were identified and analyzed using systematic gene ontology tools. This analysis revealed Notch signaling as the most significantly regulated signaling pathway (p = .009). This result is consistent with the hypothesis that Ngn3 expression makes the cell competent for Notch signaling to be activated and, conversely, more sensitive to Notch signaling inhibition. Indeed, EBs induced to express Ngn3 were significantly more sensitive to gamma-secretase inhibitor-mediated Notch signaling inhibition (p < .0001) when compared with uninduced EBs. Moreover, we find that Ngn3 induction in differentiating ESCs results in significant increases in insulin, glucagon, and somatostatin expression. PMID:16809427

  13. Glucagon-Like Peptide-1 Strengthens the Barrier Integrity in Primary Cultures of Rat Brain Endothelial Cells Under Basal and Hyperglycemia Conditions.

    PubMed

    Fukuda, Shuji; Nakagawa, Shinsuke; Tatsumi, Rie; Morofuji, Yoichi; Takeshita, Tomonori; Hayashi, Kentaro; Tanaka, Kunihiko; Matsuo, Takayuki; Niwa, Masami

    2016-06-01

    The objective of the present study was to determine the effects of glucagon-like peptide-1 (GLP-1) on barrier functions and to assess the underlying mechanism using an in vitro blood-brain barrier (BBB) model comprised of a primary culture of rat brain capillary endothelial cells (RBECs). GLP-1 increased transendothelial electrical resistance and decreased the permeability of sodium fluorescein in RBECs in a dose- and time-dependent manner. The effects on these barrier functions were significantly reduced in the presence of the GLP-1 receptor antagonist exendin-3 (9-39) and the protein kinase A (PKA) inhibitor H-89. Western blot analysis showed that GLP-1 increased the amount of occludin and claudin-5. GLP-1 analogs are approved for treatment of type 2 diabetes mellitus, and thus, we examined the effects of GLP-1 on hyperglycemia-induced BBB damage. GLP-1 inhibited the increase in production of reactive oxygen species under hyperglycemia conditions and improved the BBB integrity induced by hyperglycemia. As GLP-1 stabilized the integrity of the BBB, probably via cAMP/PKA signaling, the possibility that GLP-1 acts as a BBB-protective drug should be considered. PMID:26659380

  14. Design, synthesis and crystallization of a novel glucagon analog as a therapeutic agent

    SciTech Connect

    Li, Pengyun; Rogers, Tanya; Smiley, David; DiMarchi, Richard D.; Zhang, Faming

    2007-07-01

    The synthesis and crystallization of glucagon-Cex are reported. Glucagon and glucagon-like peptide 1 (GLP-1) are drugs or drug candidates for the treatment of metabolic diseases such as diabetes and obesity. The native hormones have pharmacological deficiencies such as short half-life and poor solubility. A novel glucagon receptor agonist named glucagon-Cex has been designed, synthesized and crystallized. This peptide was highly soluble under physiological conditions and crystallized readily. The crystal diffracted X-rays to 2.2 Å resolution and the diffraction was consistent with space group P23, with unit-cell parameters a = b = c = 48.20 Å, α = β = γ = 90.0°. The crystals were suitable for a full structural determination to reveal the conformational differences between glucagon-Cex and the native hormone.

  15. Somatostatin receptor expression and biological functions in endocrine pancreatic cells: review based on a doctoral thesis.

    PubMed

    Ludvigsen, Eva

    2007-01-01

    Type 1 diabetes is resulting from the selective destruction of insulin-producing betacells within the pancreatic islets. Somatostatin acts as an inhibitor of hormone secretion through specific receptors (sst1-5). All ssts were expressed in normal rat and mouse pancreatic islets, although the expression intensity and the co-expression pattern varied between ssts as well as between species. This may reflect a difference in response to somatostatin in islet cells of the two species. The Non-Obese Diabetic (NOD) mouse model is an experimental model of type 1 diabetes, with insulitis accompanied by spontaneous hyperglycaemia. Pancreatic specimens from NOD mice at different age and stage of disease were stained for ssts. The islet cells of diabetic NOD mice showed increased islet expression of sst2-5 compared to normoglycemic NOD mice. The increase in sst2-5 expression in the islets cells may suggest either a contributing factor in the process leading to diabetes, or a defense response against ongoing beta-cell destruction. Somatostatin analogues were tested on a human endocrine pancreatic tumour cell line and cultured pancreatic islets. Somatostatin analogues had an effect on cAMP accumulation, chromogranin A secretion and MAP kinase activity in the cell line. Treatment of rat pancreatic islets with somatostatin analogues with selective receptor affinity was not sufficient to induce an inhibition of insulin and glucagon secretion. However, a combination of selective analogues or non-selective analogues via costimulation of receptors can cause inhibition of hormone production. For insulin and glucagon, combinations of sst2 + sst5 and sst1 + sst2, respectively, showed a biological effect. In summary, knowledge of islet cell ssts expression and the effect of somatostatin analogues with high affinity to ssts may be valuable in the future attempts to influence beta-cell function in type 1 diabetes mellitus, since down-regulation of beta-cell function may promote survival of

  16. Peptide YY expression is an early event in colonic endocrine cell differentiation: evidence from normal and transgenic mice.

    PubMed

    Upchurch, B H; Fung, B P; Rindi, G; Ronco, A; Leiter, A B

    1996-04-01

    The hormone peptide YY is produced by endocrine cells in the pancreas, ileum and colon. We have previously shown that peptide YY is coexpressed in all four islet cell types in the murine pancreas when they first appear, suggesting a common peptide YY-producing progenitor. In the colon, peptide YY has been frequently identified in glucagon-expressing L-type endocrine cells. Characterization of colonic endocrine tumors in transgenic mice expressing simian virus 40 large T antigen under the control of the peptide YY gene 5' flanking region revealed tumor cells producing not only peptide YY and glucagon, but also neurotensin, cholecystokinin, substance P, serotonin, secretin, and gastrin. This suggested that multiple enteroendocrine lineages were related to peptide YY-producing cells. Subsequent examination of the ontogeny of colonic endocrine differentiation in nontransgenic mice revealed that peptide YY was the first hormone to appear during development, at embryonic day 15.5. Between embryonic days 16.5 and 18.5, cells expressing glucagon, cholecystokinin, substance P, serotonin, secretin, neurotensin, gastrin and somatostatin first appeared and peptide YY was coexpressed in each cell type at this time. Peptide YY coexpression continued in a significant fraction of most enteroendocrine cell types throughout fetal and postnatal development and into adulthood, with the exception of serotonin-producing cells. This latter population of cells expanded dramatically after birth with rare coexpression of peptide YY. These studies indicate that expression of peptide YY is an early event in colonic endocrine differentiation and support the existence of a common progenitor for all endocrine cells in the colon. PMID:8620842

  17. Glucose control of glucagon secretion—‘There’s a brand-new gimmick every year’

    PubMed Central

    2016-01-01

    Glucagon from the pancreatic α-cells is a major blood glucose-regulating hormone whose most important role is to prevent hypoglycaemia that can be life-threatening due to the brain’s strong dependence on glucose as energy source. Lack of blood glucose-lowering insulin after malfunction or autoimmune destruction of the pancreatic β-cells is the recognized cause of diabetes, but recent evidence indicates that diabetic hyperglycaemia would not develop unless lack of insulin was accompanied by hypersecretion of glucagon. Glucagon release has therefore become an increasingly important target in diabetes management. Despite decades of research, an understanding of how glucagon secretion is regulated remains elusive, and fundamentally different mechanisms continue to be proposed. The autonomous nervous system is an important determinant of glucagon release, but it is clear that secretion is also directly regulated within the pancreatic islets. The present review focuses on pancreatic islet mechanisms involved in glucose regulation of glucagon release. It will be argued that α-cell-intrinsic processes are most important for regulation of glucagon release during recovery from hypoglycaemia and that paracrine inhibition by somatostatin from the δ-cells shapes pulsatile glucagon release in hyperglycaemia. The electrically coupled β-cells ultimately determine islet hormone pulsatility by releasing synchronizing factors that affect the α- and δ-cells. PMID:27044660

  18. Glucagon in the Artificial Pancreas

    PubMed Central

    2014-01-01

    The use of glucagon, in conjunction with insulin, in a dual chamber pump (artificial pancreas, AP) is a working goal for multiple companies and researchers. However, capital investment to create, operate, and maintain facilities with sufficient scale to produce enough glucagon to treat millions of patients, at a level of profit that makes it feasible, will be substantial. It can be assumed that the marketplace will expect the daily cost of glucagon (to the consumer) to be similar to the daily cost of insulin. After one subtracts wholesaler and pharmacy markup, there may be very few dollars remaining for the drug company to cover profit, capital expenditures, marketing, burden, and other costs. Without the potential for adequate margins, manufacturers may not be willing to take the risk. Assuming that the projections discussed in this article are in the right ballpark, advance planning for the supply for glucagon needs to start today and not wait for the AP to come to market. PMID:25139825

  19. Effects of the glucagon-like polypeptide-1 analogue (Val8)GLP-1 on learning, progenitor cell proliferation and neurogenesis in the C57B/16 mouse brain.

    PubMed

    McGovern, Stephen F J; Hunter, Kerry; Hölscher, Christian

    2012-09-14

    Type 2 diabetes (T2DM) has been identified as a risk factor for Alzheimer's disease. Here, we tested the properties of the glucagon-like polypetide-1 (GLP-1) analogue (Val8)GLP-1, a drug originally developed as a treatment for T2DM at a range of doses (2.5 nmol; 25 nmol; 100 nmol; or 250 nmol/kg bw ip.) in an acute memory study in wild type C57B/l6 mice. We also tested (Val8)GLP-1 and the GLP-1 receptor antagonist exendin (9-39) in a chronic study (3 weeks at 25 nmol/kg bw ip. once-daily). We found that (Val8)GLP-1 crossed the blood brain barrier readily and that peripheral injection increased levels in the brain 30 min post-injection ip. but not 2h post-injection in rats. In the acute study, the low dose of 2.5 nmol/kg ip. enhanced motor activity in the open field task, while total distance travelled, exploratory behaviour and anxiety was not affected at any dose. Learning an object recognition task was not affected either. In the chronic study, no effect was observed in the open field assessment. The antagonist exendin (9-39) impaired object recognition learning and spatial learning in a water maze task, demonstrating the importance of GLP-1 signalling in memory formation. Locomotor activity was also affected in some cases. Blood sugar levels and insulin sensitivity was not affected in chronically treated mice. Neuronal stem cells and neurogenesis was enhanced by (Val8)GLP-1 in the dentate gyrus of wild type mice. The results demonstrate that (Val8)GLP-1 is safe in a range of doses, crosses the BBB and has potentially beneficial effects in the CNS by enhancing neurogenesis. PMID:22867941

  20. Glucagon Release Induced by Pancreatic Nerve Stimulation in the Dog

    PubMed Central

    Marliss, Errol B.; Girardier, Lucien; Seydoux, Josiane; Wollheim, Claes B.; Kanazawa, Yasunori; Orci, Lelio; Renold, Albert E.; Porte, Daniel

    1973-01-01

    A direct neural role in the regulation of immunoreactive glucagon (IRG) secretion has been investigated during stimulation of mixed autonomic nerves to the pancreas in anesthetized dogs. The responses were evaluated by measurement of blood flow and hormone concentration in the venous effluent from the stimulated region of pancreas. Electrical stimulation of the distal end of the discrete bundles of nerve fibers isolated along the superior pancreaticoduodenal artery was invariably followed by an increase in IRG output. With 10-min periods of nerve stimulation, the integrated response showed that the higher the control glucagon output, the greater was the increment. Atropinization did not influence the response to stimulation. That the preparation behaved in physiologic fashion was confirmed by a fall in IRG output, and a rise in immunoreactive insulin (IRI) output, during hyperglycemia induced by intravenous glucose (0.1 g/kg). The kinetics of this glucose effect on IRG showed characteristics opposite to those of nerve stimulation: the lower the control output, the less the decrement. Furthermore, during the control steady state, blood glucose concentration was tightly correlated with the IRI/IRG molar output ratio, the function relating the two parameters being markedly nonlinear. Injection or primed infusion of glucose diminished the IRG response to simultaneous nerve stimulation. Measurement of IRG was inferred to reflect response of pancreatic glucagon secretion on the basis of the site of sample collection (the superior pancreaticoduodenal vein), the absence of changes in arterial IRG, and similar responses being obtained using an antibody specific for pancreatic glucagon. These studies support a role for the autonomic nervous system in the control of glucagon secretion: direct nerve stimulation induces glucagon release. Such sympathetic activation may be interpreted as capable of shifting the sensitivity of the A cell to glucose in the direction of higher

  1. REVIEW: Role of cyclic AMP signaling in the production and function of the incretin hormone glucagon-like peptide-1

    NASA Astrophysics Data System (ADS)

    Yu, Zhiwen; Jin, Tianru

    2008-01-01

    Pancreatic cells express the proglucagon gene (gcg) and thereby produce the peptide hormone glucagon, which stimulates hepatic glucose production and thereby increases blood glucose levels. The same gcg gene is also expressed in the intestinal endocrine L cells and certain neural cells in the brain. In the gut, gcg expression leads to the production of glucagon-like peptide-1 (GLP-1). This incretin hormone stimulates insulin secretion when blood glucose level is high. In addition, GLP-1 stimulates pancreatic cell proliferation, inhibits cell apoptosis, and has been utilized in the trans-differentiation of insulin producing cells. Today, a long-term effective GLP-1 receptor agonist has been developed as a drug in treating diabetes and potentially other metabolic disorders. Extensive investigations have shown that the expression of gcg and the production of GLP-1 can be activated by the elevation of the second messenger cyclic AMP (cAMP). Recent studies suggest that in addition to protein kinase A (PKA), exchange protein activated by cAMP (Epac), another effector of cAMP signaling, and the crosstalk between PKA and Wnt signaling pathway, are also involved in cAMP-stimulated gcg expression and GLP-1 production. Furthermore, functions of GLP-1 in pancreatic cells are mainly mediated by cAMP-PKA, cAMP-Epac and Wnt signaling pathways as well.

  2. Glucagon action in the brain.

    PubMed

    Abraham, Mona A; Lam, Tony K T

    2016-07-01

    In recent years, novel discoveries have reshaped our understanding of the biology of brain glucagon in the regulation of peripheral homeostasis. Here we compare and contrast brain glucagon action in feeding vs glucose regulation and depict the physiological relevance of brain glucagon by reviewing their actions in two key regions of the central nervous system: the mediobasal hypothalamus and the dorsal vagal complex. These novel findings pave the way to future therapeutic strategies aimed at enhancing brain glucagon action for the treatment of diabetes and obesity. This review summarises a presentation given at the 'Novel data on glucagon' symposium at the 2015 annual meeting of the EASD. It is accompanied by two other reviews on topics from this symposium (by Young Lee and colleagues, DOI: 10.1007/s00125-016-3965-9 ), and by Russell Miller and Morris Birnbaum, DOI: 10.1007/s00125-016-3955-y ) and an overview by the Session Chair, Isabel Valverde (DOI: 10.1007/s00125-016-3946-z ). PMID:27115416

  3. Differential effects of once-weekly glucagon-like peptide-1 receptor agonist dulaglutide and metformin on pancreatic β-cell and insulin sensitivity during a standardized test meal in patients with type 2 diabetes.

    PubMed

    Mari, A; Del Prato, S; Ludvik, B; Milicevic, Z; de la Peña, A; Shurzinske, L; Karanikas, C A; Pechtner, V

    2016-08-01

    This substudy of the AWARD-3 trial evaluated the effects of the once-weekly glucagon-like peptide-1 receptor agonist, dulaglutide, versus metformin on glucose control, pancreatic function and insulin sensitivity, after standardized test meals in patients with type 2 diabetes. Meals were administered at baseline, 26 and 52 weeks to patients randomized to monotherapy with dulaglutide 1.5 mg/week (n = 133), dulaglutide 0.75 mg/week (n = 136), or metformin ≥1500 mg/day (n = 140). Fasting and postprandial serum glucose, insulin, C-peptide and glucagon levels were measured up to 3 h post-meal. β-cell function and insulin sensitivity were assessed using empirical variables and mathematical modelling. At 26 weeks, similar decreases in area under the curve for glucose [AUCglucose (0-3 h)] were observed among all groups. β-cell function [AUCinsulin /AUCglucose (0-3 h)] increased with dulaglutide and was unchanged with metformin (p ≤ 0.005, both doses). Dulaglutide improved insulin secretion rate at 9 mmol/l glucose (p ≤ 0.04, both doses) and β-cell glucose sensitivity (p = 0.004, dulaglutide 1.5 mg). Insulin sensitivity increased more with metformin versus dulaglutide. In conclusion, dulaglutide improves postprandial glycaemic control after a standardized test meal by enhancing β-cell function, while metformin exerts a greater effect on insulin sensitivity. PMID:27059816

  4. Analysis of the glucagon receptor first extracellular loop by the substituted cysteine accessibility method.

    PubMed

    Roberts, David J; Vertongen, Pascale; Waelbroeck, Magali

    2011-08-01

    Glucagon is an important hormone for the prevention of hypoglycemia, and contributes to the hyperglycemia observed in diabetic patients, yet very little is known about its receptor structure and the receptor-glucagon interaction. In related receptors, the first extracellular loop, ECL1, is highly variable in length and sequence, suggesting that it might participate in ligand recognition. We applied a variant of the SCAM (Substituted Cysteine Accessibility Method) to the glucagon receptor ECL1 and sequentially mutated positions 197 to 223 to cysteine. Most of the mutations (15/27) affected the glucagon potency, due either to a modification of the glucagon binding site, or to the destabilization of the active receptor conformation. We reasoned that side chains accessible to glucagon must also be accessible to large, hydrophilic cysteine reagents. We therefore evaluated the accessibility of the introduced cysteines to maleimide-PEO(2)-biotin ((+)-biotinyl-3-maleimido-propionamidyl-3,6-dioxa-octanediamine), and tested the effect of pretreatment of intact cells with a large cationic cysteine reagent, MTSET ([2-(trimethylammonium)ethyl]methanethiosulfonate bromide), on glucagon potency. Our results suggest that the second and third transmembrane helices (TM2 and TM3) are extended to position 202 and from position 215, respectively, and separated by a short β stretch (positions 203-209). Glucagon binding induced a conformational change close to TM2: L198C was accessible to the biotin reagent only in the presence of glucagon. Most other mutations affected the receptor activation rather than glucagon recognition, but S217 and D218 (at the top of TM3) were good candidates for glucagon recognition and V221 was very close to the binding site. PMID:21704096

  5. Glucagon regulation of energy metabolism.

    PubMed

    Heppner, Kristy M; Habegger, Kirk M; Day, Jonathan; Pfluger, Paul T; Perez-Tilve, Diego; Ward, Brian; Gelfanov, Vasily; Woods, Steve C; DiMarchi, Richard; Tschöp, Matthias

    2010-07-14

    Glucagon has long been known as a counter-regulatory hormone to insulin of fundamental importance to glucose homeostasis. Its prominent ability to stimulate glycogenolysis and gluconeogenesis, has historically cast this peptide as one hormone where the metabolic consequences of increasing blood glucose levels, especially in obesity, are viewed largely as being deleterious. This perspective may be changing in light of emerging data and reconsideration of historic studies, which suggest that glucagon has beneficial effects on body fat mass, food intake, and energy expenditure. In this review, we discuss the mechanisms of glucagon-mediated body weight regulation as well as possible novel therapeutic approaches in the treatment of obesity and glucose intolerance that may arise from these findings. The paper represents an invited review by a symposium, award winner or keynote speaker at the Society for the Study of Ingestive Behavior [SSIB] Annual Meeting in Portland, July 2009. PMID:20381509

  6. Glucagon Metabolism in the Rat

    PubMed Central

    Emmanouel, D. S.; Jaspan, J. B.; Rubenstein, A. H.; Huen, A. H-J.; Fink, E.; Katz, A. I.

    1978-01-01

    The renal handling of the biologically active glucagon component (the 3,500-mol wt fraction of immunoreactive glucagon [IRG]) and the contribution of the kidney to its overall peripheral metabolism were studied in normal and uremic rats. The metabolic clearance rate of glucagon was 31.8 ± 1.2 ml/min per kg in normal animals and was diminished by approximately one-third in each of three groups of rats with compromized renal function: 22.3±1.6 ml/min per kg in partially (70%) nephrectomized; 22.9±3.3 ml/min per kg in bilaterally ureteral ligated; and 23.2±1.2 ml/min per kg in bilaterally nephrectomized animals. In normal rats the kidney contributed 30% to the overall metabolic clearance of the hormone and the renal extraction of endogenous and exogenous glucagon was similar, averaging 22.9±1.6% and was independent of plasma IRG levels over a wide range of arterial concentrations. The remnant kidney of partially (70%) nephrectomized animals continued to extract substantial amounts (16.6±4.2%) of the hormone, but accounted for only 8% of the total peripheral catabolism of IRG. In the two groups of animals with filtering kidneys, renal glucagon uptake was linearly related to its filtered load and could be accounted for by glomerular filtration and tubular reabsorption. However, the kidneys of animals with both ureters ligated (renal extraction of inulin = 3.2±1.8%) and hence virtual absence of glomerular filtration, continued to extract 11.5±1.9% of the renal arterial glucagon, contributing by 9% to its overall metabolic clearance, indicating that IRG uptake occurs also from the post glomerular capillaries. PMID:659638

  7. Glucagon-Like Peptide-1 Receptor Agonist Treatment Prevents Glucocorticoid-Induced Glucose Intolerance and Islet-Cell Dysfunction in Humans

    PubMed Central

    van Raalte, Daniël H.; van Genugten, Renate E.; Linssen, Margot M.L.; Ouwens, D. Margriet; Diamant, Michaela

    2011-01-01

    OBJECTIVE Glucocorticoids (GCs) are regarded as diabetogenic because they impair insulin sensitivity and islet-cell function. This study assessed whether treatment with the glucagon-like peptide receptor agonist (GLP-1 RA) exenatide (EXE) could prevent GC-induced glucose intolerance. RESEARCH DESIGN AND METHODS A randomized, placebo-controlled, double-blind, crossover study in eight healthy men (age: 23.5 [20.0–28.3] years; BMI: 26.4 [24.3–28.0] kg/m2) was conducted. Participants received three therapeutic regimens for 2 consecutive days: 1) 80 mg of oral prednisolone (PRED) every day (q.d.) and intravenous (IV) EXE infusion (PRED+EXE); 2) 80 mg of oral PRED q.d. and IV saline infusion (PRED+SAL); and 3) oral placebo-PRED q.d. and intravenous saline infusion (PLB+SAL). On day 1, glucose tolerance was assessed during a meal challenge test. On day 2, participants underwent a clamp procedure to measure insulin secretion and insulin sensitivity. RESULTS PRED+SAL treatment increased postprandial glucose levels (vs. PLB+SAL, P = 0.012), which was prevented by concomitant EXE (vs. PLB+SAL, P = NS). EXE reduced PRED-induced hyperglucagonemia during the meal challenge (P = 0.018) and decreased gastric emptying (vs. PRED+SAL, P = 0.028; vs. PLB+SAL, P = 0.046). PRED+SAL decreased first-phase glucose- and arginine-stimulated C-peptide secretion (vs. PLB+SAL, P = 0.017 and P = 0.05, respectively), whereas PRED+EXE improved first- and second-phase glucose- and arginine-stimulated C-peptide secretion (vs. PLB+SAL; P = 0.017, 0.012, and 0.093, respectively). CONCLUSIONS The GLP-1 RA EXE prevented PRED-induced glucose intolerance and islet-cell dysfunction in healthy humans. Incretin-based therapies should be explored as a potential strategy to prevent steroid diabetes. PMID:21216851

  8. Exendin-4, a glucagon-like peptide-1 receptor agonist, reduces intimal thickening after vascular injury

    SciTech Connect

    Goto, Hiromasa; Nomiyama, Takashi; Mita, Tomoya; Yasunari, Eisuke; Azuma, Kosuke; Komiya, Koji; Arakawa, Masayuki; Jin, Wen Long; Kanazawa, Akio; Kawamori, Ryuzo; Fujitani, Yoshio; Hirose, Takahisa; Watada, Hirotaka

    2011-02-04

    Research highlights: {yields} Exendin-4 reduces neointimal formation after vascular injury in a mouse model. {yields} Exendin-4 dose not alter metabolic parameters in non-diabetic, non-obese mouse model. {yields} Exendin-4 reduces PDGF-induced cell proliferation in cultured SMCs. {yields} Exendin-4 may reduces neointimal formation after vascular injury at least in part through its direct action on SMCs. -- Abstract: Glucagon-like peptide-1 is a hormone secreted by L cells of the small intestine and stimulates glucose-dependent insulin response. Glucagon-like peptide-1 receptor agonists such as exendin-4 are currently used in type 2 diabetes, and considered to have beneficial effects on the cardiovascular system. To further elucidate the effect of glucagon-like peptide-1 receptor agonists on cardiovascular diseases, we investigated the effects of exendin-4 on intimal thickening after endothelial injury. Under continuous infusion of exendin-4 at 24 nmol/kg/day, C57BL/6 mice were subjected to endothelial denudation injury of the femoral artery. Treatment of mice with exendin-4 reduced neointimal formation at 4 weeks after arterial injury without altering body weight or various metabolic parameters. In addition, in vitro studies of isolated murine, rat and human aortic vascular smooth muscle cells showed the expression of GLP-1 receptor. The addition of 10 nM exendin-4 to cultured smooth muscle cells significantly reduced their proliferation induced by platelet-derived growth factor. Our results suggested that exendin-4 reduced intimal thickening after vascular injury at least in part by the suppression of platelet-derived growth factor-induced smooth muscle cells proliferation.

  9. Glucagon-Like Peptide-1 Gene Therapy

    PubMed Central

    Rowzee, Anne M.; Cawley, Niamh X.; Chiorini, John A.; Di Pasquale, Giovanni

    2011-01-01

    Glucagon-like peptide 1 (GLP-1) is a small peptide component of the prohormone, proglucagon, that is produced in the gut. Exendin-4, a GLP-1 receptor agonist originally isolated from the saliva of H. suspectum or Gila monster, is a peptide that shares sequence and functional homology with GLP-1. Both peptides have been demonstrated to stimulate insulin secretion, inhibit glucagon secretion, promote satiety and slow gastric emptying. As such, GLP-1 and Exendin-4 have become attractive pharmaceutical targets as an adjunctive therapy for individuals with type II diabetes mellitus, with several products currently available clinically. Herein we summarize the cell biology leading to GLP-1 production and secretion from intestinal L-cells and the endocrine functions of this peptide and Exendin-4 in humans. Additionally, gene therapeutic applications of GLP-1 and Exendin-4 are discussed with a focus on recent work using the salivary gland as a gene therapy target organ for the treatment of diabetes mellitus. PMID:21747830

  10. Transgenic Expression of a Single Transcription Factor Pdx1 Induces Transdifferentiation of Pancreatic Acinar Cells to Endocrine Cells in Adult Mice

    PubMed Central

    Miyazaki, Satsuki; Tashiro, Fumi; Miyazaki, Jun-ichi

    2016-01-01

    A promising approach to new diabetes therapies is to generate β cells from other differentiated pancreatic cells in vivo. Because the acinar cells represent the most abundant cell type in the pancreas, an attractive possibility is to reprogram acinar cells into β cells. The transcription factor Pdx1 (Pancreas/duodenum homeobox protein 1) is essential for pancreatic development and cell lineage determination. Our objective is to examine whether exogenous expression of Pdx1 in acinar cells of adult mice might induce reprogramming of acinar cells into β cells. We established a transgenic mouse line in which Pdx1 and EGFP (enhanced green fluorescent protein) could be inducibly expressed in the acinar cells. After induction of Pdx1, we followed the acinar cells for their expression of exocrine and endocrine markers using cell-lineage tracing with EGFP. The acinar cell-specific expression of Pdx1 in adult mice reprogrammed the acinar cells as endocrine precursor cells, which migrated into the pancreatic islets and differentiated into insulin-, somatostatin-, or PP (pancreatic polypeptide)-producing endocrine cells, but not into glucagon-producing cells. When the mice undergoing such pancreatic reprogramming were treated with streptozotocin (STZ), the newly generated insulin-producing cells were able to ameliorate STZ-induced diabetes. This paradigm of in vivo reprogramming indicates that acinar cells hold promise as a source for new islet cells in regenerative therapies for diabetes. PMID:27526291

  11. Transgenic Expression of a Single Transcription Factor Pdx1 Induces Transdifferentiation of Pancreatic Acinar Cells to Endocrine Cells in Adult Mice.

    PubMed

    Miyazaki, Satsuki; Tashiro, Fumi; Miyazaki, Jun-Ichi

    2016-01-01

    A promising approach to new diabetes therapies is to generate β cells from other differentiated pancreatic cells in vivo. Because the acinar cells represent the most abundant cell type in the pancreas, an attractive possibility is to reprogram acinar cells into β cells. The transcription factor Pdx1 (Pancreas/duodenum homeobox protein 1) is essential for pancreatic development and cell lineage determination. Our objective is to examine whether exogenous expression of Pdx1 in acinar cells of adult mice might induce reprogramming of acinar cells into β cells. We established a transgenic mouse line in which Pdx1 and EGFP (enhanced green fluorescent protein) could be inducibly expressed in the acinar cells. After induction of Pdx1, we followed the acinar cells for their expression of exocrine and endocrine markers using cell-lineage tracing with EGFP. The acinar cell-specific expression of Pdx1 in adult mice reprogrammed the acinar cells as endocrine precursor cells, which migrated into the pancreatic islets and differentiated into insulin-, somatostatin-, or PP (pancreatic polypeptide)-producing endocrine cells, but not into glucagon-producing cells. When the mice undergoing such pancreatic reprogramming were treated with streptozotocin (STZ), the newly generated insulin-producing cells were able to ameliorate STZ-induced diabetes. This paradigm of in vivo reprogramming indicates that acinar cells hold promise as a source for new islet cells in regenerative therapies for diabetes. PMID:27526291

  12. A novel, stable, aqueous glucagon formulation using ferulic acid as an excipient.

    PubMed

    Bakhtiani, Parkash A; Caputo, Nicholas; Castle, Jessica R; El Youssef, Joseph; Carroll, Julie M; David, Larry L; Roberts, Charles T; Ward, W Kenneth

    2015-01-01

    Commercial glucagon is unstable due to aggregation and degradation. In closed-loop studies, it must be reconstituted frequently. For use in a portable pump for 3 days, a more stable preparation is required. At alkaline pH, curcumin inhibited glucagon aggregation. However, curcumin is not sufficiently stable for long-term use. Here, we evaluated ferulic acid, a stable breakdown product of curcumin, for its ability to stabilize glucagon. Ferulic acid-formulated glucagon (FAFG), composed of ferulic acid, glucagon, L-methionine, polysorbate-80, and human serum albumin in glycine buffer at pH 9, was aged for 7 days at 37°C. Glucagon aggregation was assessed by transmission electron microscopy (TEM) and degradation by high-performance liquid chromatography (HPLC). A cell-based protein kinase A (PKA) assay was used to assess in vitro bioactivity. Pharmacodynamics (PD) of unaged FAFG, 7-day aged FAFG, and unaged synthetic glucagon was determined in octreotide-treated swine. No fibrils were observed in TEM images of fresh or aged FAFG. Aged FAFG was 94% intact based on HPLC analysis and there was no loss of bioactivity. In the PD swine analysis, the rise over baseline of glucose with unaged FAFG, aged FAFG, and synthetic native glucagon (unmodified human sequence) was similar. After 7 days of aging at 37°C, an alkaline ferulic acid formulation of glucagon exhibited significantly less aggregation and degradation than that seen with native glucagon and was bioactive in vitro and in vivo. Thus, this formulation may be stable for 3-7 days in a portable pump for bihormonal closed-loop treatment of T1D. PMID:25253164

  13. Glucagon-like peptide-1 receptor agonists increase pancreatic mass by induction of protein synthesis.

    PubMed

    Koehler, Jacqueline A; Baggio, Laurie L; Cao, Xiemin; Abdulla, Tahmid; Campbell, Jonathan E; Secher, Thomas; Jelsing, Jacob; Larsen, Brett; Drucker, Daniel J

    2015-03-01

    Glucagon-like peptide-1 (GLP-1) controls glucose homeostasis by regulating secretion of insulin and glucagon through a single GLP-1 receptor (GLP-1R). GLP-1R agonists also increase pancreatic weight in some preclinical studies through poorly understood mechanisms. Here we demonstrate that the increase in pancreatic weight following activation of GLP-1R signaling in mice reflects an increase in acinar cell mass, without changes in ductal compartments or β-cell mass. GLP-1R agonists did not increase pancreatic DNA content or the number of Ki67(+) cells in the exocrine compartment; however, pancreatic protein content was increased in mice treated with exendin-4 or liraglutide. The increased pancreatic mass and protein content was independent of cholecystokinin receptors, associated with a rapid increase in S6 phosphorylation, and mediated through the GLP-1R. Rapamycin abrogated the GLP-1R-dependent increase in pancreatic mass but had no effect on the robust induction of Reg3α and Reg3β gene expression. Mass spectrometry analysis identified GLP-1R-dependent upregulation of Reg family members, as well as proteins important for translation and export, including Fam129a, eIF4a1, Wars, and Dmbt1. Hence, pharmacological GLP-1R activation induces protein synthesis, leading to increased pancreatic mass, independent of changes in DNA content or cell proliferation in mice. PMID:25277394

  14. The metabolic actions of glucagon revisited

    PubMed Central

    Habegger, Kirk M.; Heppner, Kristy M.; Geary, Nori; Bartness, Timothy J.; DiMarchi, Richard; Tschöp, Matthias H.

    2013-01-01

    The initial identification of glucagon as a counter-regulatory hormone to insulin revealed this hormone to be of largely singular physiological and pharmacological purpose. Glucagon agonism, however, has also been shown to exert effects on lipid metabolism, energy balance, body adipose tissue mass and food intake. The ability of glucagon to stimulate energy expenditure, along with its hypolipidemic and satiating effects, in particular, make this hormone an attractive pharmaceutical agent for the treatment of dyslipidemia and obesity. Studies that describe novel preclinical applications of glucagon, alone and in concert with glucagon-like peptide 1 agonism, have revealed potential benefits of glucagon agonism in the treatment of the metabolic syndrome. Collectively, these observations challenge us to thoroughly investigate the physiology and therapeutic potential of insulin's long-known opponent. PMID:20957001

  15. Endocrine-committed progenitor cells retain their differentiation potential in the absence of neurogenin-3 expression

    PubMed Central

    Prasadan, Krishna; Tulachan, Sidhartha; Guo, Ping; Shiota, Chiyo; Shah, Sohail; Gittes, George

    2016-01-01

    Neurogenin-3 (ngn-3) expression is critical for endocrine development in the developing pancreas. We found that when ngn-3 was inhibited in an E11.5 pancreas, using either morpholino antisense or siRNA, it led to a significant decrease in endocrine differentiation after seven days in culture. Endocrine differentiation was rescued when ngn-3 inhibition was withdrawn after three days of culture, suggesting that the embryonic pancreas retains progenitor cells with the ability to differentiate into endocrine cell types when ngn-3 expression recurs. To determine whether the rescue phenomenon observed after withdrawing ngn-3 antisense treatment was the result of the original endocrine-committed cells reinitiating endocrine differentiation, or was instead due to new recruitment of later progenitor cells, we blocked ngn-3 expression for only the last four days of a seven-day culture. Here, insulin-positive differentiation was slightly reduced, but there was a normal number of glucagon-positive cells. In addition, there was an increase in SOX9-positive cells in ngn-3 inhibited, as well as in ngn-3 rescued pancreata, with a significant proportion of these SOX9-positive cells co-localized with DBA, an early ductal marker. This increased number of cells with co-localization of SOX9 and DBA could indicate an increased numbers of endocrine progenitor cells. PMID:20471370

  16. 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'. PMID:26483393

  17. Improving function and survival of pancreatic islets by endogenous production of glucagon-like peptide 1 (GLP-1).

    PubMed

    Wideman, Rhonda D; Yu, Irene L Y; Webber, Travis D; Verchere, C Bruce; Johnson, James D; Cheung, Anthony T; Kieffer, Timothy J

    2006-09-01

    Glucagon-like peptide 1 (GLP-1) is a hormone that has received significant attention as a therapy for diabetes because of its ability to stimulate insulin biosynthesis and release and to promote growth and survival of insulin-producing beta cells. While GLP-1 is produced from the proglucagon precursor by means of prohormone convertase (PC) 1/3 activity in enteroendocrine L cells, the same precursor is differentially processed by PC2 in pancreatic islet alpha cells to release glucagon, leaving GLP-1 trapped within a larger fragment with no known function. We hypothesized that we could induce GLP-1 production directly within pancreatic islets by means of delivery of PC1/3 and, further, that this intervention would improve the viability and function of islets. Here, we show that adenovirus-mediated expression of PC1/3 in alpha cells increases islet GLP-1 secretion, resulting in improved glucose-stimulated insulin secretion and enhanced survival in response to cytokine treatment. PC1/3 expression in alpha cells also improved performance after islet transplantation in a mouse model of type 1 diabetes, possibly by enhancing nuclear Pdx1 and insulin content of islet beta cells. These results demonstrate a unique strategy for liberating GLP-1 from directly within the target organ and highlight the potential for up-regulating islet GLP-1 production as a means of treating diabetes. PMID:16938896

  18. Identification of microRNAs expressed highly in pancreatic islet-like cell clusters differentiated from human embryonic stem cells.

    PubMed

    Chen, Bo-Zhi; Yu, Sung-Liang; Singh, Sher; Kao, Li-Pin; Tsai, Zong-Yun; Yang, Pan-Chyr; Chen, Bai-Hsiun; Shoei-Lung Li, Steven

    2011-01-01

    Type 1 diabetes is an autoimmune destruction of pancreatic islet beta cell disease, making it important to find a new alternative source of the islet beta cells to replace the damaged cells. hES (human embryonic stem) cells possess unlimited self-renewal and pluripotency and thus have the potential to provide an unlimited supply of different cell types for tissue replacement. The hES-T3 cells with normal female karyotype were first differentiated into EBs (embryoid bodies) and then induced to generate the T3pi (pancreatic islet-like cell clusters derived from T3 cells), which expressed pancreatic islet cell-specific markers of insulin, glucagon and somatostatin. The expression profiles of microRNAs and mRNAs from the T3pi were analysed and compared with those of undifferentiated hES-T3 cells and differentiated EBs. MicroRNAs negatively regulate the expression of protein-coding mRNAs. The T3pi showed very high expression of microRNAs, miR-186, miR-199a and miR-339, which down-regulated the expression of LIN28, PRDM1, CALB1, GCNT2, RBM47, PLEKHH1, RBPMS2 and PAK6. Therefore, these microRNAs and their target genes are very likely to play important regulatory roles in the development of pancreas and/or differentiation of islet cells, and they may be manipulated to increase the proportion of beta cells and insulin synthesis in the differentiated T3pi for cell therapy of type I diabetics. PMID:20735361

  19. 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

  20. α-Cell Dysfunctions and Molecular Alterations in Male Insulinopenic Diabetic Mice Are Not Completely Corrected by Insulin.

    PubMed

    Dusaulcy, Rodolphe; Handgraaf, Sandra; Heddad-Masson, Mounia; Visentin, Florian; Vesin, Christian; Reimann, Franck; Gribble, Fiona; Philippe, Jacques; Gosmain, Yvan

    2016-02-01

    Glucagon and α-cell dysfunction are critical in the development of hyperglycemia during diabetes both in humans and rodents. We hypothesized that α-cell dysfunction leading to dysregulated glucagon secretion in diabetes is due to both a lack of insulin and intrinsic defects. To characterize α-cell dysfunction in diabetes, we used glucagon-Venus transgenic male mice and induced insulinopenic hyperglycemia by streptozotocin administration leading to alterations of glucagon secretion. We investigated the in vivo impact of insulinopenic hyperglycemia on glucagon-producing cells using FACS-sorted α-cells from control and diabetic mice. We demonstrate that increased glucagonemia in diabetic mice is mainly due to increases of glucagon release and biosynthesis per cell compared with controls without changes in α-cell mass. We identified genes coding for proteins involved in glucagon biosynthesis and secretion, α-cell differentiation, and potential stress markers such as the glucagon, Arx, MafB, cMaf, Brain4, Foxa1, Foxa3, HNF4α, TCF7L2, Glut1, Sglt2, Cav2.1, Cav2.2, Nav1.7, Kir6.2/Sur1, Pten, IR, NeuroD1, GPR40, and Sumo1 genes, which were abnormally regulated in diabetic mice. Importantly, insulin treatment partially corrected α-cell function and expression of genes coding for proglucagon, or involved in glucagon secretion, glucose transport and insulin signaling but not those coding for cMAF, FOXA1, and α-cell differentiation markers as well as GPR40, NEUROD1, CAV2.1, and SUMO1. Our results indicate that insulinopenic diabetes induce marked α-cell dysfunction and molecular alteration, which are only partially corrected by in vivo insulin treatment. PMID:26696123

  1. Sotalol-induced bradycardia reversed by glucagon.

    PubMed Central

    Fernandes, C. M.; Daya, M. R.

    1995-01-01

    Glucagon is considered the drug of choice for treating bradycardia and hypotension encountered during beta-blocker poisoning. Its potential usefulness in reversing adverse effects encountered during therapeutic dosing with beta-blockers has not been well characterized. We present a case of sotalol-induced bradycardia reversed by glucagon. PMID:7787496

  2. Glucagon Receptor Blockade With a Human Antibody Normalizes Blood Glucose in Diabetic Mice and Monkeys.

    PubMed

    Okamoto, Haruka; Kim, Jinrang; Aglione, JohnPaul; Lee, Joseph; Cavino, Katie; Na, Erqian; Rafique, Ashique; Kim, Jee Hae; Harp, Joyce; Valenzuela, David M; Yancopoulos, George D; Murphy, Andrew J; Gromada, Jesper

    2015-08-01

    Antagonizing glucagon action represents an attractive therapeutic option for reducing hepatic glucose production in settings of hyperglycemia where glucagon excess plays a key pathophysiological role. We therefore generated REGN1193, a fully human monoclonal antibody that binds and inhibits glucagon receptor (GCGR) signaling in vitro. REGN1193 administration to diabetic ob/ob and diet-induced obese mice lowered blood glucose to levels observed in GCGR-deficient mice. In diet-induced obese mice, REGN1193 reduced food intake, adipose tissue mass, and body weight. REGN1193 increased circulating levels of glucagon and glucagon-like peptide 1 and was associated with reversible expansion of pancreatic α-cell area. Hyperglucagonemia and α-cell hyperplasia was observed in fibroblast growth factor 21-deficient mice treated with REGN1193. Single administration of REGN1193 to diabetic cynomolgus monkeys normalized fasting blood glucose and glucose tolerance and increased circulating levels of glucagon and amino acids. Finally, administration of REGN1193 for 8 weeks to normoglycemic cynomolgus monkeys did not cause hypoglycemia or increase pancreatic α-cell area. In summary, the GCGR-blocking antibody REGN1193 normalizes blood glucose in diabetic mice and monkeys but does not produce hypoglycemia in normoglycemic monkeys. Thus, REGN1193 provides a potential therapeutic modality for diabetes mellitus and acute hyperglycemic conditions. PMID:26020795

  3. Pancreatic small cells: Analysis of quiescence, long-term maintenance and insulin expression in vitro

    SciTech Connect

    Petropavlovskaia, M. . E-mail: maria.petropavlovskaia@mail.mcgill.ca; Bodnar, C.A.; Behie, L.A.; Rosenberg, L.

    2007-03-10

    We have previously identified a novel population of small cells in human and canine pancreas characterized by immature morphology, quiescence, and a glucose-responsive insulin secretion. Based on their immature phenotype and predominant presence in small islets, we have hypothesized that small cells serve as islet progenitors. This hypothesis remains untested, however, due to persistent quiescence and scarcity of small cells in vitro. We have recently developed a culture medium that allowed for modest small cell proliferation. In this study we characterized the expression of genes potentially involved in small cell growth regulation by Q-RT-PCR. Our results suggest that quiescence of small cells correlates with up-regulation of Cdk inhibitors p27{sup Kip1}, p16{sup INK4a} and p21{sup CIP1}, PTEN, Hep27 and Foxo1a and with down-regulation of c-Myc and the receptors for EGF, FGF2 and HGF. The exit from quiescence correlates with activation of EGFR expression and down-regulation of p27{sup Kip1} and p16{sup INK4a}. We also report here that small cells can be maintained in long-term non-adherent cultures preserving insulin and glucagon production for up to 208 days. Therefore, expansion of small cells in vitro may have a significant potential for the treatment of diabetes. This study is an important step in understanding the mechanisms involved in small cell growth regulation, which is required to fully evaluate their functional potential.

  4. Protein kinase C pathway mediates the protective effects of glucagon-like peptide-1 on the apoptosis of islet β-cells.

    PubMed

    Zhang, Lihai; Wang, Yuesheng; Wang, Jiao; Liu, Yinglan; Yin, Yanbin

    2015-11-01

    The incidence of diabetes has been increasing over previous years. It is hypothesized that promoting the survival of islet β-cells is a key direction for the treatment of diabetes. Although gastric bypass surgery improves certain types of diabetes and attenuates its progression, there are certain associated disadvantages (including intestinal obstruction and anastomotic leakage), and quality of life and physical status (such as malnutrition) are significantly affected by gastric bypass surgery. Therefore, it is important to determine the mechanisms underlying the improvement of diabetes by gastric bypass surgery and identify novel gene targets for diabetes therapeutics. In the present study, glucagon‑like peptide‑1 (GLP‑1), whose secretion was markedly increased following gastric bypass surgery, increased the activity of protein kinase C (PKC) in islet β‑cells in a dose‑dependent manner. Additionally, treatment with GLP‑1 boosted cell viability and decreased cell death in starved islet β‑cells, and inhibited mitochondria‑dependent apoptosis by regulating the expression levels of Bcl‑2/Bax. These effects were reversed by inhibiting the PKC pathway using hypericin. Therefore, the present study concluded that GLP‑1 may promote the survival and inhibit the apoptosis of islet β‑cells at least in part by activating the PKC pathway, which is an important underlying mechanism and may be exploited in the treatment of diabetes. PMID:26459881

  5. The G Protein-coupled Receptor Family C Group 6 Subtype A (GPRC6A) Receptor Is Involved in Amino Acid-induced Glucagon-like Peptide-1 Secretion from GLUTag Cells*

    PubMed Central

    Oya, Manami; Kitaguchi, Tetsuya; Pais, Ramona; Reimann, Frank; Gribble, Fiona; Tsuboi, Takashi

    2013-01-01

    Although amino acids are dietary nutrients that evoke the secretion of glucagon-like peptide 1 (GLP-1) from intestinal L cells, the precise molecular mechanism(s) by which amino acids regulate GLP-1 secretion from intestinal L cells remains unknown. Here, we show that the G protein-coupled receptor (GPCR), family C group 6 subtype A (GPRC6A), is involved in amino acid-induced GLP-1 secretion from the intestinal L cell line GLUTag. Application of l-ornithine caused an increase in intracellular Ca2+ concentration ([Ca2+]i) in GLUTag cells. Application of a GPRC6A receptor antagonist, a phospholipase C inhibitor, or an IP3 receptor antagonist significantly suppressed the l-ornithine-induced [Ca2+]i increase. We found that the increase in [Ca2+]i stimulated by l-ornithine correlated with GLP-1 secretion and that l-ornithine stimulation increased exocytosis in a dose-dependent manner. Furthermore, depletion of endogenous GPRC6A by a specific small interfering RNA (siRNA) inhibited the l-ornithine-induced [Ca2+]i increase and GLP-1 secretion. Taken together, these findings suggest that the GPRC6A receptor functions as an amino acid sensor in GLUTag cells that promotes GLP-1 secretion. PMID:23269670

  6. Purification and Characterization of a Population of EGFP-Expressing Cells from the Developing Pancreas of a Neurogenin3/EGFP Transgenic Mouse

    PubMed Central

    Barrow, J; Bernardo, A S; Hay, C W; Blaylock, M; Duncan, L; MacKenzie, A; McCreath, K; Kind, A J; Schnieke, A E; Colman, A; Hart, A W

    2005-01-01

    Neurogenin 3 (ngn3) is a basic helix loop helix transcription factor that is transiently expressed in the developing mouse pancreas with peak expression around E15. In mice lacking the ngn3 gene the endocrine cells of the pancreas fail to develop suggesting that the ngn3-positive cell may represent a progenitor cell for the endocrine pancreas. In order to purify and characterize this cell in detail we have generated a transgenic mouse, in which the ngn3 promoter drives expression of enhanced green fluorescent protein (EGFP). In the E15.5 embryo EGFP was expressed in the dorsal and ventral pancreas, the duodenum, and lower intestine as well as in the brain. This pattern of expression was in keeping with the known expression profile of the endogenous ngn3 gene. Within the pancreas EGFP was localized in close proximity to cells that stained positive for ngn3, insulin, and glucagon, but was absent from regions of the pancreas that stained positive for amylase. EGFP was also present in the pancreas at E18.5, although there was no detectable expression of ngn3. At this stage EGFP did not colocalize with any of the hormones or exocrine markers. EGFP+ cells were FACS purified (96%) from the E15 pancreas yielding ∼ 10,000 cells or 1.6% of the total pancreatic cells from one litter. RT/PCR analysis confirmed that the purified cells expressed EGFP, ngn3, insulin, glucagon, somatostatin and pancreatic polypeptide. The ability to purify ngn3+ cells provides an invaluable source of material for charactering in detail their properties. PMID:19521525

  7. Strategies for multigene expression in eukaryotic cells.

    PubMed

    Mansouri, Maysam; Berger, Philipp

    2014-09-01

    Multigene delivery systems for heterologous multiprotein expression in mammalian cells are a key technology in contemporary biological research. Multiprotein expression is essential for a variety of applications, including multiparameter analysis of living cells in vitro, changing the fate of stem cells, or production of multiprotein complexes for structural biology. Depending on the application, these expression systems have to fulfill different requirements. For some applications, homogenous expression in all cells with defined stoichiometry is necessary, whereas other applications need long term expression or require that the proteins are not modified at the N- and C-terminus. Here we summarize available multiprotein expression systems and discuss their advantages and disadvantages. PMID:25034976

  8. Chicken glucagon: sequence and potency in receptor assay.

    PubMed

    Huang, J; Eng, J; Yalow, R S

    1987-11-01

    Glucagon is a 29 amino acid peptide that is generally highly conserved. Among mammalian glucagons the only one that has been shown to differ significantly is that of the guinea pig which differs from the others in 5 of the 9 COOH-terminus amino acids. The amino acid content and partial sequencing of chicken glucagon had been reported earlier. This report describes the purification and complete amino acid sequencing of chicken glucagon and demonstrates that it differs from the usual mammalian glucagon by the replacement of asparagine at position 28 with serine. Chicken glucagon is indistinguishable from porcine glucagon in the rat liver receptor assay system. PMID:2828209

  9. Regulation of Glucagon Secretion in Normal and Diabetic Human Islets by γ-Hydroxybutyrate and Glycine*

    PubMed Central

    Li, Changhong; Liu, Chengyang; Nissim, Itzhak; Chen, Jie; Chen, Pan; Doliba, Nicolai; Zhang, Tingting; Nissim, Ilana; Daikhin, Yevgeny; Stokes, David; Yudkoff, Marc; Bennett, Michael J.; Stanley, Charles A.; Matschinsky, Franz M.; Naji, Ali

    2013-01-01

    Paracrine signaling between pancreatic islet β-cells and α-cells has been proposed to play a role in regulating glucagon responses to elevated glucose and hypoglycemia. To examine this possibility in human islets, we used a metabolomic approach to trace the responses of amino acids and other potential neurotransmitters to stimulation with [U-13C]glucose in both normal individuals and type 2 diabetics. Islets from type 2 diabetics uniformly showed decreased glucose stimulation of insulin secretion and respiratory rate but demonstrated two different patterns of glucagon responses to glucose: one group responded normally to suppression of glucagon by glucose, but the second group was non-responsive. The non-responsive group showed evidence of suppressed islet GABA levels and of GABA shunt activity. In further studies with normal human islets, we found that γ-hydroxybutyrate (GHB), a potent inhibitory neurotransmitter, is generated in β-cells by an extension of the GABA shunt during glucose stimulation and interacts with α-cell GHB receptors, thus mediating the suppressive effect of glucose on glucagon release. We also identified glycine, acting via α-cell glycine receptors, as the predominant amino acid stimulator of glucagon release. The results suggest that glycine and GHB provide a counterbalancing receptor-based mechanism for controlling α-cell secretory responses to metabolic fuels. PMID:23266825

  10. Regulation of glucagon secretion in normal and diabetic human islets by γ-hydroxybutyrate and glycine.

    PubMed

    Li, Changhong; Liu, Chengyang; Nissim, Itzhak; Chen, Jie; Chen, Pan; Doliba, Nicolai; Zhang, Tingting; Nissim, Ilana; Daikhin, Yevgeny; Stokes, David; Yudkoff, Marc; Bennett, Michael J; Stanley, Charles A; Matschinsky, Franz M; Naji, Ali

    2013-02-01

    Paracrine signaling between pancreatic islet β-cells and α-cells has been proposed to play a role in regulating glucagon responses to elevated glucose and hypoglycemia. To examine this possibility in human islets, we used a metabolomic approach to trace the responses of amino acids and other potential neurotransmitters to stimulation with [U-(13)C]glucose in both normal individuals and type 2 diabetics. Islets from type 2 diabetics uniformly showed decreased glucose stimulation of insulin secretion and respiratory rate but demonstrated two different patterns of glucagon responses to glucose: one group responded normally to suppression of glucagon by glucose, but the second group was non-responsive. The non-responsive group showed evidence of suppressed islet GABA levels and of GABA shunt activity. In further studies with normal human islets, we found that γ-hydroxybutyrate (GHB), a potent inhibitory neurotransmitter, is generated in β-cells by an extension of the GABA shunt during glucose stimulation and interacts with α-cell GHB receptors, thus mediating the suppressive effect of glucose on glucagon release. We also identified glycine, acting via α-cell glycine receptors, as the predominant amino acid stimulator of glucagon release. The results suggest that glycine and GHB provide a counterbalancing receptor-based mechanism for controlling α-cell secretory responses to metabolic fuels. PMID:23266825

  11. Intranasal glucagon: a promising approach for treatment of severe hypoglycemia.

    PubMed

    Pontiroli, Antonio E

    2015-01-01

    Prevention of diabetic complications is mainly obtained through optimal control of blood glucose levels. With hypoglycemic drugs like beta-cell stimulating drugs and especially insulin, the limit to treatment is represented by hypoglycemia, a life-threatening occurrence that is dangerous itself and can induce fear of other episodes. Glucagon, injected subcutaneously (SC) or intramuscularly (IM), is the treatment of choice for severe hypoglycemia outside of the hospital setting. However, due to practical aspects such as preparation of solutions for administration and injection by untrained persons, there are obstacles to its routine use. This review focuses on the current status of alternative routes of administration of peptide hormones, and in particular the intranasal (IN) route of glucagon, as a promising approach for the treatment of severe hypoglycemia. PMID:25385946

  12. Extra-helical binding site of a glucagon receptor antagonist.

    PubMed

    Jazayeri, Ali; Doré, Andrew S; Lamb, Daniel; Krishnamurthy, Harini; Southall, Stacey M; Baig, Asma H; Bortolato, Andrea; Koglin, Markus; Robertson, Nathan J; Errey, James C; Andrews, Stephen P; Teobald, Iryna; Brown, Alastair J H; Cooke, Robert M; Weir, Malcolm; Marshall, Fiona H

    2016-05-12

    Glucagon is a 29-amino-acid peptide released from the α-cells of the islet of Langerhans, which has a key role in glucose homeostasis. Glucagon action is transduced by the class B G-protein-coupled glucagon receptor (GCGR), which is located on liver, kidney, intestinal smooth muscle, brain, adipose tissue, heart and pancreas cells, and this receptor has been considered an important drug target in the treatment of diabetes. Administration of recently identified small-molecule GCGR antagonists in patients with type 2 diabetes results in a substantial reduction of fasting and postprandial glucose concentrations. Although an X-ray structure of the transmembrane domain of the GCGR has previously been solved, the ligand (NNC0640) was not resolved. Here we report the 2.5 Å structure of human GCGR in complex with the antagonist MK-0893 (ref. 4), which is found to bind to an allosteric site outside the seven transmembrane (7TM) helical bundle in a position between TM6 and TM7 extending into the lipid bilayer. Mutagenesis of key residues identified in the X-ray structure confirms their role in the binding of MK-0893 to the receptor. The unexpected position of the binding site for MK-0893, which is structurally similar to other GCGR antagonists, suggests that glucagon activation of the receptor is prevented by restriction of the outward helical movement of TM6 required for G-protein coupling. Structural knowledge of class B receptors is limited, with only one other ligand-binding site defined--for the corticotropin-releasing hormone receptor 1 (CRF1R)--which was located deep within the 7TM bundle. We describe a completely novel allosteric binding site for class B receptors, providing an opportunity for structure-based drug design for this receptor class and furthering our understanding of the mechanisms of activation of these receptors. PMID:27111510

  13. Optimizing Reduction in Basal Hyperglucagonaemia to Repair Defective Glucagon Counterregulation in Insulin Deficiency

    PubMed Central

    Farhy, Leon S.; McCall, Anthony L.

    2012-01-01

    In health, the pancreatic islet cells work as a network with highly coordinated signals over time to balance glycaemia within a narrow range. In type 1 diabetes (T1DM), with autoimmune destruction of the β-cells, lack of insulin is considered the primary abnormality and is the primary therapy target. However, replacing insulin alone does not achieve adequate glucose control and recent studies have focused on controlling the endogenous glucagon release as well. In T1DM, glucagon secretion is disordered but not absolutely deficient; it may be excessive postprandially yet it is characteristically insufficient and delayed in response to hypoglycaemia. We review our system-level analysis of the pancreatic endocrine network mechanisms of glucagon counterregulation (GCR) and their dysregulation in T1DM and focus on possible use of α-cell inhibitors (ACI) to manipulate the glucagon axis to repair the defective GCR. Our results indicate that the GCR abnormalities are of “network origin”. The lack of β-cell signalling is the primary deficiency which contributes to two separate network abnormalities: (i) absence of a β-cell switch-off trigger and (ii) increase intraislet basal glucagon. A strategy to repair these abnormalities with ACI is proposed which could achieve better control of glycaemia with reduced hypoglycaemia risk. PMID:21824267

  14. Glucagon: acute actions on hepatic metabolism.

    PubMed

    Miller, Russell A; Birnbaum, Morris J

    2016-07-01

    Type 2 diabetes mellitus is the result of impaired systemic control of glucose homeostasis, in part through the dysregulation of the hormone glucagon. Glucagon acts on the liver to increase glucose production through alterations in hepatic metabolism, and reducing the elevated glucagon signalling in diabetic patients is an attractive strategy for the treatment of hyperglycaemia. Here we review the actions of the hormone in the liver, focusing on the acute alterations of metabolic pathways. This review summarises a presentation given at the 'Novel data on glucagon' symposium at the 2015 annual meeting of the EASD. It is accompanied by two other reviews on topics from this symposium (by Mona Abraham and Tony Lam, DOI: 10.1007/s00125-016-3950-3 , and by Young Lee and colleagues, DOI: 10.1007/s00125-016-3965-9 ) and an overview by the Session Chair, Isabel Valverde (DOI: 10.1007/s00125-016-3946-z ). PMID:27115415

  15. Minireview: Glucagon in Stress and Energy Homeostasis

    PubMed Central

    Jones, B. J.; Tan, T.

    2012-01-01

    Glucagon is traditionally thought of as an antihypoglycemic hormone, for example in response to starvation. However, it actually increases energy expenditure and has other actions not in line with protection from hypoglycemia. Furthermore, it is often found to be elevated when glucose is also raised, for example in circumstances of psychological and metabolic stress. These findings seem more in keeping with glucagon having some role as a hormone enhancing the response to stress. PMID:22294753

  16. Effects of Alterations of Plasma Free Fatty Acid Levels on Pancreatic Glucagon Secretion in Man

    PubMed Central

    Gerich, John E.; Langlois, Maurice; Schneider, Victor; Karam, John H.; Noacco, Claudio

    1974-01-01

    The present investigation was undertaken to ascertain whether alterations in plasma free fatty acids (FFA) affect pancreatic glucagon secretion in man since FFA have been reported to influence pancreatic alpha cell function in other species. Elevation of plasma FFA from a mean (±SE) basal level of 0.478±0.036 mM to 0.712±0.055 mM after heparin administration caused plasma glucagon levels to fall approximately 50%, from a basal value of 122±15 pg/ml to 59±14 pg/ml (P < 0.001). Lowering of plasma FFA from a basal level of 0.520±0.046 mM to 0.252±0.041 mM after nicotinic acid administration raised plasma glucagon from a basal level of 113±18 pg/ml to 168±12 pg/ml (P < 0.005). Infusion of glucose elevated plasma glucose levels to the same degree that heparin raised plasma FFA levels. This resulted in suppression of plasma glucagon despite the fact that plasma FFA levels also were suppressed. Glucagon responses to arginine were diminished after elevation of plasma FFA (P < 0.01) and during infusion of glucose (P < 0.01). Diminution of plasma FFA by nicotinic acid did not augment glucagon responses to arginine. These results thus demonstrate that rather small alterations in plasma FFA within the physiologic range have a significant effect on glucagon secretion in man. Although the effects of glucose appear to predominate over those of FFA, alterations in plasma FFA may nevertheless exert an important physiologic influence over human pancreatic alpha cell function, especially in the postabsorptive state. PMID:4825225

  17. Cross-talk between glucagon- and adenosine-mediated signalling systems in rat hepatocytes: effects on cyclic AMP-phosphodiesterase activity.

    PubMed Central

    Robles-Flores, M; Allende, G; Piña, E; García-Sáinz, J A

    1995-01-01

    The effect of adenosine analogues on glucagon-stimulated cyclic AMP accumulation in rat hepatocytes was explored. N6-Cyclopentyladenosine (CPA), 5'-N-ethylcarboxamidoadenosine and N6-(R-phenylisopropyl)adenosine inhibited in a dose-dependent manner the cyclic AMP accumulation induced by glucagon. This effect seems to be mediated through A1 adenosine receptors. Pertussis toxin completely abolished the effect of CPA on glucagon-stimulated cyclic AMP accumulation in whole cells which suggested that a pertussis-toxin-sensitive G-protein was involved. On the other hand, this action of adenosine analogues on glucagon-induced cyclic AMP accumulation was reverted by the selective low-Km cyclic AMP-phosphodiesterase inhibitor Ro 20-1724. Analysis of cyclic AMP-phosphodiesterase activity in purified hepatocyte plasma membranes showed that glucagon in the presence of GTP inhibited basal PDE activity by 45% and that CPA reverted this inhibition in dose-dependent manner. In membranes derived from pertussis-toxin-treated rats, we observed no inhibition of cyclic AMP-phosphodiesterase activity by glucagon in the absence or presence of CPA. Our results indicate that in hepatocyte plasma membranes, stimulation of adenylate cyclase activity and inhibition of a low-Km cyclic AMP phosphodiesterase activity are co-ordinately regulated by glucagon, and that A1 adenosine receptors can inhibit glucagon-stimulated cyclic AMP accumulation by blocking glucagon's effect on phosphodiesterase activity. Images Figure 2 PMID:8554517

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

    PubMed Central

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

    2013-01-01

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

  19. Deoxynivalenol (Vomitoxin)-Induced Cholecystokinin and Glucagon-Like Peptide-1 Release in the STC-1 Enteroendocrine Cell Model Is Mediated by Calcium-Sensing Receptor and Transient Receptor Potential Ankyrin-1 Channel.

    PubMed

    Zhou, Hui-Ren; Pestka, James J

    2015-06-01

    Food refusal is a hallmark of exposure of experimental animals to the trichothecene mycotoxin deoxynivalenol (DON), a common foodborne contaminant. Although studies in the mouse suggest that DON suppresses food intake by aberrantly inducing the release of satiety hormones from enteroendocrine cells (EECs) found in the gut epithelium, the underlying mechanisms for this effect are not understood. To address this gap, we employed the murine neuroendocrine tumor STC-1 cell line, a widely used EEC model, to test the hypothesis that DON-induced hormone exocytosis is mediated by G protein-coupled receptor (GPCR)-mediated Ca(2+) signaling. The results indicate for the first time that DON elicits Ca(2)-dependent secretion of cholecystokinin (CCK) and glucagon-like peptide-1(7-36) amide (GLP-1), hormones that regulate food intake and energy homeostasis and that are products of 2 critical EEC populations--I cells of the small intestine and L cells of the large intestine, respectively. Furthermore, these effects were mediated by the GPCR Ca(2+)-sensing receptor (CaSR) and involved the following serial events: (1)PLC-mediated activation of the IP3 receptor and mobilization of intracellular Ca(2+) stores, (2) activation of transient receptor potential melastatin-5 ion channel and resultant L-type voltage-sensitive Ca(2+) channel-facilitated extracellular Ca(2+) entry, (3) amplification of extracellular Ca(2+) entry by transient receptor potential ankyrin-1 channel activation, and finally (4) Ca(2+)-driven CCK and GLP-1 excytosis. These in vitro findings provide a foundation for future investigation of mechanisms by which DON and other trichothecenes modulate EEC function in ex vivo and in vivo models. PMID:25787141

  20. Deoxynivalenol (Vomitoxin)-Induced Cholecystokinin and Glucagon-Like Peptide-1 Release in the STC-1 Enteroendocrine Cell Model Is Mediated by Calcium-Sensing Receptor and Transient Receptor Potential Ankyrin-1 Channel

    PubMed Central

    Zhou, Hui-Ren; Pestka, James J.

    2015-01-01

    Food refusal is a hallmark of exposure of experimental animals to the trichothecene mycotoxin deoxynivalenol (DON), a common foodborne contaminant. Although studies in the mouse suggest that DON suppresses food intake by aberrantly inducing the release of satiety hormones from enteroendocrine cells (EECs) found in the gut epithelium, the underlying mechanisms for this effect are not understood. To address this gap, we employed the murine neuroendocrine tumor STC-1 cell line, a widely used EEC model, to test the hypothesis that DON-induced hormone exocytosis is mediated by G protein-coupled receptor (GPCR)-mediated Ca2+ signaling. The results indicate for the first time that DON elicits Ca2-dependent secretion of cholecystokinin (CCK) and glucagon-like peptide-17-36 amide (GLP-1), hormones that regulate food intake and energy homeostasis and that are products of 2 critical EEC populations—I cells of the small intestine and L cells of the large intestine, respectively. Furthermore, these effects were mediated by the GPCR Ca2+-sensing receptor (CaSR) and involved the following serial events: (1)PLC-mediated activation of the IP3 receptor and mobilization of intracellular Ca2+ stores, (2) activation of transient receptor potential melastatin-5 ion channel and resultant L-type voltage-sensitive Ca2+ channel-facilitated extracellular Ca2+ entry, (3) amplification of extracellular Ca2+ entry by transient receptor potential ankyrin-1 channel activation, and finally (4) Ca2+-driven CCK and GLP-1 excytosis. These in vitro findings provide a foundation for future investigation of mechanisms by which DON and other trichothecenes modulate EEC function in ex vivo and in vivo models. PMID:25787141

  1. Sodium-glucose cotransporter-2 inhibition and the insulin: Glucagon ratio: Unexplored dimensions.

    PubMed

    Kalra, Sanjay; Gupta, Yashdeep; Patil, Shiva

    2015-01-01

    The sodium-glucose cotransporter 2 (SGLT-2) inhibitors are a novel class of glucose-lowering drugs which act by inhibiting the reabsorption of filtered glucose from the kidneys. Their effect on insulin and glucagon levels has recently been studied but is not fully explained. This communication proposes various hypotheses: A direct effect of SGLT-2 inhibition on the alpha cell receptors, a paracrine or intra-islet mediated effect on alpha cell sensitivity to glucose, and a calorie restriction mimetic action, to explain the impact of these drugs on the insulin glucagon ratio. PMID:25932403

  2. Human neuroepithelial cells express NMDA receptors.

    PubMed

    Sharp, Christopher D; Fowler, M; Jackson, T H; Houghton, J; Warren, A; Nanda, A; Chandler, I; Cappell, B; Long, A; Minagar, A; Alexander, J S

    2003-11-13

    L-glutamate, an excitatory neurotransmitter, binds to both ionotropic and metabotropic glutamate receptors. In certain parts of the brain the BBB contains two normally impermeable barriers: 1) cerebral endothelial barrier and 2) cerebral epithelial barrier. Human cerebral endothelial cells express NMDA receptors; however, to date, human cerebral epithelial cells (neuroepithelial cells) have not been shown to express NMDA receptor message or protein. In this study, human hypothalamic sections were examined for NMDA receptors (NMDAR) expression via immunohistochemistry and murine neuroepithelial cell line (V1) were examined for NMDAR via RT-PCR and Western analysis. We found that human cerebral epithelium express protein and cultured mouse neuroepithelial cells express both mRNA and protein for the NMDA receptor. These findings may have important consequences for neuroepithelial responses during excitotoxicity and in disease. PMID:14614784

  3. Centrally administered glucagon stimulates sympathetic nerve activity in rat.

    PubMed

    Krzeski, R; Czyzyk-Krzeska, M F; Trzebski, A; Millhorn, D E

    1989-12-18

    The effect of pancreatic glucagon given intravenously, intracerebroventricularly and microinjected into the nucleus of the solitary tract on sympathetic activity in the cervical trunk and adrenal nerve was examined in rat. In each case glucagon caused a relatively long-lasting substantial increase in discharge of both nerves. This finding shows that glucagon can act centrally to stimulate sympathetic activity. The most probable site for the sympathoexcitatory effect of glucagon is the nucleus of the solitary tract. PMID:2598031

  4. Foxp3 expression in human cancer cells

    PubMed Central

    Karanikas, Vaios; Speletas, Matthaios; Zamanakou, Maria; Kalala, Fani; Loules, Gedeon; Kerenidi, Theodora; Barda, Angeliki K; Gourgoulianis, Konstantinos I; Germenis, Anastasios E

    2008-01-01

    Objective Transcription factor forkhead box protein 3 (Foxp3) specifically characterizes the thymically derived naturally occurring regulatory T cells (Tregs). Limited evidence indicates that it is also expressed, albeit to a lesser extent, in tissues other than thymus and spleen, while, very recently, it was shown that Foxp3 is expressed by pancreatic carcinoma. This study was scheduled to investigate whether expression of Foxp3 transcripts and mature protein occurs constitutively in various tumor types. Materials and methods Twenty five tumor cell lines of different tissue origins (lung cancer, colon cancer, breast cancer, melanoma, erythroid leukemia, acute T-cell leukemia) were studied. Detection of Foxp3 mRNA was performed using both conventional RT-PCR and quantitative real-time PCR while protein expression was assessed by immunocytochemistry and flow cytometry, using different antibody clones. Results Foxp3 mRNA as well as Foxp3 protein was detected in all tumor cell lines, albeit in variable levels, not related to the tissue of origin. This expression correlated with the expression levels of IL-10 and TGFb1. Conclusion We offer evidence that Foxp3 expression, characterizes tumor cells of various tissue origins. The biological significance of these findings warrants further investigation in the context of tumor immune escape, and especially under the light of current anti-cancer efforts interfering with Foxp3 expression. PMID:18430198

  5. The Insulin:Glucagon Ratio and the Choice of Glucose-Lowering Drugs.

    PubMed

    Kalra, Sanjay; Gupta, Yashdeep

    2016-03-01

    The influence of alpha and beta cells, through glucagon and insulin, on energy metabolism is well known. The insulin:glucagon ratio (IGR) is a frequently discussed entity in the medical literature. However, in recent years, focus has shifted to other pathways and markers of health and disease. This communication revisits the insulin:glucagon bipolar axis and describes the significance of the IGR. It reviews the effects of various glucose-lowering drugs on this ratio, and hypothesizes that the ratio can be used to predict the appropriate choice of drugs for managing diabetes. Drugs which increase the IGR may be beneficial in insulinopenic conditions, while those which decrease IGR may be of help in the setting of hyperinsulinemia or insulin resistance. PMID:26965024

  6. Glucose-dependent insulinotropic polypeptide: effects on insulin and glucagon secretion in humans.

    PubMed

    Christensen, Mikkel Bring

    2016-04-01

    The hormones glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are secreted by enteroendocrine cells in the intestinal mucosa in response to nutrient ingestion. They are called incretin hormones because of their ability to enhance insulin secretion. However, in recent years it has become clear that the incretin hormones also affect glucagon secretion. While GLP-1 decreases glucagon levels, the effect of GIP on glucagon levels has been unclear. The regulation of glucagon secretion is interesting, as the combination of inadequate insulin secretion and excessive glucagon secretion are essential contributors to the hyperglycaemia that characterise patients with type 2 diabetes. Moreover, the near absence of a well-timed glucagon response contributes to an increased risk of hypoglycaemia in patients with type 1 diabetes. The overall aim of this PhD thesis was to investigate how the blood glucose level affects the glucagon and insulin responses to GIP in healthy subjects (Study 1) and patients with Type 2 diabetes (Study 2), and more specifically to investigate the effects of GIP and GLP-1 at low blood glucose in patients with Type 1 diabetes without endogenous insulin secretion (Study 3). The investigations in the three mentioned study populations have been described in three original articles. The employed study designs were in randomised, placebo-controlled, crossover set-up, in which the same research subject is subjected to several study days thereby acting as his own control. Interventions were intravenous administration of hormones GIP, GLP-1 and placebo (saline) during different blood glucose levels maintained (clamped) at a certain level. The end-points were plasma concentrations of glucagon and insulin as well as the amount of glucose used to clamp the blood glucose levels. In Study 3, we also used stable glucose isotopes to estimate the endogenous glucose production and assessed symptoms and cognitive function during

  7. Decorin expression in quiescent myogenic cells

    SciTech Connect

    Nishimura, Takanori Nozu, Kenjiro; Kishioka, Yasuhiro; Wakamatsu, Jun-ichi; Hattori, Akihito

    2008-06-06

    Satellite cells are quiescent muscle stem cells that promote postnatal muscle growth and repair. When satellite cells are activated by myotrauma, they proliferate, migrate, differentiate, and ultimately fuse to existing myofibers. The remainder of these cells do not differentiate, but instead return to quiescence and remain in a quiescent state until activation begins the process again. This ability to maintain their own population is important for skeletal muscle to maintain the capability to repair during postnatal life. However, the mechanisms by which satellite cells return to quiescence and maintain the quiescent state are still unclear. Here, we demonstrated that decorin mRNA expression was high in cell cultures containing a higher ratio of quiescent satellite cells when satellite cells were stimulated with various concentrations of hepatocyte growth factor. This result suggests that quiescent satellite cells express decorin at a high level compared to activated satellite cells. Furthermore, we examined the expression of decorin in reserve cells, which were undifferentiated myoblasts remaining after induction of differentiation by serum-deprivation. Decorin mRNA levels in reserve cells were higher than those in differentiated myotubes and growing myoblasts. These results suggest that decorin participates in the quiescence of myogenic cells.

  8. Treatment of Type 1 Diabetes With Adipose Tissue–Derived Stem Cells Expressing Pancreatic Duodenal Homeobox 1

    PubMed Central

    Lin, Ching-Shwun

    2009-01-01

    Due to the limited supply of donor pancreas, it is imperative that we identify alternative cell sources that can be used to treat diabetes mellitus (DM). Multipotent adipose tissue–derived stem cells (ADSC) can be abundantly and safely isolated for autologous transplantation and therefore are an ideal candidate. Here, we report the derivation of insulin-producing cells from human or rat ADSC by transduction with the pancreatic duodenal homeobox 1 (Pdx1) gene. RT-PCR analyses showed that native ADSC expressed insulin, glucagon, and NeuroD genes that were up-regulated following Pdx1 transduction. ELISA analyses showed that the transduced cells secreted increasing amount of insulin in response to increasing concentration of glucose. Transplantation of these cells under the renal capsule of streptozotocin-induced diabetic rats resulted in lowered blood glucose, higher glucose tolerance, smoother fur, and less cataract. Histological examination showed that the transplanted cells formed tissue-like structures and expressed insulin. Thus, ADSC-expressing Pdx1 appear to be suitable for treatment of DM. PMID:19245309

  9. Vertebrate Cells Express Protozoan Antigen after Hybridization

    NASA Astrophysics Data System (ADS)

    Crane, Mark St. J.; Dvorak, James A.

    1980-04-01

    Epimastigotes, the invertebrate host stage of Trypanosoma cruzi, the protozoan parasite causing Chagas' disease in man, were fused with vertebrate cells by using polyethylene glycol. Hybrid cells were selected on the basis of T. cruzi DNA complementation of biochemical deficiencies in the vertebrate cells. Some clones of the hybrid cells expressed T. cruzi-specific antigen. It might be possible to use selected antigens obtained from the hybrids as vaccines for immunodiagnosis or for elucidation of the pathogenesis of Chagas' disease.

  10. Imaging gene expression in single living cells

    PubMed Central

    Shav-Tal, Yaron; Singer, Robert H.; Darzacq, Xavier

    2016-01-01

    Technical advances in the field of live-cell imaging have introduced the cell biologist to a new, dynamic, subcellular world. The static world of molecules in fixed cells has now been extended to the time dimension. This allows the visualization and quantification of gene expression and intracellular trafficking events of the studied molecules and the associated enzymatic processes in individual cells, in real time. PMID:15459666

  11. A monomer-trimer model supports intermittent glucagon fibril growth

    NASA Astrophysics Data System (ADS)

    Košmrlj, Andrej; Cordsen, Pia; Kyrsting, Anders; Otzen, Daniel E.; Oddershede, Lene B.; Jensen, Mogens H.

    2015-03-01

    We investigate in vitro fibrillation kinetics of the hormone peptide glucagon at various concentrations using confocal microscopy and determine the glucagon fibril persistence length 60μm. At all concentrations we observe that periods of individual fibril growth are interrupted by periods of stasis. The growth probability is large at high and low concentrations and is reduced for intermediate glucagon concentrations. To explain this behavior we propose a simple model, where fibrils come in two forms, one built entirely from glucagon monomers and one entirely from glucagon trimers. The opposite building blocks act as fibril growth blockers, and this generic model reproduces experimental behavior well.

  12. Energy-Dependent Modulation of Glucagon-Like Signaling in Drosophila via the AMP-Activated Protein Kinase

    PubMed Central

    Braco, Jason T.; Gillespie, Emily L.; Alberto, Gregory E.; Brenman, Jay E.; Johnson, Erik C.

    2012-01-01

    Adipokinetic hormone (AKH) is the equivalent of mammalian glucagon, as it is the primary insect hormone that causes energy mobilization. In Drosophila, current knowledge of the mechanisms regulating AKH signaling is limited. Here, we report that AMP-activated protein kinase (AMPK) is critical for normal AKH secretion during periods of metabolic challenges. Reduction of AMPK in AKH cells causes a suite of behavioral and physiological phenotypes resembling AKH cell ablations. Specifically, reduced AMPK function increases life span during starvation and delays starvation-induced hyperactivity. Neither AKH cell survival nor gene expression is significantly impacted by reduced AMPK function. AKH immunolabeling was significantly higher in animals with reduced AMPK function; this result is paralleled by genetic inhibition of synaptic release, suggesting that AMPK promotes AKH secretion. We observed reduced secretion in AKH cells bearing AMPK mutations employing a specific secretion reporter, confirming that AMPK functions in AKH secretion. Live-cell imaging of wild-type AKH neuroendocrine cells shows heightened excitability under reduced sugar levels, and this response was delayed and reduced in AMPK-deficient backgrounds. Furthermore, AMPK activation in AKH cells increases intracellular calcium levels in constant high sugar levels, suggesting that the underlying mechanism of AMPK action is modification of ionic currents. These results demonstrate that AMPK signaling is a critical feature that regulates AKH secretion, and, ultimately, metabolic homeostasis. The significance of these findings is that AMPK is important in the regulation of glucagon signaling, suggesting that the organization of metabolic networks is highly conserved and that AMPK plays a prominent role in these networks. PMID:22798489

  13. Energy-dependent modulation of glucagon-like signaling in Drosophila via the AMP-activated protein kinase.

    PubMed

    Braco, Jason T; Gillespie, Emily L; Alberto, Gregory E; Brenman, Jay E; Johnson, Erik C

    2012-10-01

    Adipokinetic hormone (AKH) is the equivalent of mammalian glucagon, as it is the primary insect hormone that causes energy mobilization. In Drosophila, current knowledge of the mechanisms regulating AKH signaling is limited. Here, we report that AMP-activated protein kinase (AMPK) is critical for normal AKH secretion during periods of metabolic challenges. Reduction of AMPK in AKH cells causes a suite of behavioral and physiological phenotypes resembling AKH cell ablations. Specifically, reduced AMPK function increases life span during starvation and delays starvation-induced hyperactivity. Neither AKH cell survival nor gene expression is significantly impacted by reduced AMPK function. AKH immunolabeling was significantly higher in animals with reduced AMPK function; this result is paralleled by genetic inhibition of synaptic release, suggesting that AMPK promotes AKH secretion. We observed reduced secretion in AKH cells bearing AMPK mutations employing a specific secretion reporter, confirming that AMPK functions in AKH secretion. Live-cell imaging of wild-type AKH neuroendocrine cells shows heightened excitability under reduced sugar levels, and this response was delayed and reduced in AMPK-deficient backgrounds. Furthermore, AMPK activation in AKH cells increases intracellular calcium levels in constant high sugar levels, suggesting that the underlying mechanism of AMPK action is modification of ionic currents. These results demonstrate that AMPK signaling is a critical feature that regulates AKH secretion, and, ultimately, metabolic homeostasis. The significance of these findings is that AMPK is important in the regulation of glucagon signaling, suggesting that the organization of metabolic networks is highly conserved and that AMPK plays a prominent role in these networks. PMID:22798489

  14. Pancreatic signals controlling food intake; insulin, glucagon and amylin

    PubMed Central

    Woods, Stephen C; Lutz, Thomas A; Geary, Nori; Langhans, Wolfgang

    2006-01-01

    The control of food intake and body weight by the brain relies upon the detection and integration of signals reflecting energy stores and fluxes, and their interaction with many different inputs related to food palatability and gastrointestinal handling as well as social, emotional, circadian, habitual and other situational factors. This review focuses upon the role of hormones secreted by the endocrine pancreas: hormones, which individually and collectively influence food intake, with an emphasis upon insulin, glucagon and amylin. Insulin and amylin are co-secreted by B-cells and provide a signal that reflects both circulating energy in the form of glucose and stored energy in the form of visceral adipose tissue. Insulin acts directly at the liver to suppress the synthesis and secretion of glucose, and some plasma insulin is transported into the brain and especially the mediobasal hypothalamus where it elicits a net catabolic response, particularly reduced food intake and loss of body weight. Amylin reduces meal size by stimulating neurons in the hindbrain, and there is evidence that amylin additionally functions as an adiposity signal controlling body weight as well as meal size. Glucagon is secreted from A-cells and increases glucose secretion from the liver. Glucagon acts in the liver to reduce meal size, the signal being relayed to the brain via the vagus nerves. To summarize, hormones of the endocrine pancreas are collectively at the crossroads of many aspects of energy homeostasis. Glucagon and amylin act in the short term to reduce meal size, and insulin sensitizes the brain to short-term meal-generated satiety signals; and insulin and perhaps amylin as well act over longer intervals to modulate the amount of fat maintained and defended by the brain. Hormones of the endocrine pancreas interact with receptors at many points along the gut–brain axis, from the liver to the sensory vagus nerve to the hindbrain to the hypothalamus; and their signals are

  15. Fundamentals of Expression in Mammalian Cells.

    PubMed

    Dyson, Michael R

    2016-01-01

    Expression of proteins in mammalian cells is a key technology important for many functional studies on human and higher eukaryotic genes. Studies include the mapping of protein interactions, solving protein structure by crystallization and X-ray diffraction or solution phase NMR and the generation of antibodies to enable a range of studies to be performed including protein detection in vivo. In addition the production of therapeutic proteins and antibodies, now a multi billion dollar industry, has driven major advances in cell line engineering for the production of grams per liter of active proteins and antibodies. Here the key factors that need to be considered for successful expression in HEK293 and CHO cells are reviewed including host cells, expression vector design, transient transfection methods, stable cell line generation and cultivation conditions. PMID:27165328

  16. Glucagon-like peptide 2 may mediate growth and development of the bovine gastrointestinal tract

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Glucagon-like peptide 2 (GLP-2), secreted by enteroendocrine cells, promotes growth, reduces apoptosis, and enhances blood flow, nutrient absorption, and barrier function in intestinal epithelium of monogastric species. Regulatory functions of GLP-2 in the ruminant gastrointestinal tract (GIT) are u...

  17. Novel dual agonist peptide analogues derived from dogfish glucagon show promising in vitro insulin releasing actions and antihyperglycaemic activity in mice.

    PubMed

    O'Harte, F P M; Ng, M T; Lynch, A M; Conlon, J M; Flatt, P R

    2016-08-15

    The antidiabetic potential of thirteen novel dogfish glucagon derived analogues were assessed in vitro and in acute in vivo studies. Stable peptide analogues enhanced insulin secretion from BRIN-BD11 β-cells (p < 0.001) and reduced acute glycaemic responses following intraperitoneal glucose (25 nmol/kg) in healthy NIH Swiss mice (p < 0.05-p<0.001). The in vitro insulinotropic actions of [S2a]dogfish glucagon, [S2a]dogfish glucagon-exendin-4(31-39) and [S2a]dogfish glucagon-Lys(30)-γ-glutamyl-PAL, were blocked (p < 0.05-p<0.001) by the specific GLP-1 and glucagon receptor antagonists, exendin-4(9-39) and (desHis(1)Pro(4)Glu(9))glucagon amide but not by (Pro(3))GIP, indicating lack of GIP receptor involvement. These analogues dose-dependently stimulated cAMP production in GLP-1 and glucagon (p < 0.05-p<0.001) but not GIP-receptor transfected cells. They improved acute glycaemic and insulinotropic responses in high-fat fed diabetic mice and in wild-type C57BL/6J and GIPR-KO mice (p < 0.05-p<0.001), but not GLP-1R-KO mice, confirming action on GLP-1 but not GIP receptors. Overall, dogfish glucagon analogues have potential for diabetes therapy, exerting beneficial metabolic effects via GLP-1 and glucagon receptors. PMID:27179756

  18. 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 Central

    Gault, Victor A.; Bhat, Vikas K.; Irwin, Nigel; Flatt, Peter R.

    2013-01-01

    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, [dA2]GLP-1/GcG, stimulated cAMP production in GIP, GLP-1, and glucagon receptor-transfected cells. Acute administration of [dA2]GLP-1/GcG in combination with glucose significantly lowered plasma glucose and increased plasma insulin in normal and obese diabetic (ob/ob) mice. Furthermore, [dA2]GLP-1/GcG elicited a protracted glucose-lowering and insulinotropic effect in high fat-fed mice. Twice daily administration of [dA2]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, [dA2]GLP-1/GcG significantly improved glucose tolerance and insulin sensitivity by day 21. Interestingly, locomotor activity was increased in [dA2]GLP-1/GcG mice, without appreciable changes in aspects of metabolic rate. Studies in knock-out mice confirmed the biological action of [dA2]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. PMID:24165127

  19. The forgotten members of the glucagon family.

    PubMed

    Bataille, Dominique; Dalle, Stéphane

    2014-10-01

    From proglucagon, at least six final biologically active peptides are produced by tissue-specific post-translational processing. While glucagon and GLP-1 are the subject of permanent studies, the four others are usually left in the shadow, in spite of their large biological interest. The present review is devoted to oxyntomodulin and miniglucagon, not forgetting glicentin, although much less is known about it. Oxyntomodulin (OXM) and glicentin are regulators of gastric acid and hydromineral intestinal secretions. OXM is also deeply involved in the control of food intake and energy expenditure, properties that make this peptide a credible treatment of obesity if the question of administration is solved, as for any peptide. Miniglucagon, the C-terminal undecapeptide of glucagon which results from a secondary processing of original nature, displays properties antagonistic to that of the mother-hormone glucagon: (a) it inhibits glucose-, glucagon- and GLP-1-stimulated insulin release at sub-picomolar concentrations, (b) it reduces the in vivo insulin response to glucose with no change in glycemia, (c) it displays insulin-like properties at the cellular level using only a part of the pathway used by insulin, making it a good basis for developing a pharmacological workaround of insulin resistance. PMID:25115338

  20. Glucagon orchestrates stress-induced hyperglycaemia.

    PubMed

    Harp, J B; Yancopoulos, G D; Gromada, J

    2016-07-01

    Hyperglycaemia is commonly observed on admission and during hospitalization for medical illness, traumatic injury, burn and surgical intervention. This transient hyperglycaemia is referred to as stress-induced hyperglycaemia (SIH) and frequently occurs in individuals without a history of diabetes. SIH has many of the same underlying hormonal disturbances as diabetes mellitus, specifically absolute or relative insulin deficiency and glucagon excess. SIH has the added features of elevated blood levels of catecholamines and cortisol, which are not typically present in people with diabetes who are not acutely ill. The seriousness of SIH is highlighted by its greater morbidity and mortality rates compared with those of hospitalized patients with normal glucose levels, and this increased risk is particularly high in those without pre-existing diabetes. Insulin is the treatment standard for SIH, but new therapies that reduce glucose variability and hypoglycaemia are desired. In the present review, we focus on the key role of glucagon in SIH and discuss the potential use of glucagon receptor blockers and glucagon-like peptide-1 receptor agonists in SIH to achieve target glucose control. PMID:27027662

  1. Glucagon-like peptide-1 binding to rat skeletal muscle.

    PubMed

    Delgado, E; Luque, M A; Alcántara, A; Trapote, M A; Clemente, F; Galera, C; Valverde, I; Villanueva-Peñacarrillo, M L

    1995-01-01

    We have found [125I]glucagon-like peptide-1(7-36)-amide-specific binding activity in rat skeletal muscle plasma membranes, with an estimated M(r) of 63,000 by cross-linking and SDS-PAGE. The specific binding was time and membrane protein concentration dependent, and displaceable by unlabeled GLP-1(7-36)-amide with an ID50 of 3 x 10(-9) M of the peptide; GLP-1(1-36)-amide also competed, whereas glucagon and insulin did not. GLP-1(7-36)-amide did not modify the basal adenylate cyclase activity in skeletal muscle plasma membranes. These data, together with our previous finding of a potent glycogenic effect of GLP-1(7-36)-amide in rat soleus muscle, and also in isolated hepatocytes, which was not accompanied by a rise in the cell cyclic AMP content, lead use to believe that the insulin-like effects of this peptide on glucose metabolism in the muscle could be mediated by a type of receptor somehow different to that described for GLP-1 in pancreatic B cells, where GLP-1 action is mediated by the cyclic AMP-adenylate cyclase system. PMID:7784253

  2. Glucagon-like peptide-1 is specifically involved in sweet taste transmission.

    PubMed

    Takai, Shingo; Yasumatsu, Keiko; Inoue, Mayuko; Iwata, Shusuke; Yoshida, Ryusuke; Shigemura, Noriatsu; Yanagawa, Yuchio; Drucker, Daniel J; Margolskee, Robert F; Ninomiya, Yuzo

    2015-06-01

    Five fundamental taste qualities (sweet, bitter, salty, sour, umami) are sensed by dedicated taste cells (TCs) that relay quality information to gustatory nerve fibers. In peripheral taste signaling pathways, ATP has been identified as a functional neurotransmitter, but it remains to be determined how specificity of different taste qualities is maintained across synapses. Recent studies demonstrated that some gut peptides are released from taste buds by prolonged application of particular taste stimuli, suggesting their potential involvement in taste information coding. In this study, we focused on the function of glucagon-like peptide-1 (GLP-1) in initial responses to taste stimulation. GLP-1 receptor (GLP-1R) null mice had reduced neural and behavioral responses specifically to sweet compounds compared to wild-type (WT) mice. Some sweet responsive TCs expressed GLP-1 and its receptors were expressed in gustatory neurons. GLP-1 was released immediately from taste bud cells in response to sweet compounds but not to other taste stimuli. Intravenous administration of GLP-1 elicited transient responses in a subset of sweet-sensitive gustatory nerve fibers but did not affect other types of fibers, and this response was suppressed by pre-administration of the GLP-1R antagonist Exendin-4(3-39). Thus GLP-1 may be involved in normal sweet taste signal transmission in mice. PMID:25678625

  3. Glucagon like peptide-2 induces intestinal restitution through VEGF release from subepithelial myofibroblasts.

    PubMed

    Bulut, Kerem; Pennartz, Christian; Felderbauer, Peter; Meier, Juris J; Banasch, Matthias; Bulut, Daniel; Schmitz, Frank; Schmidt, Wolfgang E; Hoffmann, Peter

    2008-01-14

    Glucagon like peptide-2 (GLP-2) exerts intestinotrophic actions, but the underlying mechanisms are still a matter of debate. Recent studies demonstrated the expression of the GLP-2 receptor on fibroblasts located in the subepithelial tissue, where it might induce the release of growth factors such as keratinocyte growth factor (KGF) or vascular endothelial growth factor (VEGF). Therefore, in the present studies we sought to elucidate the downstream mechanisms involved in improved intestinal adaptation by GLP-2. Human colonic fibroblasts (CCD-18Co), human colonic cancer cells (Caco-2 cells) and rat ileum IEC-18 cells were used. GLP-2 receptor mRNA expression was determined using real time RT-PCR. Conditioned media from CCD-18Co cells were obtained following incubation with GLP-2 (50-250 nM) for 24 h. Cell viability was assessed by a 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide (MTT)-assay, and wound healing was determined with an established migration-assay. Transforming Growth Factor beta (TGF-beta), VEGF and KGF mRNA levels were determined by RT-PCR. Protein levels of VEGF and TGF-beta in CCD-18Co cells following GLP-2 stimulation were determined using ELISA. Neutralizing TGF-beta and VEGF-A antibodies were utilized to assess the role of TGF-beta and VEGF-A in the process of wound healing. GLP-2 receptor expression was detected in CCD-18Co cells. Conditioned media from CCD-18Co cells dose-dependently induced proliferation in Caco-2 cells, but not in IEC-18 cells. Conditioned media also enhanced cell migration in IEC-18 cells (P<0.01), while migration was even inhibited in Caco-2 cells (P<0.0012). GLP-2 significantly stimulated mRNA expression of VEGF and TGF-beta, but not of KGF in CCD-18Co. The migratory effects of GLP-2 were completely abolished in the presence of TGF-beta and VEGF-A antibodies. GLP-2 exerts differential effects on the epithelium of the small intestine and the colon. Thus, in small intestinal cells GLP-2 stimulates wound

  4. Exenatide Alters Gene Expression of Neural Cell Adhesion Molecule (NCAM), Intercellular Cell Adhesion Molecule (ICAM), and Vascular Cell Adhesion Molecule (VCAM) in the Hippocampus of Type 2 Diabetic Model Mice

    PubMed Central

    Gumuslu, Esen; Cine, Naci; Gökbayrak, Merve Ertan; Mutlu, Oguz; Celikyurt, Ipek Komsuoglu; Ulak, Guner

    2016-01-01

    Background Glucagon-like peptide-1 (GLP-1), a potent and selective agonist for the GLP-1 receptor, ameliorates the symptoms of diabetes through stimulation of insulin secretion. Exenatide is a potent and selective agonist for the GLP-1 receptor. Cell adhesion molecules are members of the immunoglobulin superfamily and are involved in synaptic rearrangements in the mature brain. Material/Methods The present study demonstrated the effects of exenatide treatment (0.1 μg/kg, subcutaneously, twice daily for 2 weeks) on the gene expression levels of cell adhesion molecules, neural cell adhesion molecule (NCAM), intercellular cell adhesion molecule (ICAM), and vascular cell adhesion molecule (VCAM) in the brain tissue of diabetic BALB/c male mice by real-time quantitative polymerase chain reaction (PCR). Diabetes was induced by streptozotocin/nicotinamide (STZ-NA) injection to male mice. Results The results of this study revealed that hippocampal gene expression of NCAM, ICAM, and VCAM were found to be up-regulated in STZ-NA-induced diabetic mice compared to those of controls. A significant decrease in the gene expression levels of NCAM, ICAM, and VCAM were determined after 2 weeks of exenatide administration. Conclusions Cell adhesion molecules may be involved in the molecular mechanism of diabetes. Exenatide has a strong beneficial action in managing diabetes induced by STZ/NA by altering gene expression of NCAM, ICAM, and VCAM. PMID:27465247

  5. Glucagon-like peptide-1 binding to rat hepatic membranes.

    PubMed

    Villanueva-Peñacarrillo, M L; Delgado, E; Trapote, M A; Alcántara, A; Clemente, F; Luque, M A; Perea, A; Valverde, I

    1995-07-01

    We have found [125I]glucagon-like peptide (GLP)-1(7-36)amide specific binding activity in rat liver and isolated hepatocyte plasma membranes, with an M(r) of approximately 63,000, estimated by cross-linking and SDS-PAGE. The specific binding was time- and membrane protein concentration-dependent, and equally displaced by unlabelled GLP-1(7-36)amide and by GLP-1(1-36)amide, achieving its ID50 at 3 x 10(-9) M of the peptides. GLP-1(7-36)amide did not modify the basal or the glucagon (10(-8) M)-stimulated adenylate cyclase in the hepatocyte plasma membranes. These data, together with our previous findings of a potent glycogenic effect of GLP-1(7-36)amide in isolated rat hepatocytes, led us to postulate that the insulin-like effects of this peptide on glucose liver metabolism could be mediated by a type of receptor probably different from that described for GLP-1 in pancreatic B-cells or, alternatively, by the same receptor which, in this tissue as well as in muscle, uses a different transduction system. PMID:7561616

  6. Opposite Regulation of Ghrelin and Glucagon-like Peptide-1 by Metabolite G-Protein-Coupled Receptors.

    PubMed

    Engelstoft, M S; Schwartz, T W

    2016-09-01

    Gut hormones send information about incoming nutrients to the rest of the body and thereby control many aspects of metabolism. The secretion of ghrelin and glucagon-like protein (GLP)-1, two hormones with opposite secretory patterns and opposite actions on multiple targets, is controlled by a limited number of G-protein coupled receptors (GPCRs); half of which recognize and bind dietary nutrient metabolites, metabolites generated by gut microbiota, and metabolites of the host's intermediary metabolism. Most metabolite GPCRs controlling ghrelin secretion are inhibitory, whereas all metabolite receptors controlling GLP-1 secretion are stimulatory. This dichotomy in metabolite sensor function, which is obtained through a combination of differential expression and cell-dependent signaling bias, offers pharmacological targets to stimulate GLP-1 and inhibit ghrelin through the same mechanism. PMID:27474997

  7. Quantitative Electron Microscopic Autoradiography of Insulin, Glucagon, and Somatostatin Binding Sites on Islets

    NASA Astrophysics Data System (ADS)

    Patel, Yogesh C.; Amherdt, Mylene; Orci, Lelio

    1982-09-01

    After monolayer cultures of rat islets were exposed to [125I]insulin, [125I]glucagon, and [125I]tyrosinyl somatostatin, specific autoradiographic grains associated with each radioactively labeled ligand were found on B, A, and D cells. The density of labeling of the B, A, and D cells with each labeled ligand correlated well with the known actions of the three hormones on each of the islet cells.

  8. Glucagon-Like Peptide 1 Receptor Activation Attenuates Platelet Aggregation and Thrombosis.

    PubMed

    Cameron-Vendrig, Alison; Reheman, Adili; Siraj, M Ahsan; Xu, Xiaohong Ruby; Wang, Yiming; Lei, Xi; Afroze, Talat; Shikatani, Eric; El-Mounayri, Omar; Noyan, Hossein; Weissleder, Ralph; Ni, Heyu; Husain, Mansoor

    2016-06-01

    Short-term studies in subjects with diabetes receiving glucagon-like peptide 1 (GLP-1)-targeted therapies have suggested a reduced number of cardiovascular events. The mechanisms underlying this unexpectedly rapid effect are not known. We cloned full-length GLP-1 receptor (GLP-1R) mRNA from a human megakaryocyte cell line (MEG-01), and found expression levels of GLP-1Rs in MEG-01 cells to be higher than those in the human lung but lower than in the human pancreas. Incubation with GLP-1 and the GLP-1R agonist exenatide elicited a cAMP response in MEG-01 cells, and exenatide significantly inhibited thrombin-, ADP-, and collagen-induced platelet aggregation. Incubation with exenatide also inhibited thrombus formation under flow conditions in ex vivo perfusion chambers using human and mouse whole blood. In a mouse cremaster artery laser injury model, a single intravenous injection of exenatide inhibited thrombus formation in normoglycemic and hyperglycemic mice in vivo. Thrombus formation was greater in mice transplanted with bone marrow lacking a functional GLP-1R (Glp1r(-/-)), compared with those receiving wild-type bone marrow. Although antithrombotic effects of exenatide were partly lost in mice transplanted with bone marrow from Glp1r(-/-) mice, they were undetectable in mice with a genetic deficiency of endothelial nitric oxide synthase. The inhibition of platelet function and the prevention of thrombus formation by GLP-1R agonists represent potential mechanisms for reduced atherothrombotic events. PMID:26936963

  9. Glucagon, ureteral colic and ureteral peristalsis.

    PubMed

    Boyarsky, S; Labay, P C

    1978-01-01

    Experimentally, glucagon is an effective ureteral relaxant that induces a moderate diuresis. Our data suggest that in specified cases of ureteral colic, before the development of a ureteral bar, flaccid dilatation of the pelvis and renal shutdown, glucagon may facilitate the expulsion of small ureteral calculi. When the ureteral pain is due to hyperperistalsis the drug has promise in the alleviation of pain. The complexities of the pathophysiology of ureteral colic in an experimental model support the treatment of ureteral edema in colic with anti-inflammatory drugs but suggest certain precautions in the design of any investigation of drug therapy for colic, lest a good drug be found ineffective for the wrong reason-that it was used in an impossible situation. PMID:753018

  10. Glucagon-like peptide-1: glucose homeostasis and beyond.

    PubMed

    Cho, Young Min; Fujita, Yukihiro; Kieffer, Timothy J

    2014-01-01

    Glucagon-like peptide-1 (GLP-1), an incretin hormone secreted primarily from the intestinal L-cells in response to meals, modulates nutrient homeostasis via actions exerted in multiple tissues and cell types. GLP-1 and its analogs, as well as compounds that inhibit endogenous GLP-1 breakdown, have become an effective therapeutic strategy for many subjects with type 2 diabetes. Here we review the discovery of GLP-1; its synthesis, secretion, and elimination from the circulation; and its multiple pancreatic and extrapancreatic effects. Finally, we review current options for GLP-1-based diabetes therapy, including GLP-1 receptor agonism and inhibition of GLP-1 breakdown, as well as the benefits and drawbacks of different modes of therapy and the potential for new therapeutic avenues. PMID:24245943

  11. CD43 expression in B cell lymphoma.

    PubMed Central

    Treasure, J.; Lane, A.; Jones, D. B.; Wright, D. H.

    1992-01-01

    AIMS: To determine the expression of CD43 in frozen sections in a range of B cell lymphomas. METHODS: The monoclonal antibody WR14, clustered provisionally in the Fourth Leucocyte Typing Workshop as a CD43 reagent, was investigated by epitope blocking studies on formalin fixed reactive lymph node tissue, using the established CD43 antibody MT1, to validate its use as a CD43 reagent. CD43 expression was studied in 131 immunophenotypically defined B cell lymphomas, including lymphocytic lymphoma (Lc, n = 13), centrocytic lymphoma (Cc, n = 14), and a range of follicle centre cell lymphomas (FCC) including centroblastic/centrocytic follicular (CbCcF, n = 48), centroblastic diffuse (CbD, n = 39), centroblastic/centrocytic diffuse (CbCcD, n = 4), centroblastic follicular and diffuse (Cb FD, n = 3) and centroblastic/centrocytic follicular and diffuse (CbCc FD, n = 1). Nine lymphomas of mucosa associated lymphoid tissue (MALT) were also examined. RESULTS: Epitope blocking studies showed that WR14 is a CD43 reagent that binds to an epitope identical with or close to that recognised by MT1. Eleven of 13 (84%) cases of Lc and 11 of 14 (78%) cases of Cc expressed CD43; 87 of 95 (91%) cases of FCC did not. All eight low grade lymphomas of MALT were negative. One high grade lymphoma, transformed from a low grade MALT lymphoma, was positive for CD43. The expression of CD43 by tumours of B cell lineage was associated with the expression of CD5 (p < 0.001) although either antigen could occasionally be found in the absence of the other. CONCLUSION: CD43 reagents can be used in conjunction with CD5 antibodies for the immunophenotypic discrimination of follicle centre cell lymphomas from non-follicle centre cell lymphomas. Images PMID:1280654

  12. Exendin-4 enhances expression of Neurod1 and Glut2 in insulin-producing cells derived from mouse embryonic stem cells

    PubMed Central

    Zhao, Qiaoshi; Yang, Yuzhi; Hu, Jing; Shan, Zhiyan; Wu, Yanshuang

    2016-01-01

    Introduction Stem cells involved cell replacement therapies for type 1 diabetes mellitus is promising, yet time-consuming and inefficient. Exendin-4 is a glucagon-like peptide-1 (GLP-1) receptor agonist which has been reported to possess anti-apoptotic effects, thereby increasing β-cell mass and improving β-cell function. The present study aimed to investigate whether exendin-4 would enhance the differentiation of embryonic stem cells into insulin-secreting cells and improve the pancreatic differentiation strategy. Material and methods R1 embryonic stem cells were treated with different concentrations of exendin-4 and divided into three groups. In the high dosage group (group H), exendin-4 was added at the dosage of 10 nmol/l. In the low dosage group (group L), exendin-4 was added at the dosage of 0.1 nmol/l. Group C was a control. Expression of genes related to the β-cell phenotype and immunofluorescence staining of insulin and C-peptide were detected. Results Compared with groups L and C, group H had the highest mRNA expression levels of Isl1, Pdx1, Ngn3, and Insulin1 (p < 0.05). Neurod1 and Glut2 only emerged at the final stage of differentiation in group H. Immunofluorescence analysis revealed that exendin-4 upregulated the protein expression of insulin and C-peptide. Conclusions Exendin-4 remarkably facilitated Neurod1 and Glut2 gene transcription, and was able to induce differentiation of embryonic stem cells into endocrine and insulin-producing cells. PMID:26925137

  13. Pharmacological Actions of Glucagon-Like Peptide-1, Gastric Inhibitory Polypeptide, and Glucagon.

    PubMed

    Sekar, R; Singh, K; Arokiaraj, A W R; Chow, B K C

    2016-01-01

    Glucagon family of peptide hormones is a group of structurally related brain-gut peptides that exert their pleiotropic actions through interactions with unique members of class B1 G protein-coupled receptors (GPCRs). They are key regulators of hormonal homeostasis and are important drug targets for metabolic disorders such as type-2 diabetes mellitus (T2DM), obesity, and dysregulations of the nervous systems such as migraine, anxiety, depression, neurodegeneration, psychiatric disorders, and cardiovascular diseases. The current review aims to provide a detailed overview of the current understanding of the pharmacological actions and therapeutic advances of three members within this family including glucagon-like peptide-1 (GLP-1), gastric inhibitory polypeptide (GIP), and glucagon. PMID:27572131

  14. Cardiovascular responses to glucagon - Physiologic measurement by external recordings.

    NASA Technical Reports Server (NTRS)

    Byrne, M. J.; Pigott, V.; Spodick, D. H.

    1972-01-01

    Assessment by noninvasive polygraphic techniques of the cardiovascular responses of normal subjects to intravenous injections of glucagon and glucagon diluent. A blinding procedure which eliminated observer bias was used during the reading of tracings. Analysis of group results showed that glucagon provoked uniformly significant changes, including increase in heart rate, blood pressure, pressure-rate product, and ejection time index, and decrease in prejection period, mechanical and electromechanical systole, left ventricular ejection time, and the ratio PEP/LVET. The principal results correlated well with those of previous studies of the hemodynamic effects of glucagon.

  15. Dental enamel cells express functional SOCE channels

    PubMed Central

    Nurbaeva, Meerim K.; Eckstein, Miriam; Concepcion, Axel R.; Smith, Charles E.; Srikanth, Sonal; Paine, Michael L.; Gwack, Yousang; Hubbard, Michael J.; Feske, Stefan; Lacruz, Rodrigo S.

    2015-01-01

    Dental enamel formation requires large quantities of Ca2+ yet the mechanisms mediating Ca2+ dynamics in enamel cells are unclear. Store-operated Ca2+ entry (SOCE) channels are important Ca2+ influx mechanisms in many cells. SOCE involves release of Ca2+ from intracellular pools followed by Ca2+ entry. The best-characterized SOCE channels are the Ca2+ release-activated Ca2+ (CRAC) channels. As patients with mutations in the CRAC channel genes STIM1 and ORAI1 show abnormal enamel mineralization, we hypothesized that CRAC channels might be an important Ca2+ uptake mechanism in enamel cells. Investigating primary murine enamel cells, we found that key components of CRAC channels (ORAI1, ORAI2, ORAI3, STIM1, STIM2) were expressed and most abundant during the maturation stage of enamel development. Furthermore, inositol 1,4,5-trisphosphate receptor (IP3R) but not ryanodine receptor (RyR) expression was high in enamel cells suggesting that IP3Rs are the main ER Ca2+ release mechanism. Passive depletion of ER Ca2+ stores with thapsigargin resulted in a significant raise in [Ca2+]i consistent with SOCE. In cells pre-treated with the CRAC channel blocker Synta-66 Ca2+ entry was significantly inhibited. These data demonstrate that enamel cells have SOCE mediated by CRAC channels and implicate them as a mechanism for Ca2+ uptake in enamel formation. PMID:26515404

  16. Cell cycle gene expression under clinorotation

    NASA Astrophysics Data System (ADS)

    Artemenko, Olga

    2016-07-01

    Cyclins and cyclin-dependent kinase (CDK) are main regulators of the cell cycle of eukaryotes. It's assumes a significant change of their level in cells under microgravity conditions and by other physical factors actions. The clinorotation use enables to determine the influence of gravity on simulated events in the cell during the cell cycle - exit from the state of quiet stage and promotion presynthetic phase (G1) and DNA synthesis phase (S) of the cell cycle. For the clinorotation effect study on cell proliferation activity is the necessary studies of molecular mechanisms of cell cycle regulation and development of plants under altered gravity condition. The activity of cyclin D, which is responsible for the events of the cell cycle in presynthetic phase can be controlled by the action of endogenous as well as exogenous factors, but clinorotation is one of the factors that influence on genes expression that regulate the cell cycle.These data can be used as a model for further research of cyclin - CDK complex for study of molecular mechanisms regulation of growth and proliferation. In this investigation we tried to summarize and analyze known literature and own data we obtained relatively the main regulators of the cell cycle in altered gravity condition.

  17. Stimulation of glucagon-like peptide-1 secretion downstream of the ligand-gated ion channel TRPA1

    PubMed Central

    Emery, Edward C.; Diakogiannaki, Eleftheria; Gentry, Clive; Psichas, Arianna; Habib, Abdella M.; Bevan, Stuart; Fischer, Michael J. M.; Reimann, Frank; Gribble, Fiona M.

    2015-01-01

    Stimulus-coupled incretin secretion from enteroendocrine cells plays a fundamental role in glucose homeostasis, and could be targeted for the treatment of type-2 diabetes. Here, we investigated the expression and function of transient receptor potential (TRP) ion channels in enteroendocrine L-cells producing glucagon-like peptide-1 (GLP-1). By microarray and qPCR analysis we identified trpa1 as an L-cell enriched transcript in the small intestine. Calcium imaging of primary L-cells and the model cell line GLUTag revealed responses triggered by the TRPA1 agonists allyl-isothiocyanate (AITC, mustard oil), carvacrol and polyunsaturated fatty acids, that were blocked by TRPA1 antagonists. Electrophysiology in GLUTag cells showed that carvacrol induced a current with characteristics typical of TRPA1 and triggered the firing of action potentials. TRPA1 activation caused an increase in GLP-1 secretion from primary murine intestinal cultures and GLUTag cells; an effect that was abolished in cultures from trpa1−/− mice or by pharmacological TRPA1 inhibition. These findings present TRPA1 as a novel sensory mechanism in enteroendocrine L-cells, coupled to the facilitation of GLP-1 release, which may be exploitable as a target for treating diabetes. PMID:25325736

  18. The glucagon-like peptide 2 pathway may mediate growth and development of the bovine gastrointestinal tract

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Glucagon-like peptide 2 (GLP-2), secreted by enteroendocrine cells, has a number of physiological effects on the intestine of monogastric species, including promotion of growth of intestinal epithelium, reduction of epithelial cell apoptosis, and enhancement of intestinal blood flow, nutrient absorp...

  19. Glucagon Like Peptide-1 Promotes Adipocyte Differentiation via the Wnt4 Mediated Sequestering of Beta-Catenin

    PubMed Central

    Liu, Rui; Li, Na; Lin, Yi; Wang, Mei; Peng, Yongde; Lewi, Keidren; Wang, Qinghua

    2016-01-01

    Glucagon-like peptide-1 (GLP-1) plays a role in the regulation of adipogenesis; however, the precise underlying molecular mechanism has not been fully defined. Wnt was recently identified as an important regulator of adipogenesis. This study aimed to investigate the involvement of the Wnt signaling pathway in the effects of GLP-1 on adipocyte differentiation. 3T3-L1 cells were induced to differentiate. The changes in the expression levels of adipogenic transcription factors and Wnts and the phosphorylation level and subcellular localization of β-catenin were quantified after GLP-1 treatment. GLP-1 stimulated adipocyte differentiation and lipid accumulation, which were accompanied by the expression of adipocyte marker genes. The expression of Wnt4 was upregulated in the process of adipocyte differentiation, which was further enhanced by treatment with GLP-1. β-catenin, an important mediator of the Wnt pathway, was immediately dephosphorylated and translocated from cytoplasm to nucleus when differentiation was induced. In the presence of GLP-1, however, β-catenin was redirected to the cell plasma membrane leading to its decreased accumulation in the nucleus. Knockdown of Wnt4 blocked the effect of GLP-1 on the cellular localization of β-catenin and expression level of adipogenic transcription factors. Our findings showed that GLP-1 promoted adipogenesis through the modulation of the Wnt4/β-catenin signaling pathway, suggesting that the GLP-1-Wntβ-catenin system might be a new target for the treatment of metabolic disease. PMID:27504979

  20. Pax6 and Pdx1 are required for production of glucose-dependent insulinotropic polypeptide in proglucagon-expressing L cells.

    PubMed

    Fujita, Yukihiro; Chui, Jeannie W Y; King, David S; Zhang, Tianjiao; Seufert, Jochen; Pownall, Scott; Cheung, Anthony T; Kieffer, Timothy J

    2008-09-01

    Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are incretin hormones that play important roles in maintaining glucose homeostasis and are being actively pursued as novel therapeutic agents for diabetes. GIP is produced by dispersed enteroendocrine cells and interestingly at times is coexpressed with GLP-1. We sought to determine the factors that selectively define GIP- vs. GLP-1-expressing cells. We performed comparative immunostaining of Pax6 and Pdx1 in GIP- and GLP-1-secreting cells. We investigated whether Pax6 and Pdx1 activate the human GIP promoter in control IEC-6 cells and GIP-expressing STC-1 cells. EMSA was performed to assess the binding of these transcription factors to the GIP promoter. Pax6 and Pdx1 consistently colocalized in GIP-immunoreactive cells. Cells that coexpress GIP and GLP-1 were Pax6 and Pdx1 positive, whereas cells expressing only GLP-1 were Pax6 positive but did not express Pdx1. GIP promoter activity was enhanced in IEC-6 cells by exogenous Pax6 or Pdx1 and diminished in STC-1 cells by inhibition of endogenous Pax6 or Pdx1 by dominant-negative forms. Promoter truncation analysis revealed a major loss of promoter activity when the sequence between -184 to -145 bp was deleted. EMSA studies indicated that Pax6 and Pdx1 bind to this proximal sequence of the human GIP promoter. Our findings indicate that concomitant expression of Pax6 and Pdx1 is important for GIP expression. Our results also suggest that the presence of Pdx1 defines whether GLP-1-expressing gastrointestinal L cells also coexpress GIP. PMID:18593849

  1. Inhibition of acetyl-CoA carboxylase activity in isolated rat adipocytes incubated with glucagon. Interactions with the effects of insulin, adrenaline and adenosine deaminase

    PubMed Central

    Zammit, Victor A.; Corstorphine, Clark G.

    1982-01-01

    1. Adipocytes isolated from epididymal fat-pads of fed rats were incubated with different concentrations of glucagon, insulin, adrenaline and adenosine deaminase, and the effects of these agents on the `initial' activity of acetyl-CoA carboxylase in the cells were studied. 2. Glucagon (at concentrations between 0.1 and 10nm) inhibited acetyl-CoA carboxylase activity. Maximal inhibition was approx. 70% of the `control' activity in the absence of added hormone, and the concentration of hormone required for half-maximal inhibition was 0.3–0.5nm-glucagon. 3. Incubation of cells with adenosine deaminase resulted in a similar inhibition of acetyl-CoA carboxylase activity. Preincubation of adipocytes with adenosine deaminase did not alter either the sensitivity of carboxylase activity to increasing concentrations of glucagon or the maximal extent of inhibition. 4. Adrenaline inhibited acetyl-CoA carboxylase to the same extent as glucagon. Preincubation of the cells with glucagon did not alter the sensitivity of enzyme activity to adrenaline or the degree of maximal inhibition. 5. Insulin activated the enzyme by 70–80% of `control' activity. Preincubation of the cells with glucagon did not alter the concentration of insulin required to produce half the maximal stimulatory effect (about 12μunits of insulin/ml). The effects of insulin and glucagon appeared to be mediated completely independently, and were approximately quantitatively similar but opposite. These characteristics resulted in the mutual cancellation of the effects of the two hormones when they were both present at equally effective concentrations. 6. The implications of these findings with regard to current concepts about the mechanism of regulation of acetyl-CoA carboxylase and to the regulation of the enzyme in vivo are discussed. PMID:6131671

  2. Glucagon levels and metabolic effects in fasting man

    PubMed Central

    Marliss, Errol B.; Aoki, Thomas T.; Unger, Roger H.; Soeldner, J. Stuart; Cahill, George F.

    1970-01-01

    The role of glucagon in the metabolic adaptation to prolonged fasting in man has been examined. Plasma immunoreactive glucagon was determined during 6-wk fasts and during infusion of exogenous glucagon using an assay which minimized nonpancreatic immunoreactivity. Plasma glucagon concentrations rose twofold to a peak on the 3rd day of fasting and then declined thereafter to a level maintained at or above postabsorptive. Insulin concentration declined to a plateau by the 3rd day. Thus a persisting altered relationship of glucagon and insulin concentrations characterized the fasted state. A synergism of low insulin and relative or absolute elevation of glucagon levels is viewed as a hormonal mechanism controlling the rate of hepatic substrate extraction for gluconeogenesis. Glucagon was infused systemically into 4-6 wk fasted subjects at three dose levels. A marked sensitivity of individual plasma free amino acids to the induced elevations of plasma glucagon within the physiologic range was demonstrated. At higher concentrations, equivalent to those present in the portal vein, stimulation of hepatic gluconeogenesis occurred, and the effects on glucose, insulin, and growth hormone levels and on ketone metabolism were induced. Images PMID:5480852

  3. Glucagon and heart in type 2 diabetes: new perspectives.

    PubMed

    Ceriello, Antonio; Genovese, Stefano; Mannucci, Edoardo; Gronda, Edoardo

    2016-01-01

    Increased levels of glucagon in type 2 diabetes are well known and, until now, have been considered deleterious. However, glucagon has an important role in the maintenance of both heart and kidney function. Moreover, in the past, glucagon has been therapeutically used for heart failure treatment. The new antidiabetic drugs, dipeptidyl peptidase-4 inhibitors and sodium-glucose co-transporter-2 inhibitors, are able to decrease and to increase glucagon levels, respectively, while contrasting data have been reported regarding the glucagon like peptide 1 receptors agonists. The cardiovascular outcome trials, requested by the FDA, raised some concerns about the possibility that the dipeptidyl peptidase-4 inhibitors can precipitate the heart failure, while, at least for empagliflozin, a positive effect has been shown in decreasing both cardiovascular death and heart failure. The recent LEADER Trial, showed a significant reduction of cardiovascular death with liraglutide, but a neutral effect on heart failure. A possible explanation of the results with the DPPIV inhibitors and empagliflozin might be related to their divergent effect on glucagon levels. Due to unclear effects of glucagon like peptide 1 receptor agonists on glucagon, the possible role of this hormone in the Leader trial remains unclear. PMID:27568179

  4. Dynamics of glucagon secretion in mice and rats revealed using a validated sandwich ELISA for small sample volumes.

    PubMed

    Wewer Albrechtsen, Nicolai J; Kuhre, Rune E; Windeløv, Johanne A; Ørgaard, Anne; Deacon, Carolyn F; Kissow, Hannelouise; Hartmann, Bolette; Holst, Jens J

    2016-08-01

    Glucagon is a metabolically important hormone, but many aspects of its physiology remain obscure, because glucagon secretion is difficult to measure in mice and rats due to methodological inadequacies. Here, we introduce and validate a low-volume, enzyme-linked immunosorbent glucagon assay according to current analytical guidelines, including tests of sensitivity, specificity, and accuracy, and compare it, using the Bland-Altman algorithm and size-exclusion chromatography, with three other widely cited assays. After demonstrating adequate performance of the assay, we measured glucagon secretion in response to intravenous glucose and arginine in anesthetized mice (isoflurane) and rats (Hypnorm/midazolam). Glucose caused a long-lasting suppression to very low values (1-2 pmol/l) within 2 min in both species. Arginine stimulated secretion 8- to 10-fold in both species, peaking at 1-2 min and returning to basal levels at 6 min (mice) and 12 min (rats). d-Mannitol (osmotic control) was without effect. Ketamine/xylazine anesthesia in mice strongly attenuated (P < 0.01) α-cell responses. Chromatography of pooled plasma samples confirmed the accuracy of the assay. In conclusion, dynamic analysis of glucagon secretion in rats and mice with the novel accurate sandwich enzyme-linked immunosorbent assay revealed extremely rapid and short-lived responses to arginine and rapid and profound suppression by glucose. PMID:27245336

  5. Glucagon receptor antibody completely suppresses type 1 diabetes phenotype without insulin by disrupting a novel diabetogenic pathway

    PubMed Central

    Wang, May-Yun; Yan, Hai; Shi, Zhiqing; Evans, Matthew R.; Yu, Xinxin; Lee, Young; Chen, Shiuhwei; Williams, Annie; Philippe, Jacques; Roth, Michael G.; Unger, Roger H.

    2015-01-01

    Insulin monotherapy can neither maintain normoglycemia in type 1 diabetes (T1D) nor prevent the long-term damage indicated by elevated glycation products in blood, such as glycated hemoglobin (HbA1c). Here we find that hyperglycemia, when unaccompanied by an acute increase in insulin, enhances itself by paradoxically stimulating hyperglucagonemia. Raising glucose from 5 to 25 mM without insulin enhanced glucagon secretion ∼two- to fivefold in InR1-G9 α cells and ∼18-fold in perfused pancreata from insulin-deficient rats with T1D. Mice with T1D receiving insulin treatment paradoxically exhibited threefold higher plasma glucagon during hyperglycemic surges than during normoglycemic intervals. Blockade of glucagon action with mAb Ac, a glucagon receptor (GCGR) antagonizing antibody, maintained glucose below 100 mg/dL and HbA1c levels below 4% in insulin-deficient mice with T1D. In rodents with T1D, hyperglycemia stimulates glucagon secretion, up-regulating phosphoenolpyruvate carboxykinase and enhancing hyperglycemia. GCGR antagonism in mice with T1D normalizes glucose and HbA1c, even without insulin. PMID:25675519

  6. Oral Delivery of Pentameric Glucagon-Like Peptide-1 by Recombinant Lactobacillus in Diabetic Rats.

    PubMed

    Lin, Yin; Krogh-Andersen, Kasper; Pelletier, Julien; Marcotte, Harold; Östenson, Claes-Göran; Hammarström, Lennart

    2016-01-01

    Glucagon-like peptide-1 (GLP-1) is an incretin hormone produced by intestinal cells and stimulates insulin secretion from the pancreas in a glucose-dependent manner. Exogenously supplied GLP-1 analogues are used in the treatment of type 2 diabetes. An anti-diabetic effect of Lactobacillus in lowering plasma glucose levels and its use as a vehicle for delivery of protein and antibody fragments has been shown previously. The aim of this study was to employ lactobacilli as a vehicle for in situ production and delivery of GLP-1 analogue to normalize blood glucose level in diabetic GK (Goto-Kakizaki) rats. In this study, we designed pentameric GLP-1 (5×GLP-1) analogues which were both expressed in a secreted form and anchored to the surface of lactobacilli. Intestinal trypsin sites were introduced within 5×GLP-1, leading to digestion of the pentamer into an active monomeric form. The E. coli-produced 5×GLP-1 peptides delivered by intestinal intubation to GK rats resulted in a significant improvement of glycemic control demonstrated by an intraperitoneal glucose tolerance test. Meanwhile, the purified 5×GLP-1 (trypsin-digested) from the Lactobacillus cultures stimulated insulin secretion from HIT-T15 cells, similar to the E. coli-produced 5×GLP-1 peptides. When delivered by gavage to GK rats, non-expressor L. paracasei significantly lowered the blood glucose level but 5×GLP-1 expression did not provide an additional anti-diabetic effect, possibly due to the low levels produced. Our results indicate that lactobacilli themselves might be used as an alternative treatment method for type 2 diabetes, but further work is needed to increase the expression level of GLP-1 by lactobacilli in order to obtain a significant insulinotropic effect in vivo. PMID:27610615

  7. Increased glucagon secretion during hyperthermia in a sauna.

    PubMed

    Tatár, P; Vigas, M; Jurcovicová, J; Kvetnanský, R; Strec, V

    1986-01-01

    Plasma glucagon, adrenaline, noradrenaline, insulin and glucose concentrations were measured in 7 healthy young males during hyperthermia in a sauna bath: plasma glucagon levels increased from baseline values of 127.0 +/- 12.9 (SEM) pg X ml-1 to a maximum of 173.6 +/- 16.1 (SEM) pg X ml-1 at the 20th min of exposure. No change in plasma insulin and a slight increase in plasma glucose concentration were seen. Since a concomitant moderate increase in plasma catecholamine levels was also present, the adrenergic stimulus is believed to trigger glucagon release during hyperthermia. Diminished visceral blood flow, known to occur in sauna baths, may cause a decrease in the degradation of plasma glucagon and thus contribute to the elevated plasma glucagon levels. PMID:3525154

  8. Glucagon-like peptide-2 protects impaired intestinal mucosal barriers in obstructive jaundice rats

    PubMed Central

    Chen, Jun; Dong, Jia-Tian; Li, Xiao-Jing; Gu, Ye; Cheng, Zhi-Jian; Cai, Yuan-Kun

    2015-01-01

    AIM: To observe the protective effect of glucagon-like peptide-2 (GLP-2) on the intestinal barrier of rats with obstructive jaundice and determine the possible mechanisms of action involved in the protective effect. METHODS: Thirty-six Sprague-Dawley rats were randomly divided into a sham operation group, an obstructive jaundice group, and a GLP-2 group; each group consisted of 12 rats. The GLP-2 group was treated with GLP-2 after the day of surgery, whereas the other two groups were treated with the same concentration of normal saline. Alanine aminotransferase (ALT), total bilirubin, and endotoxin levels were recorded at 1, 3, 7, 10 and 14 d. Furthermore, on the 14th day, body weight, the wet weight of the small intestine, pathological changes of the small intestine and the immunoglobulin A (IgA) expressed by plasma cells located in the small intestinal lamina propria were recorded for each group. RESULTS: In the rat model, jaundice was obvious, and the rats’ activity decreased 4-6 d post bile duct ligation. Compared with the sham operation group, the obstructive jaundice group displayed increased yellow staining of abdominal visceral serosa, decreased small intestine wet weight, thinning of the intestinal muscle layer and villi, villous atrophy, uneven height, fusion, partial villous epithelial cell shedding, substantial inflammatory cell infiltration and significantly reduced IgA expression. However, no significant gross changes were noted between the GLP-2 and sham groups. With time, the levels of ALT, endotoxin and bilirubin in the GLP-2 group were significantly increased compared with the sham group (P < 0.01). The increasing levels of the aforementioned markers were more significant in the obstructive jaundice group than in the GLP-2 group (P < 0.01). CONCLUSION: GLP-2 reduces intestinal mucosal injuries in obstructive jaundice rats, which might be attributed to increased intestinal IgA and reduced bilirubin and endotoxin. PMID:25593463

  9. Physiology and pharmacology of the enteroendocrine hormone glucagon-like peptide-2.

    PubMed

    Drucker, Daniel J; Yusta, Bernardo

    2014-01-01

    Glucagon-like peptide-2 (GLP-2) is a 33-amino-acid proglucagon-derived peptide secreted from enteroendocrine L cells. GLP-2 circulates at low basal levels in the fasting period, and plasma levels rise rapidly after food ingestion. Renal clearance and enzymatic inactivation control the elimination of bioactive GLP-2. GLP-2 increases mesenteric blood flow and activates proabsorptive pathways in the gut, facilitating nutrient absorption. GLP-2 also enhances gut barrier function and induces proliferative and cytoprotective pathways in the small bowel. The actions of GLP-2 are transduced via a single G protein-coupled receptor (GLP-2R), expressed predominantly within the gastrointestinal tract. Disruption of GLP-2R signaling increases susceptibility to gut injury and impairs the adaptive mucosal response to refeeding. Sustained augmentation of GLP-2R signaling reduces the requirement for parenteral nutrition in human subjects with short-bowel syndrome. Hence GLP-2 integrates nutrient-derived signals to optimize mucosal integrity and energy absorption. PMID:24161075

  10. Expression of bacterial genes in plant cells.

    PubMed Central

    Fraley, R T; Rogers, S G; Horsch, R B; Sanders, P R; Flick, J S; Adams, S P; Bittner, M L; Brand, L A; Fink, C L; Fry, J S; Galluppi, G R; Goldberg, S B; Hoffmann, N L; Woo, S C

    1983-01-01

    Chimeric bacterial genes conferring resistance to aminoglycoside antibiotics have been inserted into the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid and introduced into plant cells by in vitro transformation techniques. The chimeric genes contain the nopaline synthase 5' and 3' regulatory regions joined to the genes for neomycin phosphotransferase type I or type II. The chimeric genes were cloned into an intermediate vector, pMON120, and inserted into pTiB6S3 by recombination and then introduced into petunia and tobacco cells by cocultivating A. tumefaciens cells with protoplast-derived cells. Southern hybridization was used to confirm the presence of the chimeric genes in the transformed plant tissues. Expression of the chimeric genes was determined by the ability of the transformed cells to proliferate on medium containing normally inhibitory levels of kanamycin (50 micrograms/ml) or other aminoglycoside antibiotics. Plant cells transformed by wild-type pTiB6S3 or derivatives carrying the bacterial neomycin phosphotransferase genes with their own promoters failed to grow under these conditions. The significance of these results for plant genetic engineering is discussed. Images PMID:6308651

  11. Effect of fluoride on insulin level of rats and insulin receptor expression in the MC3T3-E1 cells.

    PubMed

    Hu, Chun-Yan; Ren, Li-Qun; Li, Xi-Ning; Wu, Nan; Li, Guang-Sheng; Liu, Qin-Yi; Xu, Hui

    2012-12-01

    Studies on the role of insulin and insulin receptor (InsR) in the process of skeletal fluorosis, especially in osteogenic function, are rare. We evaluated the effect of increasing F⁻ doses on the marker of bone formation, serum insulin level and pancreatic secretion changes in vivo and mRNA expression of InsR and osteocalcin (OCN) in vitro. Wistar rats (n = 50) were divided into two groups, i.e. a control group and fluoride group. The fluoride groups were treated with fluoride by drinking tap water containing 100 mg F⁻/L. The fluoride ion-selective electrode measured the fluoride concentrations of femurs. The alkaline phosphatase (ALP), OCN, insulin and glucagon of serum were tested to observe the effect of fluoride action on them. Meantime, the pancreas pathological morphometry analysis via β cells stained by aldehyde fuchsin showed the action of fluoride on pancreas secretion. MC3T3-E1 cells (derived from newborn mouse calvaria) were exposed to varying concentrations and periods of fluoride. The mRNA expression of InsR and OCN was quantified with real-time PCR. Results showed that 1-year fluoride treatment obviously stimulated ALP activity and OCN level along with increase of bone fluoride concentration of rats, which indicated that fluoride obviously stimulated osteogenic action of rats. In vitro study, the dual effect of fluoride on osteoblast function is shown. On the other hand, there was a significant increase of serum insulin level and a general decrease of glucagon level, and the histomorphometry analysis indicated an elevated insulin-positive area and increase in islet size in rats treated with fluoride for 1 year. In addition, fluoride obviously facilitated the mRNA expression of InsR in vitro. To sum up, there existed a close relationship between insulin secretion and fluoride treatment. The insulin signal pathway might be involved in the underlying occurrence or development of skeletal fluorosis. PMID:22872571

  12. Sustained expression of GLP-1 receptor differentially modulates β-cell functions in diabetic and nondiabetic mice.

    PubMed

    Kubo, Fumiyo; Miyatsuka, Takeshi; Sasaki, Shugo; Takahara, Mitsuyoshi; Yamamoto, Yuichi; Shimo, Naoki; Watada, Hirotaka; Kaneto, Hideaki; Gannon, Maureen; Matsuoka, Taka-aki; Shimomura, Iichiro

    2016-02-26

    Glucagon-like peptide 1 (GLP-1) has been shown to play important roles in maintaining β-cell functions, such as insulin secretion and proliferation. While expression levels of GLP-1 receptor (Glp1r) are compromised in the islets of diabetic rodents, it remains unclear when and to what degree Glp1r mRNA levels are decreased during the progression of diabetes. In this study, we performed real-time PCR with the islets of db/db diabetic mice at different ages, and found that the expression levels of Glp1r were comparable to those of the islets of nondiabetic db/misty controls at the age of four weeks, and were significantly decreased at the age of eight and 12 weeks. To investigate whether restored expression of Glp1r affects the diabetic phenotypes, we generated the transgenic mouse model Pdx1(PB)-CreER(TM); CAG-CAT-Glp1r (βGlp1r) that allows for induction of Glp1r expression specifically in β cells. Whereas the expression of exogenous Glp1r had no measurable effect on glucose tolerance in nondiabetic βGlp1r;db/misty mice, βGlp1r;db/db mice exhibited higher glucose and lower insulin levels in blood on glucose challenge test than control db/db littermates. In contrast, four weeks of treatment with exendin-4 improved the glucose profiles and increased serum insulin levels in βGlp1r;db/db mice, to significantly higher levels than those in control db/db mice. These differential effects of exogenous Glp1r in nondiabetic and diabetic mice suggest that downregulation of Glp1r might be required to slow the progression of β-cell failure under diabetic conditions. PMID:26854076

  13. Defective insulin secretion by chronic glucagon receptor activation in glucose intolerant mice.

    PubMed

    Ahlkvist, Linda; Omar, Bilal; Valeur, Anders; Fosgerau, Keld; Ahrén, Bo

    2016-03-01

    Stimulation of insulin secretion by short-term glucagon receptor (GCGR) activation is well characterized; however, the effect of long-term GCGR activation on β-cell function is not known, but of interest, since hyperglucagonemia occurs early during development of type 2 diabetes. Therefore, we examined whether chronic GCGR activation affects insulin secretion in glucose intolerant mice. To induce chronic GCGR activation, high-fat diet fed mice were continuously (2 weeks) infused with the stable glucagon analog ZP-GA-1 and challenged with oral glucose and intravenous glucose±glucagon-like peptide 1 (GLP1). Islets were isolated to evaluate the insulin secretory response to glucose±GLP1 and their pancreas were collected for immunohistochemical analysis. Two weeks of ZP-GA-1 infusion reduced insulin secretion both after oral and intravenous glucose challenges in vivo and in isolated islets. These inhibitory effects were corrected for by GLP1. Also, we observed increased β-cell area and islet size. We conclude that induction of chronic ZP-GA-1 levels in glucose intolerant mice markedly reduces insulin secretion, and thus, we suggest that chronic activation of the GCGR may contribute to the failure of β-cell function during development of type 2 diabetes. PMID:26698567

  14. Gene expression profiles in irradiated cancer cells

    NASA Astrophysics Data System (ADS)

    Minafra, L.; Bravatà, V.; Russo, G.; Ripamonti, M.; Gilardi, M. C.

    2013-07-01

    Knowledge of the molecular and genetic mechanisms underlying cellular response to radiation may provide new avenues to develop innovative predictive tests of radiosensitivity of tumours and normal tissues and to improve individual therapy. Nowadays very few studies describe molecular changes induced by hadrontherapy treatments, therefore this field has to be explored and clarified. High-throughput methodologies, such as DNA microarray, allow us to analyse mRNA expression of thousands of genes simultaneously in order to discover new genes and pathways as targets of response to hadrontherapy. Our aim is to elucidate the molecular networks involved in the sensitivity/resistance of cancer cell lines subjected to hadrontherapy treatments with a genomewide approach by using cDNA microarray technology to identify gene expression profiles and candidate genes responsible of differential cellular responses.

  15. Gene expression profiles in irradiated cancer cells

    SciTech Connect

    Minafra, L.; Bravatà, V.; Russo, G.; Ripamonti, M.; Gilardi, M. C.

    2013-07-26

    Knowledge of the molecular and genetic mechanisms underlying cellular response to radiation may provide new avenues to develop innovative predictive tests of radiosensitivity of tumours and normal tissues and to improve individual therapy. Nowadays very few studies describe molecular changes induced by hadrontherapy treatments, therefore this field has to be explored and clarified. High-throughput methodologies, such as DNA microarray, allow us to analyse mRNA expression of thousands of genes simultaneously in order to discover new genes and pathways as targets of response to hadrontherapy. Our aim is to elucidate the molecular networks involved in the sensitivity/resistance of cancer cell lines subjected to hadrontherapy treatments with a genomewide approach by using cDNA microarray technology to identify gene expression profiles and candidate genes responsible of differential cellular responses.

  16. Evaluation of immunohistochemical staining for glucagon in human pancreatic tissue

    PubMed Central

    Gurlo, Tatyana; Butle, Peter C.; Butler, Alexandra E.

    2016-01-01

    Immunohistochemistry (IHC) and immunofluorescence (IF) staining techniques are important diagnostic tools of anatomic pathology in the clinical setting and widely used analytical tools in research laboratories. In diabetes research, they are routinely used for the assessment of beta- and alpha-cell mass, for assessment of endocrine cell distribution within the pancreas, for evaluation of islet composition and islet morphology. Here, we present the evaluation of IHC techniques for the detection of alpha-cells in human pancreatic tissue. We compared the Horse Radish Peroxidase (HRP)-based method utilizing DAB Peroxidase Substrate to the Alkaline Phosphatase (AP)-based method utilizing Vector Red substrate. We conclude that HRP–DAB staining is a robust and reliable method for detection of alpha-cells using either rabbit polyclonal or mouse monoclonal anti-glucagon antibodies. However, AP-Vector Red staining should be used with caution, because it is affected by the dehydration with ethanol and toluene preceding the mounting of slides with Permount mounting medium. When AP-Vector Red is a preferable method for alpha-cell labeling, slides should be mounted using aqueous mounting medium or, alternatively, they could be air-dried before permanent mounting PMID:27182095

  17. The inactivation of extracellular signal-regulated kinase by glucagon-like peptide-1 contributes to neuroprotection against oxidative stress.

    PubMed

    Nakajima, Shingo; Numakawa, Tadahiro; Adachi, Naoki; Yoon, Hyung Shin; Odaka, Haruki; Ooshima, Yoshiko; Kunugi, Hiroshi

    2016-03-11

    Glucagon-like peptide-1 (GLP-1), an insulinotropic peptide secreted from enteroendocrine cells, has been known to have a neuroprotective effect. However, it is not fully understood the intracellular mediator of GLP-1 signaling in neuronal cells. In the present study, we examined the change in intracellular signaling of cortical neurons after GLP-1 application and luminal glucose stimulation in vitro and in vivo. GLP-1 receptor was highly expressed in cultured cortical neurons and brain tissues including the prefrontal cortex and hippocampus. The activation of GLP-1 receptor (5min) significantly decreased levels of phosphorylated extracellular signal-regulated kinase (pERK), which is involved in neuronal cell survival and death, in cultured cortical neurons. Oral glucose administration also rapidly reduced pERK levels in the prefrontal cortex, while intraperitoneal glucose injection did not show such an effect. Further, GLP-1 attenuated hydrogen peroxide-induced cell death and hyperactivity of ERK in cultured cortical neurons. It is possible that increased GLP-1 by luminal glucose stimulation affects cortical system including the maintenance of neuronal cell survival. PMID:26827720

  18. Comparative physiology of glucagon-like peptide-2 – Implications and applications for production and health of ruminants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Glucagon-like peptide-2 (GLP-2) is a 33-amino acid peptide derived from proteolytic cleavage of proglucagon by prohormone convertase 1/3 in enteroendocrine L-cells. Studies conducted in humans, rodent models, and in vitro indicate that GLP-2 is secreted in response to the presence of molecules in th...

  19. Comparative physiology of glucagon-like peptide 2 - Implications and applications for production and health of ruminants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Glucagon-like peptide 2 (GLP-2) is a 33-amino acid peptide derived from proteolytic cleavage of proglucagon by prohormone convertase 1/3 in enteroendocrine L-cells. Studies conducted in humans, rodent models, and in vitro indicate that GLP-2 is secreted in response to the presence of molecules in th...

  20. Glucagon and adenylate cyclase: binding studies and requirements for activation.

    PubMed

    Levey, G S; Fletcher, M A; Klein, I

    1975-01-01

    Solubilization of myocardial adenylate cyclase abolished responsiveness to glucagon and catecholamines, two of the hormones which activate the membrane-bound enzyme. Adenylate cyclase freed of detergent by DEAE-cellulose chromatography continues to remain unresponsive to hormone stimulation. However, adding purified bovine brain phospholipids--phosphotidylserine and monophosphatidylinositol--restored responsiveness to glucagon and catecholamines, respectively. 125-i-glucagon binding appeared to be independent of phospholipid, since equal binding was observed in the presence or absence of detergent and in the presence or absence of phospholipids. Chromatography of the solubilized preparation on Sephadex G-100 WAS CHARACTERIZED BY 125-I-glucagon binding and fluoride-stimulatable adenylate cyclase activity appearing in the fractions consistent with the void volume, suggesting a molecular weight greater than 100,000 for the receptor-adenylate cyclase complex. Prior incubation of the binding peak with 125-I-glucagon and rechromatography of the bound glucagon on Sephadex G-100 shifted its elution to a later fraction consistent with a smaller-molecular-weight peak. The molecular weight of this material was 24,000 to 28,000, as determined by SDS polyacrylamide gel electrophoresis. The latter findings are consistent with a dissociable receptor site for glucagon on myocardial adenylate cyclase. PMID:165684

  1. Prediabetes Linked to Excess Glucagon in Transgenic Mice with Pancreatic Active AKT1

    PubMed Central

    Albury-Warren, Toya M.; Pandey, Veethika; Spinel, Lina P.; Masternak, Michal M.; Altomare, Deborah A

    2015-01-01

    Protein Kinase B/AKT, has three isoforms (AKT1-3) and is renowned for its central role in the regulation of cell growth and proliferation, due to its constitutive activation in various cancers. AKT2, which is highly expressed in insulin responsive tissues, has been identified as a primary regulator of glucose metabolism as Akt2 knockout mice (Akt2−/−) are glucose intolerant and insulin resistant. However, the role of AKT1 in glucose metabolism is not as clearly defined. We previously showed that mice with myristoylated Akt1 (AKT1Myr) expressed through a bicistronic Pdx1-TetA and TetO-MyrAkt1 system were susceptible to islet cell carcinomas, and in this study we characterized an early onset, prediabetic phenotype. Beginning at weaning (3 weeks of age), the glucose intolerant AKT1Myr mice exhibited non-fasted hyperglycemia, which progressed to fasted hyperglycemia by 5 months of age. The glucose intolerance was attributed to a fasted hyperglucagonemia, and hepatic insulin resistance detectable by reduced phosphorylation of the insulin receptor following insulin injection into the inferior vena cava. In contrast, treatment with doxycycline diet to turn-off the transgene, caused attenuation of the non-fasted and fasted hyperglycemia, thus affirming AKT1 hyperactivation as the trigger. Collectively, this model highlights a novel glucagon-mediated mechanism by which AKT1 hyperactivation affects glucose homeostasis, and provides an avenue to better delineate the molecular mechanisms responsible for diabetes mellitus and the potential association with pancreatic cancer. PMID:26487674

  2. Prediabetes linked to excess glucagon in transgenic mice with pancreatic active AKT1.

    PubMed

    Albury-Warren, Toya M; Pandey, Veethika; Spinel, Lina P; Masternak, Michal M; Altomare, Deborah A

    2016-01-01

    Protein kinase B/AKT has three isoforms (AKT1-3) and is renowned for its central role in the regulation of cell growth and proliferation, due to its constitutive activation in various cancers. AKT2, which is highly expressed in insulin-responsive tissues, has been identified as a primary regulator of glucose metabolism as Akt2 knockout mice (Akt2(-/-)) are glucose-intolerant and insulin-resistant. However, the role of AKT1 in glucose metabolism is not as clearly defined. We previously showed that mice with myristoylated Akt1 (AKT1(Myr)) expressed through a bicistronic Pdx1-TetA and TetO-MyrAkt1 system were susceptible to islet cell carcinomas, and in this study we characterized an early onset, prediabetic phenotype. Beginning at weaning (3 weeks of age), the glucose-intolerant AKT1(Myr) mice exhibited non-fasted hyperglycemia, which progressed to fasted hyperglycemia by 5 months of age. The glucose intolerance was attributed to a fasted hyperglucagonemia, and hepatic insulin resistance detectable by reduced phosphorylation of the insulin receptor following insulin injection into the inferior vena cava. In contrast, treatment with doxycycline diet to turn off the transgene caused attenuation of the non-fasted and fasted hyperglycemia, thus affirming AKT1 hyperactivation as the trigger. Collectively, this model highlights a novel glucagon-mediated mechanism by which AKT1 hyperactivation affects glucose homeostasis and provides an avenue to better delineate the molecular mechanisms responsible for diabetes mellitus and the potential association with pancreatic cancer. PMID:26487674

  3. Na+ current properties in islet α- and β-cells reflect cell-specific Scn3a and Scn9a expression

    PubMed Central

    Zhang, Quan; Chibalina, Margarita V; Bengtsson, Martin; Groschner, Lukas N; Ramracheya, Reshma; Rorsman, Nils J G; Leiss, Veronika; Nassar, Mohammed A; Welling, Andrea; Gribble, Fiona M; Reimann, Frank; Hofmann, Franz; Wood, John N; Ashcroft, Frances M; Rorsman, Patrik

    2014-01-01

    Mouse pancreatic β- and α-cells are equipped with voltage-gated Na+ currents that inactivate over widely different membrane potentials (half-maximal inactivation (V0.5) at −100 mV and −50 mV in β- and α-cells, respectively). Single-cell PCR analyses show that both α- and β-cells have Nav1.3 (Scn3) and Nav1.7 (Scn9a) α subunits, but their relative proportions differ: β-cells principally express Nav1.7 and α-cells Nav1.3. In α-cells, genetically ablating Scn3a reduces the Na+ current by 80%. In β-cells, knockout of Scn9a lowers the Na+ current by >85%, unveiling a small Scn3a-dependent component. Glucagon and insulin secretion are inhibited in Scn3a−/− islets but unaffected in Scn9a-deficient islets. Thus, Nav1.3 is the functionally important Na+ channel α subunit in both α- and β-cells because Nav1.7 is largely inactive at physiological membrane potentials due to its unusually negative voltage dependence of inactivation. Interestingly, the Nav1.7 sequence in brain and islets is identical and yet the V0.5 for inactivation is >30 mV more negative in β-cells. This may indicate the presence of an intracellular factor that modulates the voltage dependence of inactivation. PMID:25172946

  4. Treatment of Aluminium Phosphide Poisoning with a Combination of Intravenous Glucagon, Digoxin and Antioxidant Agents.

    PubMed

    Oghabian, Zohreh; Mehrpour, Omid

    2016-08-01

    Aluminium phosphide (AlP) is used to protect stored grains from rodents. It produces phosphine gas (PH3), a mitochondrial poison thought to cause toxicity by blocking the cytochrome c oxidase enzyme and inhibiting oxidative phosphorylation, which results in cell death. AlP poisoning has a high mortality rate among humans due to the rapid onset of cardiogenic shock and metabolic acidosis, despite aggressive treatment. We report a 21-year-old male who was referred to the Afzalipour Hospital, Kerman, Iran, in 2015 after having intentionally ingested a 3 g AlP tablet. He was successfully treated with crystalloid fluids, vasopressors, sodium bicarbonate, digoxin, glucagon and antioxidant agents and was discharged from the hospital six days after admission in good clinical condition. For the treatment of AlP poisoning, the combination of glucagon and digoxin with antioxidant agents should be considered. However, evaluation of further cases is necessary to optimise treatment protocols. PMID:27606117

  5. Treatment of Aluminium Phosphide Poisoning with a Combination of Intravenous Glucagon, Digoxin and Antioxidant Agents

    PubMed Central

    Oghabian, Zohreh; Mehrpour, Omid

    2016-01-01

    Aluminium phosphide (AlP) is used to protect stored grains from rodents. It produces phosphine gas (PH3), a mitochondrial poison thought to cause toxicity by blocking the cytochrome c oxidase enzyme and inhibiting oxidative phosphorylation, which results in cell death. AlP poisoning has a high mortality rate among humans due to the rapid onset of cardiogenic shock and metabolic acidosis, despite aggressive treatment. We report a 21-year-old male who was referred to the Afzalipour Hospital, Kerman, Iran, in 2015 after having intentionally ingested a 3 g AlP tablet. He was successfully treated with crystalloid fluids, vasopressors, sodium bicarbonate, digoxin, glucagon and antioxidant agents and was discharged from the hospital six days after admission in good clinical condition. For the treatment of AlP poisoning, the combination of glucagon and digoxin with antioxidant agents should be considered. However, evaluation of further cases is necessary to optimise treatment protocols. PMID:27606117

  6. Gene Expression by Mouse Inner Ear Hair Cells during Development

    PubMed Central

    Scheffer, Déborah I.; Shen, Jun

    2015-01-01

    Hair cells of the inner ear are essential for hearing and balance. As a consequence, pathogenic variants in genes specifically expressed in hair cells often cause hereditary deafness. Hair cells are few in number and not easily isolated from the adjacent supporting cells, so the biochemistry and molecular biology of hair cells can be difficult to study. To study gene expression in hair cells, we developed a protocol for hair cell isolation by FACS. With nearly pure hair cells and surrounding cells, from cochlea and utricle and from E16 to P7, we performed a comprehensive cell type-specific RNA-Seq study of gene expression during mouse inner ear development. Expression profiling revealed new hair cell genes with distinct expression patterns: some are specific for vestibular hair cells, others for cochlear hair cells, and some are expressed just before or after maturation of mechanosensitivity. We found that many of the known hereditary deafness genes are much more highly expressed in hair cells than surrounding cells, suggesting that genes preferentially expressed in hair cells are good candidates for unknown deafness genes. PMID:25904789

  7. Abnormal Secretion of Insulin and Glucagon by the In Vitro Perfused Pancreas of the Genetically Diabetic Chinese Hamster

    PubMed Central

    Frankel, Barbara J.; Gerich, John E.; Hagura, Ryoko; Fanska, Rudy E.; Gerritsen, George C.; Grodsky, Gerold M.

    1974-01-01

    Hereditary insulin-deficient diabetes mellitus occurs in certain sublines of nonobese Chinese hamsters. Several characteristics of this syndrome are similar to those seen in insulin-deficient human diabetics. Therefore, to characterize pancreatic islet function, dynamic insulin and glucagon release from normal and nonketotic diabetic hamster pancreases in response to glucose (300 mg/100 ml) and theophylline (10 mM), infused singly and together, was studied in vitro. 20-min glucose infusions of normal hamster pancreases caused biphasic insulin release, consisting of a rapid first peak and a gradually rising second phase, similar to that reported for man in vivo. Both phases were significantly reduced in the diabetic pancreases. Theophylline alone stimulated similar nonphasic insulin release in both the normal and the diabetic pancreases. Glucose and theophylline together caused greater insulin release than either stimulant alone in both normals and diabetics; however, the diabetic response was still subnormal. Glucose suppressed glucagon release from normal pancreases; suppression was significantly impaired in diabetics. Theophylline stimulated nonphasic glucagon release in both the normals and diabetics. Glucose partially suppressed the theophylline-stimulated release in both groups. Insulin/glucagon molar ratios of the diabetics were consistently subnormal, although individual hormone levels often overlapped into the normal range. In summary, the pancreases of genetically diabetic Chinese hamsters perfused in vitro showed: (a) decreased first and second phase insulin release in response to glucose-containing stimuli—only partially ameliorated by theophylline—, and (b) impaired suppression of glucagon in response to glucose, resulting in (c) a decreased insulin/glucagon molar ratio. These data support the suggestion that both alpha and beta cells of diabetic pancreases may be insensitive to glucose. Images PMID:4830228

  8. T cells stimulate catabolic gene expression by the stromal cells from giant cell tumor of bone

    SciTech Connect

    Cowan, Robert W.; Ghert, Michelle; Singh, Gurmit

    2012-03-23

    Highlights: Black-Right-Pointing-Pointer Two T cell lines stimulate PTHrP, RANKL, MMP13 gene expression in GCT cell cultures. Black-Right-Pointing-Pointer CD40 expressed by stromal cells; CD40L detected in whole tumor but not cultures. Black-Right-Pointing-Pointer Effect of CD40L treatment on GCT cells increased PTHrP and MMP13 gene expression. Black-Right-Pointing-Pointer PTHrP treatment increased MMP13 expression, while inhibition decreased expression. Black-Right-Pointing-Pointer T cells may stimulate GCT stromal cells and promote the osteolysis of the tumor. -- Abstract: The factors that promote the localized bone resorption by giant cell tumor of bone (GCT) are not fully understood. We investigated whether T cells could contribute to bone resorption by stimulating expression of genes for parathyroid hormone-related protein (PTHrP), matrix metalloproteinase (MMP)-13, and the receptor activator of nuclear-factor {kappa}B ligand (RANKL). Two cell lines, Jurkat clone E6-1 and D1.1, were co-cultured with isolated GCT stromal cells. Real-time PCR analyses demonstrated a significant increase of all three genes following 48 h incubation, and PTHrP and MMP-13 gene expression was also increased at 24 h. Further, we examined the expression of CD40 ligand (CD40L), a protein expressed by activated T cells, and its receptor, CD40, in GCT. Immunohistochemistry results revealed expression of the CD40 receptor in both the stromal cells and giant cells of the tumor. RNA collected from whole GCT tissues showed expression of CD40LG, which was absent in cultured stromal cells, and suggests that CD40L is expressed within GCT. Stimulation of GCT stromal cells with CD40L significantly increased expression of the PTHrP and MMP-13 genes. Moreover, we show that inhibition of PTHrP with neutralizing antibodies significantly decreased MMP13 expression by the stromal cells compared to IgG-matched controls, whereas stimulation with PTHrP (1-34) increased MMP-13 gene expression. These

  9. Murine somatic cell nuclear transfer using reprogrammed donor cells expressing male germ cell-specific genes.

    PubMed

    Kang, Hoin; Park, Jong Im; Roh, Sangho

    2016-01-01

    In vivo-matured mouse oocytes were enucleated, and a single murine embryonic fibroblast (control or reprogrammed by introducing extracts from murine testis tissue, which showed expression of male germ cell-specific genes) was injected into the cytoplasm of the oocytes. The rate of blastocyst development and expression levels of Oct-4, Eomes and Cdx-2 were not significantly different in both experimental groups. However, the expression levels of Nanog, Sox9 and Glut-1 were significantly increased when reprogrammed cells were used as donor nuclei. Increased expression of Nanog can be supportive of complete reprogramming of somatic cell nuclear transfer murine embryos. The present study suggested that donor cells expressing male germ cell-specific genes can be reconstructed and can develop into embryos with normal high expression of developmentally essential genes. PMID:26369430

  10. Cocaine- and amphetamine-regulated transcript: a novel regulator of energy homeostasis expressed in a subpopulation of pancreatic islet cells.

    PubMed

    Gilon, Patrick

    2016-09-01

    Type 2 diabetes is characterised by chronic hyperglycaemia and its incidence is highly increased by exaggerated food consumption. It results from a lack of insulin action/production, but growing evidence suggests that it might also involve hyperglucagonaemia and impaired control of glucose homeostasis by the brain. In recent years, the cocaine and amphetamine-regulated transcript (CART) peptides have generated a lot of interest in the battle against obesity because, via the brain, they exert anorexic effects and they increase energy expenditure. They are also localised, outside the brain, in discrete regions of the body and play a hormonal role in controlling various functions. In this issue of Diabetologia, the Wierup group (doi: 10.1007/s00125-016-4020-6 ) shows that CART peptides are expressed heterogeneously in islet cells of various species, including humans, and that their expression is upregulated in diabetes. The authors also shine a spotlight on some interesting effects of CART peptides on islet function, including stimulation of insulin secretion and inhibition of glucagon release. CART peptides would thus be at the centre of a cooperation between the brain and the endocrine pancreas to control glucose homeostasis. Although the mechanisms of action of CART peptides remain enigmatic because no specific receptor for these peptides has so far been discovered, their potential therapeutic use is evident and represents a new challenge for future research. PMID:27421727

  11. Glucagon in the artificial pancreas: supply and marketing challenges.

    PubMed

    Rylander, Dick

    2015-01-01

    The use of glucagon, in conjunction with insulin, in a dual chamber pump (artificial pancreas, AP) is a working goal for multiple companies and researchers. However, capital investment to create, operate, and maintain facilities with sufficient scale to produce enough glucagon to treat millions of patients, at a level of profit that makes it feasible, will be substantial. It can be assumed that the marketplace will expect the daily cost of glucagon (to the consumer) to be similar to the daily cost of insulin. After one subtracts wholesaler and pharmacy markup, there may be very few dollars remaining for the drug company to cover profit, capital expenditures, marketing, burden, and other costs. Without the potential for adequate margins, manufacturers may not be willing to take the risk. Assuming that the projections discussed in this article are in the right ballpark, advance planning for the supply for glucagon needs to start today and not wait for the AP to come to market. PMID:25139825

  12. The effects of over expressing aquaporins on the cryopreservation of hepatocytes.

    PubMed

    Kumar, Balasubramanian K; Coger, Robin N; Schrum, Laura W; Lee, Charles Y

    2015-10-01

    During cryopreservation, aquaporins are critical in regulating water transport across cellular membranes and preventing osmotic damages. Hepatocytes express aquaporin (AQP) 0, 8, 9, 11, and 12; this study investigates whether increasing the localization of AQP8 on the cellular membrane would improve cell viability by increasing water transport during cryopreservation. Primary rat hepatocytes were cultured and treated with dibutyryl cAMP (Bt(2)cAMP) or glucagon to increase the expression of AQP8 at the cellular membrane via translocation. This phenomenon is verified through two experiments - confocal immunofluorescence microscopy and cell shrinkage analysis. The immunofluorescence results showed increase in AQP8 on the cellular membrane of treated cells, and cell shrinkage analysis showed an increase in water transport of treated cells compared to controls. Primary rat hepatocytes were treated with Bt(2)cAMP or glucagon and cryopreserved using standard protocols in a controlled rate freezer. This resulted in a significant increase in the cell viability on warming. These results indicate that Bt(2)cAMP or glucagon treated hepatocytes had increased expression of aquaporin in the cellular membrane, increased water transport during cryopreservation, and increased post-thaw viability. PMID:26247402

  13. The fluctuation of blood glucose, insulin and glucagon concentrations before and after insulin therapy in type 1 diabetes

    NASA Astrophysics Data System (ADS)

    Arif, Idam; Nasir, Zulfa

    2015-09-01

    A dynamical-systems model of plasma glucose, insulin and glucagon concentrations has been developed to investigate the effects of insulin therapy on blood glucose, insulin and glucagon regulations in type 1 diabetic patients. Simulation results show that the normal regulation of blood glucose concentration depends on insulin and glucagon concentrations. On type 1 diabetic case, the role of insulin on regulating blood glucose is not optimal because of the destruction of β cells in pancreas. These β cells destructions cause hyperglycemic episode affecting the whole body metabolism. To get over this, type 1 diabetic patients need insulin therapy to control the blood glucose level. This research has been done by using rapid acting insulin (lispro), long-acting insulin (glargine) and the combination between them to know the effects of insulin therapy on blood glucose, insulin and glucagon concentrations. Simulation results show that these different types of insulin have different effects on blood glucose concentration. Insulin therapy using lispro shows better blood glucose control after consumption of meals. Glargin gives better blood glucose control between meals and during sleep. Combination between lispro and glargine shows better glycemic control for whole day blood glucose level.

  14. Altered mitochondrial function after acute alteration of the endogenous insulin/glucagon ratio

    SciTech Connect

    Rohweder-Dunn, G.; Aprille, J.R.

    1986-05-01

    Mannoheptulose (MH) affects pancreatic Islet cells to cause a drop in serum insulin and a rise in glucagon. This effect peaks 1 hr after injection and results in a 3-fold increase in serum glucose. Here they examined whether metabolic functions of liver mitochondria (mito) are altered by this change in hormone status. Rats fed ad lib on 12 hr light/dark cycles were given MH (2g/kg) or vehicle i.p. during the first 2 hrs of the light cycle. Liver mito were isolated 1 hr later. Acid-extracts were assayed for ATP+ADP+AMP (nmol/mg prot). Citrulline synthesis and pyruvate carboxylation rates (nmol/min/mg prot) were assayed by following H(/sup 14/C)O/sub 3//sup -/ fixation in appropriate media. State 3 and 2,4-DNP-uncoupled respiratory rates (1/2 nmol O/sub 2//min/mg prot) were assayed polarographically with succinate. The effects of MH on mito are comparable to reported effects of glucagon injection. MH evokes acute reciprocal changes in insulin and glucagon that are highly reproducible. Thus, MH offers an interesting model for studying the effect of endogenous hormones on mito functions.

  15. Mechanisms of Glucagon Degradation at Alkaline pH

    PubMed Central

    Caputo, Nicholas; Castle, Jessica R.; Bergstrom, Colin P.; Carroll, Julie M.; Bakhtiani, Parkash A.; Jackson, Melanie A.; Roberts, Charles T.; David, Larry L.; Ward, W. Kenneth

    2014-01-01

    Glucagon is unstable and undergoes degradation and aggregation in aqueous solution. For this reason, its use in portable pumps for closed loop management of diabetes is limited to very short periods. In this study, we sought to identify the degradation mechanisms and the bioactivity of specific degradation products. We studied degradation in the alkaline range, a range at which aggregation is minimized. Native glucagon and analogs identical to glucagon degradation products were synthesized. To quantify biological activity in glucagon and in the degradation peptides, a protein kinase A-based bioassay was used. Aged, fresh, and modified peptides were analyzed by liquid chromatography with mass spectrometry (LCMS). Oxidation of glucagon at the Met residue was common but did not reduce bioactivity. Deamidation and isomerization were also common and were more prevalent at pH 10 than 9. The biological effects of deamidation and isomerization were unpredictable; deamidation at some sites did not reduce bioactivity. Deamidation of Gln 3, isomerization of Asp 9, and deamidation with isomerization at Asn 28 all caused marked potency loss. Studies with molecular-weight-cutoff membranes and LCMS revealed much greater fibrillation at pH 9 than 10. Further work is necessary to determine formulations of glucagon that minimize degradation and fibrillation. PMID:23651991

  16. Activation of Transmembrane Bile Acid Receptor TGR5 Modulates Pancreatic Islet α Cells to Promote Glucose Homeostasis.

    PubMed

    Kumar, Divya P; Asgharpour, Amon; Mirshahi, Faridoddin; Park, So Hyun; Liu, Sichen; Imai, Yumi; Nadler, Jerry L; Grider, John R; Murthy, Karnam S; Sanyal, Arun J

    2016-03-25

    The physiological role of the TGR5 receptor in the pancreas is not fully understood. We previously showed that activation of TGR5 in pancreatic β cells by bile acids induces insulin secretion. Glucagon released from pancreatic α cells and glucagon-like peptide 1 (GLP-1) released from intestinal L cells regulate insulin secretion. Both glucagon and GLP-1 are derived from alternate splicing of a common precursor, proglucagon by PC2 and PC1, respectively. We investigated whether TGR5 activation in pancreatic α cells enhances hyperglycemia-induced PC1 expression thereby releasing GLP-1, which in turn increases β cell mass and function in a paracrine manner. TGR5 activation augmented a hyperglycemia-induced switch from glucagon to GLP-1 synthesis in human and mouse islet α cells by GS/cAMP/PKA/cAMP-response element-binding protein-dependent activation of PC1. Furthermore, TGR5-induced GLP-1 release from α cells was via an Epac-mediated PKA-independent mechanism. Administration of the TGR5 agonist, INT-777, to db/db mice attenuated the increase in body weight and improved glucose tolerance and insulin sensitivity. INT-777 augmented PC1 expression in α cells and stimulated GLP-1 release from islets of db/db mice compared with control. INT-777 also increased pancreatic β cell proliferation and insulin synthesis. The effect of TGR5-mediated GLP-1 from α cells on insulin release from islets could be blocked by GLP-1 receptor antagonist. These results suggest that TGR5 activation mediates cross-talk between α and β cells by switching from glucagon to GLP-1 to restore β cell mass and function under hyperglycemic conditions. Thus, INT-777-mediated TGR5 activation could be leveraged as a novel way to treat type 2 diabetes mellitus. PMID:26757816

  17. Glucagon sensitivity and clearance in type 1 diabetes: insights from in vivo and in silico experiments.

    PubMed

    Hinshaw, Ling; Mallad, Ashwini; Dalla Man, Chiara; Basu, Rita; Cobelli, Claudio; Carter, Rickey E; Kudva, Yogish C; Basu, Ananda

    2015-09-01

    Glucagon use in artificial pancreas for type 1 diabetes (T1D) is being explored for prevention and rescue from hypoglycemia. However, the relationship between glucagon stimulation of endogenous glucose production (EGP) viz., hepatic glucagon sensitivity, and prevailing glucose concentrations has not been examined. To test the hypothesis that glucagon sensitivity is increased at hypoglycemia vs. euglycemia, we studied 29 subjects with T1D randomized to a hypoglycemia or euglycemia clamp. Each subject was studied at three glucagon doses at euglycemia or hypoglycemia, with EGP measured by isotope dilution technique. The peak EGP increments and the integrated EGP response increased with increasing glucagon dose during euglycemia and hypoglycemia. However, the difference in dose response based on glycemia was not significant despite higher catecholamine concentrations in the hypoglycemia group. Knowledge of glucagon's effects on EGP was used to develop an in silico glucagon action model. The model-derived output fitted the obtained data at both euglycemia and hypoglycemia for all glucagon doses tested. Glucagon clearance did not differ between glucagon doses studied in both groups. Therefore, the glucagon controller of a dual hormone control system may not need to adjust glucagon sensitivity, and hence glucagon dosing, based on glucose concentrations during euglycemia and hypoglycemia. PMID:26152766

  18. Global gene expression analyses of hematopoietic stem cell-like cell lines with inducible Lhx2 expression

    PubMed Central

    Richter, Karin; Wirta, Valtteri; Dahl, Lina; Bruce, Sara; Lundeberg, Joakim; Carlsson, Leif; Williams, Cecilia

    2006-01-01

    Background Expression of the LIM-homeobox gene Lhx2 in murine hematopoietic cells allows for the generation of hematopoietic stem cell (HSC)-like cell lines. To address the molecular basis of Lhx2 function, we generated HSC-like cell lines where Lhx2 expression is regulated by a tet-on system and hence dependent on the presence of doxycyclin (dox). These cell lines efficiently down-regulate Lhx2 expression upon dox withdrawal leading to a rapid differentiation into various myeloid cell types. Results Global gene expression of these cell lines cultured in dox was compared to different time points after dox withdrawal using microarray technology. We identified 267 differentially expressed genes. The majority of the genes overlapping with HSC-specific databases were those down-regulated after turning off Lhx2 expression and a majority of the genes overlapping with those defined as late progenitor-specific genes were the up-regulated genes, suggesting that these cell lines represent a relevant model system for normal HSCs also at the level of global gene expression. Moreover, in situ hybridisations of several genes down-regulated after dox withdrawal showed overlapping expression patterns with Lhx2 in various tissues during embryonic development. Conclusion Global gene expression analysis of HSC-like cell lines with inducible Lhx2 expression has identified genes putatively linked to self-renewal / differentiation of HSCs, and function of Lhx2 in organ development and stem / progenitor cells of non-hematopoietic origin. PMID:16600034

  19. Expression and function of ryanodine receptors in nonexcitable cells.

    PubMed

    Bennett, D L; Cheek, T R; Berridge, M J; De Smedt, H; Parys, J B; Missiaen, L; Bootman, M D

    1996-03-15

    We have used reverse transcriptase-polymerase chain reaction to investigate the expression of ryanodine receptors in several excitable and nonexcitable cell types. Consistent with previous reports, we detected ryanodine receptor expression in brain, heart, and skeletal muscle. In addition, we detected ryanodine receptor expression in various other excitable cells including PC 12 and A7r5 cells. Several muscle cell lines (BC3H1, C2C12, L6, and Sol8) weakly expressed ryanodine receptor when undifferentiated but strongly expressed type 1 and type 3 ryanodine receptor isoforms when differentiated into a muscle phenotype. Only 2 (HeLa and LLC-PK1 cells) out of 11 nonexcitable cell types examined expressed ryanodine receptors. Expression of ryanodine receptors at the protein level in these cells was confirmed using [3H]ryanodine binding. We also investigated the function of ryanodine receptors in Ca2+ signaling in HeLa cells using single-cell Fura-2 imaging. Neither caffeine nor ryanodine caused a detectable elevation of cytoplasmic Ca2+ in single HeLa cells. However, ryanodine caused a significant decrease in the amplitude of Ca 2+ signals evoked by repetitive stimulation with ATP. These studies show that ryanodine receptors are expressed in some nonexcitable cell types and furthermore suggest that the ryanodine receptors may be involved in a subtle regulation of intracellular Ca2+ responses. PMID:8626432

  20. Systematic characterization of lncRNAs' cell-to-cell expression heterogeneity in glioblastoma cells

    PubMed Central

    Dong, Jun; Zhuang, Yan; Huang, Shuyu; Ma, Binbin; Chen, Puxiang; Li, Xiaodong; Zhang, Bo; Li, Zhiguang; Jin, Bilian

    2016-01-01

    Glioblastoma (GBM) is the most common malignant adult brain tumor generally associated with high level of cellular heterogeneity and a dismal prognosis. Long noncoding RNAs (lncRNAs) are emerging as novel mediators of tumorigenesis. Recently developed single-cell RNA-seq provides an unprecedented way for analysis of the cell-to-cell variability in lncRNA expression profiles. Here we comprehensively examined the expression patterns of 2,003 lncRNAs in 380 cells from five primary GBMs and two glioblastoma stem-like cell (GSC) lines. Employing the self-organizing maps, we displayed the landscape of the lncRNA expression dynamics for individual cells. Further analyses revealed heterogeneous nature of lncRNA in abundance and splicing patterns. Moreover, lncRNA expression variation is also ubiquitously present in the established GSC lines composed of seemingly identical cells. Through comparative analysis of GSC and corresponding differentiated cell cultures, we defined a stemness signature by the set of 31 differentially expressed lncRNAs, which can disclose stemness gradients in five tumors. Additionally, based on known classifier lncRNAs for molecular subtypes, each tumor was found to comprise individual cells representing four subtypes. Our systematic characterization of lncRNA expression heterogeneity lays the foundation for future efforts to further understand the function of lncRNA, develop valuable biomarkers, and enhance knowledge of GBM biology. PMID:26918340

  1. The Peutz-Jeghers kinase LKB1 suppresses polyp growth from intestinal cells of a proglucagon-expressing lineage in mice

    PubMed Central

    Zac-Varghese, Sagen; Trapp, Stefan; Richards, Paul; Sayers, Sophie; Sun, Gao; Bloom, Stephen R.; Reimann, Frank; Gribble, Fiona M.; Rutter, Guy A.

    2014-01-01

    Liver kinase B1 (LKB1; also known as STK11) is a serine/threonine kinase and tumour suppressor that is mutated in Peutz-Jeghers syndrome (PJS), a premalignant syndrome associated with the development of gastrointestinal polyps. Proglucagon-expressing enteroendocrine cells are involved in the control of glucose homeostasis and the regulation of appetite through the secretion of gut hormones such as glucagon-like peptide-1 (GLP-1) and peptide tyrosine tyrosine (PYY). To determine the role of LKB1 in these cells, we bred mice bearing floxed alleles of Lkb1 against animals carrying Cre recombinase under proglucagon promoter control. These mice (GluLKB1KO) were viable and displayed near-normal growth rates and glucose homeostasis. However, they developed large polyps at the gastro-duodenal junction, and displayed premature mortality (death from 120 days of age). Histological analysis of the polyps demonstrated that they had a PJS-like appearance with an arborising smooth-muscle core. Circulating GLP-1 levels were normal in GluLKB1KO mice and the polyps expressed low levels of the peptide, similar to levels in the neighbouring duodenum. Lineage tracing using a Rosa26tdRFP transgene revealed, unexpectedly, that enterocytes within the polyps were derived from non-proglucagon-expressing precursors, whereas connective tissue was largely derived from proglucagon-expressing precursors. Developmental studies in wild-type mice suggested that a subpopulation of proglucagon-expressing cells undergo epithelial-mesenchymal transition (EMT) to become smooth-muscle-like cells. Thus, it is likely that polyps in the GluLKB1KO mice developed from a unique population of smooth-muscle-like cells derived from a proglucagon-expressing precursor. The loss of LKB1 within this subpopulation seems to be sufficient to drive tumorigenesis. PMID:25190708

  2. Probing cell-free gene expression noise in femtoliter volumes

    SciTech Connect

    Karig, David K; Jung, Seung-Yong; Srijanto, Bernadeta R; Collier, Pat; Simpson, Michael L

    2013-01-01

    Cell-free systems offer a simplified and flexible context that enables important biological reactions while removing complicating factors such as fitness, division, and mutation that are associated with living cells. However, cell-free expression in unconfined spaces is missing important elements of expression in living cells. In particular, the small volume of living cells can give rise to significant stochastic effects, which are negligible in bulk cell-free reactions. Here, we confine cell-free gene expression reactions to cell relevant 20 fL volumes (between the volumes of E. coli and S. cerevisiae), in polydimethylsiloxane (PDMS) containers. We demonstrate that expression efficiency varies widely at this volume, and we analyze gene expression noise. Noise analysis reveals signatures of translational bursting while noise dynamics suggest that overall cell-free expression is limited by a diminishing translation rate. In addition to offering a unique approach to understanding noise in gene circuits, our work contributes to a deeper understanding of the biophysical properties of cell-free expression systems, thus aiding efforts to harness cell-free systems for synthetic biology applications.

  3. Estradiol Upregulates c-FLIPlong Expression in Anterior Pituitary Cells.

    PubMed

    Jaita, G; Zárate, S; Ferraris, J; Gottardo, M F; Eijo, G; Magri, M L; Pisera, D; Seilicovich, A

    2016-04-01

    Anterior pituitary cell turnover depends on a tight balance between proliferation and apoptosis. We have previously shown that estrogens sensitize anterior pituitary cells to pro-apoptotic stimuli. c-FLIP (cellular-FLICE-inhibitory-protein) isoforms are regulatory proteins of apoptosis triggered by death receptors. c-FLIPshort isoform competes with procaspase-8 inhibiting its activation. However, c-FLIPlong isoform may have a pro- or anti-apoptotic function depending on its expression level. In the present study, we explored whether estrogens modulate c-FLIP expression in anterior pituitary cells from ovariectomized (OVX) rats and in GH3 cells, a somatolactotrope cell line. Acute administration of 17β-estradiol to OVX rats increased c-FLIPlong expression in the anterior pituitary gland without changing c-FLIPshort expression as assessed by Western blot. Estradiol in vitro also increased c-FLIPlong expression in anterior pituitary cells but not in GH3 cells. As determined by flow cytometry, the percentage of anterior pituitary cells expressing c-FLIP was higher than in GH3 cells. However, c-FLIP fluorescence intensity in GH3 cells was higher than in anterior pituitary cells. FasL increased the percentage of TUNEL-positive GH3 cells incubated either with or without estradiol suggesting that the pro-apoptotic action of Fas activation is estrogen-independent. Our results show that unlike what happens in nontumoral pituitary cells, estrogens do not modulate either c-FLIPlong expression or FasL-induced apoptosis in GH3 cells. The stimulatory effect of estradiol on c-FLIPlong expression could be involved in the sensitizing effect of this steroid to apoptosis in anterior pituitary cells. The absence of this estrogenic action in tumor pituitary cells could be involved in their tumor-like behavior. PMID:26566102

  4. Pharmacologic suppression of target cell recognition by engineered T cells expressing chimeric T-cell receptors.

    PubMed

    Alvarez-Vallina, L; Yañez, R; Blanco, B; Gil, M; Russell, S J

    2000-04-01

    Adoptive therapy with autologous T cells expressing chimeric T-cell receptors (chTCRs) is of potential interest for the treatment of malignancy. To limit possible T-cell-mediated damage to normal tissues that weakly express the targeted tumor antigen (Ag), we have tested a strategy for the suppression of target cell recognition by engineered T cells. Jurkat T cells were transduced with an anti-hapten chTCR tinder the control of a tetracycline-suppressible promoter and were shown to respond to Ag-positive (hapten-coated) but not to Ag-negative target cells. The engineered T cells were then reacted with hapten-coated target cells at different effector to target cell ratios before and after exposure to tetracycline. When the engineered T cells were treated with tetracycline, expression of the chTCR was greatly decreased and recognition of the hapten-coated target cells was completely suppressed. Tetracycline-mediated suppression of target cell recognition by engineered T cells may be a useful strategy to limit the toxicity of the approach to cancer gene therapy. PMID:10811469

  5. The Cardiovascular Biology of Glucagon-like Peptide-1.

    PubMed

    Drucker, Daniel J

    2016-07-12

    Glucagon-like peptide-1, produced predominantly in enteroendocrine cells, controls glucose metabolism and energy homeostasis through regulation of islet hormone secretion, gastrointestinal motility, and food intake, enabling development of GLP-1 receptor (GLP-1R) agonists for the treatment of diabetes and obesity. GLP-1 also acts on the immune system to suppress inflammation, and GLP-1R signaling in multiple tissues impacts cardiovascular function in health and disease. Here we review how GLP-1 and clinically approved GLP-1R agonists engage mechanisms that influence the risk of developing cardiovascular disease. We discuss how GLP-1R agonists modify inflammation, cardiovascular physiology, and pathophysiology in normal and diabetic animals through direct and indirect mechanisms and review human studies illustrating mechanisms linking GLP-1R signaling to modification of the cardiovascular complications of diabetes. The risks and benefits of GLP-1R agonists are updated in light of recent data suggesting that GLP-1R agonists favorably modify outcomes in diabetic subjects at high risk for cardiovascular events. PMID:27345422

  6. TOX expression in cutaneous B-cell lymphomas.

    PubMed

    Schrader, Anne M R; Jansen, Patty M; Willemze, Rein

    2016-08-01

    Thymocyte selection-associated high-mobility group box (TOX) is aberrantly expressed in cutaneous T-cell lymphomas. In a recent study, TOX expression was noted unexpectedly in the follicle center (germinal center) B-cells of reactive lymph nodes and tonsils, used as external controls. To evaluate whether TOX is also expressed by cutaneous B-cell lymphomas, TOX immunohistochemistry was performed on skin biopsies of 44 patients with primary and secondary cutaneous B-cell proliferations. TOX was expressed not only in the reactive follicle center cells of lymph nodes, tonsils, cutaneous lymphoid hyperplasia, and primary cutaneous marginal zone lymphomas, but also by the neoplastic follicle center cells of 16/17 patients with primary cutaneous follicle center lymphoma (PCFCL) and 7/7 patients with cutaneous manifestations of systemic follicular lymphoma (FL). Notably, TOX showed a very similar expression pattern as BCL6, a marker of germinal center B-cells. In 4/10 patients with a BCL6(+) primary cutaneous diffuse large B-cell lymphoma, leg type (PCDLBCL,LT) and in 2/2 patients with a secondary cutaneous BCL6(+) diffuse large B-cell lymphoma (DLBCL), TOX was expressed by more than 50 % of the neoplastic B-cells. In contrast, in 3/3 BCL6(-) PCDLBCL,LT, TOX was completely negative or weakly expressed by a minor proportion of the neoplastic B-cells. In conclusion, TOX is expressed not only by neoplastic T-cells, but also by both reactive and neoplastic follicle center (germinal center) B-cells and a proportion of BCL6(+) PCDLBCL,LT and secondary cutaneous BCL6(+) DLBCL. The functional significance of TOX expression in reactive and neoplastic B-cells remains to be elucidated. PMID:27180090

  7. Mechanism of glucagon inhibition of liver acetyl-CoA carboxylase. Interrelationship of the effects of phosphorylation, polymer-protomer transition, and citrate on enzyme activity.

    PubMed

    Swenson, T L; Porter, J W

    1985-03-25

    The short-term regulation of rat liver acetyl-CoA carboxylase by glucagon has been studied in hepatocytes from rats that had been fasted and refed a fat-free diet. Glucagon inhibition of the activity of this enzyme can be accounted for by a direct correlation between phosphorylation, polymer-protomer ratio, and activity. Glucagon rapidly inactivates acetyl-CoA carboxylase with an accompanying 4-fold increase in the phosphorylation of the enzyme and 3-fold increase in the protomer-polymer ratio of enzyme protein. Citrate, an allosteric activator of acetyl-CoA carboxylase required for enzyme activity, has no effect on these phenomena, indicating a mechanism that is independent of citrate concentration within the cell. The observation of these effects of glucagon on acetyl-CoA carboxylase activity is absolutely dependent upon the minimization of proteolytic degradation of the enzyme after cell lysis. Therefore, for the first time, an interrelationship has been demonstrated between phosphorylation, protomer-polymer ratio, and citrate for the inactivation of acetyl-CoA carboxylase by glucagon. PMID:2857722

  8. Characteristics and EGFP expression of goat mammary gland epithelial cells.

    PubMed

    Zheng, Y-M; He, X-Y; Zhang, Y

    2010-12-01

    The aims of this study were (i) to establish a goat mammary gland epithelial (GMGE) cell line, and (ii) to determine if these GMGE cells could be maintained long-term in culture by continuous subculturing following transfection with a reporter gene, enhanced green fluorescence protein (EGFP). Primary culture of GMGE cells was achieved by outgrowth of migrating cells from the fragments of the mammary gland tissue of a lactating goat. The passage 16 GMGE cells were transfected with EGFP gene using lipofection. The expression of Cell keratins of epithelial cells in GMGE cells was test by immunofluorescence. Βeta-Casein gene mRNA was test for GMGE cells by RT-PCR. The results showed that when grown at low density on a plastic substratum, the GMGE cells formed islands, and when grown to confluency, the cells formed a monolayer and aggregated with the characteristic cobble-stone morphology of epithelial cells. GMGE cells could form dome-like structure which looked like nipple, and the lumen-like structures formed among the cells. Several blister-like structures appeared in the appearance of the cells. The GMGE cells contained different cell types, majority of the cells were short shuttle-like or polygon which were beehive-like. A part of cells were round and flat, a small number of cells were elongated. Some of the GMGE cells contained milk drops. The cell nuclei were round which had 2-4 obvious cores. The expression of Cell keratins demonstrated the property of epithelial cells in GMGE cells by immunofluorescence. The GMGE cells could express transcript encoding a Βeta-Casein protein. EGFP gene was successfully transferred into the GMGE cells, and the transfected cells could be maintained long-term in culture by continuous subculturing. In conclusion, we have established a EGFP gene transfected GMGE (ET-GMGE) cell line and maintained it long-term in culture by continuous subculturing. PMID:20113446

  9. Lack of Association Between Residual Insulin Production and Glucagon Response to Hypoglycemia in Youth With Short Duration of Type 1 Diabetes

    PubMed Central

    Sherr, Jennifer; Xing, Dongyuan; Ruedy, Katrina J.; Beck, Roy W.; Kollman, Craig; Buckingham, Bruce; White, Neil H.; Fox, Larry; Tsalikian, Eva; Weinzimer, Stuart; Arbelaez, Ana Maria; Tamborlane, William V.

    2013-01-01

    OBJECTIVE To examine the loss of glucagon response to hypoglycemia and its relationship with residual β-cell function early in the course of type 1 diabetes (T1D) in youth. RESEARCH DESIGN AND METHODS Twenty-one youth with T1D duration <1 year (ages 8–18 years, T1D duration 6–52 weeks) underwent mixed-meal tolerance tests (MMTTs) to assess residual β-cell function and hypoglycemic clamps to assess glucagon responses to hypoglycemia. Glucagon responses to hypoglycemia in T1D subjects were compared with those in 12 nondiabetic young adults (ages 19–25 years). RESULTS Peak MMTT-stimulated C-peptide levels (range 0.12–1.43) were ≥0.2 nmol/L in all but one T1D subject. As expected, the median of glucagon responses to hypoglycemia in the T1D subjects (18 pg/mL [interquartile range 7–32]) was significantly reduced compared with the responses in nondiabetic control subjects (38 pg/mL [19–66], P = 0.02). However, there was no correlation between the incremental increase in plasma glucagon during the hypoglycemic clamp and the incremental increase and peak plasma C-peptide level during the MMTT. Similarly, the seven T1D subjects who failed to achieve an increase in glucagon ≥12 pg/mL (i.e., 3 SD above baseline values) had C-peptide response ≥0.2 nmol/L (0.54–1.12), and the one T1D subject with peak stimulated <0.2 nmol/L had a 14 pg/mL increase in plasma glucagon in response to hypoglycemia. CONCLUSIONS Impaired plasma glucagon responses to hypoglycemia are evident in youth with T1D during the first year of the disease. Moreover, defective and absent glucagon responses to hypoglycemia were observed in patients who retained clinically important residual endogenous β-cell function. PMID:23288858

  10. Advantages and applications of CAR-expressing natural killer cells

    PubMed Central

    Glienke, Wolfgang; Esser, Ruth; Priesner, Christoph; Suerth, Julia D.; Schambach, Axel; Wels, Winfried S.; Grez, Manuel; Kloess, Stephan; Arseniev, Lubomir; Koehl, Ulrike

    2015-01-01

    In contrast to donor T cells, natural killer (NK) cells are known to mediate anti-cancer effects without the risk of inducing graft-versus-host disease (GvHD). In order to improve cytotoxicity against resistant cancer cells, auspicious efforts have been made with chimeric antigen receptor (CAR) expressing T- and NK cells. These CAR-modified cells express antigen receptors against tumor-associated surface antigens, thus redirecting the effector cells and enhancing tumor-specific immunosurveillance. However, many cancer antigens are also expressed on healthy tissues, potentially leading to off tumor/on target toxicity by CAR-engineered cells. In order to control such potentially severe side effects, the insertion of suicide genes into CAR-modified effectors can provide a means for efficient depletion of these cells. While CAR-expressing T cells have entered successfully clinical trials, experience with CAR-engineered NK cells is mainly restricted to pre-clinical investigations and predominantly to NK cell lines. In this review we summarize the data on CAR expressing NK cells focusing on the possible advantage using these short-lived effector cells and discuss the necessity of suicide switches. Furthermore, we address the compliance of such modified NK cells with regulatory requirements as a new field in cellular immunotherapy. PMID:25729364

  11. Transferrin receptor expression by stimulated cells in mixed lymphocyte culture.

    PubMed Central

    Salmon, M; Bacon, P A; Symmons, D P; Walton, K W

    1985-01-01

    Transferrin receptor (TRFr) expression by cells in mixed lymphocyte culture increases steadily for the first 5 days, but then reaches a plateau. By the sixth day in culture, about 20% of viable cells express TRFr in two-way mixed lymphocyte reactions. This subpopulation of TRFr-positive cells represents the proliferating population; it is heterogeneous, containing T-cell blasts and smaller cells which are a mixture of T and non-T cells. A small group of non-T cells have phenotypic similarity to natural killer (NK) cells. T cells appear to divide earlier in the course of the response than non-T cells. The biphasic nature of this response and the slower non-T reactivity may be due to a secondary stimulation of non-T cells by factors released from activated T cells (such as interleukin-2). PMID:2982734

  12. Robust Inference of Cell-to-Cell Expression Variations from Single- and K-Cell Profiling

    PubMed Central

    Narayanan, Manikandan; Martins, Andrew J.; Tsang, John S.

    2016-01-01

    Quantifying heterogeneity in gene expression among single cells can reveal information inaccessible to cell-population averaged measurements. However, the expression level of many genes in single cells fall below the detection limit of even the most sensitive technologies currently available. One proposed approach to overcome this challenge is to measure random pools of k cells (e.g., 10) to increase sensitivity, followed by computational “deconvolution” of cellular heterogeneity parameters (CHPs), such as the biological variance of single-cell expression levels. Existing approaches infer CHPs using either single-cell or k-cell data alone, and typically within a single population of cells. However, integrating both single- and k-cell data may reap additional benefits, and quantifying differences in CHPs across cell populations or conditions could reveal novel biological information. Here we present a Bayesian approach that can utilize single-cell, k-cell, or both simultaneously to infer CHPs within a single condition or their differences across two conditions. Using simulated as well as experimentally generated single- and k-cell data, we found situations where each data type would offer advantages, but using both together can improve precision and better reconcile CHP information contained in single- and k-cell data. We illustrate the utility of our approach by applying it to jointly generated single- and k-cell data to reveal CHP differences in several key inflammatory genes between resting and inflammatory cytokine-activated human macrophages, delineating differences in the distribution of ‘ON’ versus ‘OFF’ cells and in continuous variation of expression level among cells. Our approach thus offers a practical and robust framework to assess and compare cellular heterogeneity within and across biological conditions using modern multiplexed technologies. PMID:27438699

  13. Robust Inference of Cell-to-Cell Expression Variations from Single- and K-Cell Profiling.

    PubMed

    Narayanan, Manikandan; Martins, Andrew J; Tsang, John S

    2016-07-01

    Quantifying heterogeneity in gene expression among single cells can reveal information inaccessible to cell-population averaged measurements. However, the expression level of many genes in single cells fall below the detection limit of even the most sensitive technologies currently available. One proposed approach to overcome this challenge is to measure random pools of k cells (e.g., 10) to increase sensitivity, followed by computational "deconvolution" of cellular heterogeneity parameters (CHPs), such as the biological variance of single-cell expression levels. Existing approaches infer CHPs using either single-cell or k-cell data alone, and typically within a single population of cells. However, integrating both single- and k-cell data may reap additional benefits, and quantifying differences in CHPs across cell populations or conditions could reveal novel biological information. Here we present a Bayesian approach that can utilize single-cell, k-cell, or both simultaneously to infer CHPs within a single condition or their differences across two conditions. Using simulated as well as experimentally generated single- and k-cell data, we found situations where each data type would offer advantages, but using both together can improve precision and better reconcile CHP information contained in single- and k-cell data. We illustrate the utility of our approach by applying it to jointly generated single- and k-cell data to reveal CHP differences in several key inflammatory genes between resting and inflammatory cytokine-activated human macrophages, delineating differences in the distribution of 'ON' versus 'OFF' cells and in continuous variation of expression level among cells. Our approach thus offers a practical and robust framework to assess and compare cellular heterogeneity within and across biological conditions using modern multiplexed technologies. PMID:27438699

  14. Role of Central Glucagon-like Peptide-1 in Stress Regulation

    PubMed Central

    Ghosal, Sriparna; Myers, Brent; Herman, James P.

    2013-01-01

    Glucagon-like peptide 1 (GLP-1) is best known as an incretin hormone, secreted from L cells in the intestine in response to nutrient ingestion to stimulate glucose-dependent insulin secretion. However, GLP-1 is also expressed in neurons, and plays a major role in regulation of homeostatic function within the central nervous system (CNS). This review summarizes our current state of knowledge on the role GLP-1 plays in neural coordination of the organismal stress response. In brain, the primary locus of GLP-1 production is in the caudal nucleus of the solitary tract (NTS) and the ventrolateral medulla of the hindbrain. GLP-1 immunoreactive fibers directly innervate hypophysiotrophic corticotropin-releasing hormone (CRH) neurons in the hypothalamic paraventricular nucleus (PVN), placing GLP-1 in prime position to integrate hypothalamo-pituitary-adrenocortical responses. Exogenous central GLP-1 activates HPA axis stress responses, and responses to a variety of stressors can be blocked by a GLP-1 receptor (GLP-1R) antagonist, confirming an excitatory role in glucocorticoid secretion. In addition, central infusion of GLP-1R agonist increases heart rate and blood pressure, and activates hypothalamic and brainstem neurons innervating sympathetic preganglionic neurons, suggesting a sympathoexcitatory role of GLP-1 in the CNS. Bioavailability of preproglucagon (PPG) mRNA and GLP-1 peptide is reduced by exogenous or endogenous glucocorticoid secretion, perhaps as a mechanism to reduce GLP-1-mediated stress excitation. Altogether, the data suggest that GLP-1 plays a key role in activation of stress responses, which may be connected with its role in central regulation of energy homeostasis. PMID:23623992

  15. Molecular Characterisation of Small Molecule Agonists Effect on the Human Glucagon Like Peptide-1 Receptor Internalisation

    PubMed Central

    Thompson, Aiysha; Stephens, Jeffrey W.; Bain, Stephen C.

    2016-01-01

    The glucagon-like peptide receptor (GLP-1R), which is a G-protein coupled receptor (GPCR), signals through both Gαs and Gαq coupled pathways and ERK phosphorylation to stimulate insulin secretion. The aim of this study was to determine molecular details of the effect of small molecule agonists, compounds 2 and B, on GLP-1R mediated cAMP production, intracellular Ca2+ accumulation, ERK phosphorylation and its internalisation. In human GLP-1R (hGLP-1R) expressing cells, compounds 2 and B induced cAMP production but caused no intracellular Ca2+ accumulation, ERK phosphorylation or hGLP-1R internalisation. GLP-1 antagonists Ex(9–39) and JANT-4 and the orthosteric binding site mutation (V36A) in hGLP-1R failed to inhibit compounds 2 and B induced cAMP production, confirming that their binding site distinct from the GLP-1 binding site on GLP-1R. However, K334A mutation of hGLP-1R, which affects Gαs coupling, inhibited GLP-1 as well as compounds 2 and B induced cAMP production, indicating that GLP-1, compounds 2 and B binding induce similar conformational changes in the GLP-1R for Gαs coupling. Additionally, compound 2 or B binding to the hGLP-1R had significantly reduced GLP-1 induced intracellular Ca2+ accumulation, ERK phosphorylation and hGLP-1R internalisation. This study illustrates pharmacology of differential activation of GLP-1R by GLP-1 and compounds 2 and B. PMID:27100083

  16. Chemokine receptor expression by inflammatory T cells in EAE

    PubMed Central

    Mony, Jyothi Thyagabhavan; Khorooshi, Reza; Owens, Trevor

    2014-01-01

    Chemokines direct cellular infiltration to tissues, and their receptors and signaling pathways represent targets for therapy in diseases such as multiple sclerosis (MS). The chemokine CCL20 is expressed in choroid plexus, a site of entry of T cells to the central nervous system (CNS). The CCL20 receptor CCR6 has been reported to be selectively expressed by CD4+ T cells that produce the cytokine IL-17 (Th17 cells). Th17 cells and interferon-gamma (IFNγ)-producing Th1 cells are implicated in induction of MS and its animal model experimental autoimmune encephalomyelitis (EAE). We have assessed whether CCR6 identifies specific inflammatory T cell subsets in EAE. Our approach was to induce EAE, and then examine chemokine receptor expression by cytokine-producing T cells sorted from CNS at peak disease. About 7% of CNS-infiltrating CD4+ T cells produced IFNγ in flow cytometric cytokine assays, whereas less than 1% produced IL-17. About 1% of CD4+ T cells produced both cytokines. CCR6 was expressed by Th1, Th1+17 and by Th17 cells, but not by CD8+ T cells. CD8+ T cells expressed CXCR3, which was also expressed by CD4+ T cells, with no correlation to cytokine profile. Messenger RNA for IFNγ, IL-17A, and the Th1 and Th17-associated transcription factors T-bet and RORγt was detected in both CCR6+ and CXCR3+ CD4+ T cells. IFNγ, but not IL-17A mRNA expression was detected in CD8+ T cells in CNS. CCR6 and CD4 were co-localized in spinal cord infiltrates by double immunofluorescence. Consistent with flow cytometry data some but not all CD4+ T cells expressed CCR6 within infiltrates. CD4-negative CCR6+ cells included macrophage/microglial cells. Thus we have for the first time directly studied CD4+ and CD8+ T cells in the CNS of mice with peak EAE, and determined IFNγ and IL17 expression by cells expressing CCR6 and CXCR3. We show that neither CCR6 or CXCR3 align with CD4 T cell subsets, and Th1 or mixed Th1+17 predominate in EAE. PMID:25071447

  17. [The physiology of glucagon-like peptide-1 and its role in the pathophysiology of type 2 diabetes mellitus].

    PubMed

    Escalada, Francisco Javier

    2014-01-01

    The hormone glucagon-like peptide-1 (GLP-1) is synthesized and secreted by L cells in the small intestine in response to food ingestion. After reaching the general circulation it has a half-life of 2-3 minutes due to degradation by the enzyme dipeptidyl peptidase-4. Its physiological role is directed to control plasma glucose concentration, though GLP-1 also plays other different metabolic functions following nutrient absorption. Biological activities of GLP-1 include stimulation of insulin biosynthesis and glucose-dependent insulin secretion by pancreatic beta cell, inhibition of glucagon secretion, delay of gastric emptying and inhibition of food intake. GLP-1 is able to reduce plasma glucose levels in patients with type 2 diabetes and also can restore beta cell sensitivity to exogenous secretagogues, suggesting that the increasing GLP-1 concentration may be an useful therapeutic strategy for the treatment of patients with type 2 diabetes. PMID:25326836

  18. [The physiology of glucagon-like peptide-1 and its role in the pathophysiology of type 2 diabetes mellitus].

    PubMed

    Escalada, Francisco Javier

    2014-09-01

    The hormone glucagon-like peptide-1 (GLP-1) is synthesized and secreted by L cells in the small intestine in response to food ingestion. After reaching the general circulation it has a half-life of 2-3 minutes due to degradation by the enzyme dipeptidyl peptidase-4. Its physiological role is directed to control plasma glucose concentration, though GLP-1 also plays other different metabolic functions following nutrient absorption. Biological activities of GLP-1 include stimulation of insulin biosynthesis and glucose-dependent insulin secretion by pancreatic beta cell, inhibition of glucagon secretion, delay of gastric emptying and inhibition of food intake. GLP-1 is able to reduce plasma glucose levels in patients with type 2 diabetes and also can restore beta cell sensitivity to exogenous secretagogues, suggesting that the increasing GLP-1 concentration may be an useful therapeutic strategy for the treatment of patients with type 2 diabetes. PMID:25437458

  19. Geometry of the Gene Expression Space of Individual Cells

    PubMed Central

    Korem, Yael; Szekely, Pablo; Hart, Yuval; Sheftel, Hila; Hausser, Jean; Mayo, Avi; Rothenberg, Michael E.; Kalisky, Tomer; Alon, Uri

    2015-01-01

    There is a revolution in the ability to analyze gene expression of single cells in a tissue. To understand this data we must comprehend how cells are distributed in a high-dimensional gene expression space. One open question is whether cell types form discrete clusters or whether gene expression forms a continuum of states. If such a continuum exists, what is its geometry? Recent theory on evolutionary trade-offs suggests that cells that need to perform multiple tasks are arranged in a polygon or polyhedron (line, triangle, tetrahedron and so on, generally called polytopes) in gene expression space, whose vertices are the expression profiles optimal for each task. Here, we analyze single-cell data from human and mouse tissues profiled using a variety of single-cell technologies. We fit the data to shapes with different numbers of vertices, compute their statistical significance, and infer their tasks. We find cases in which single cells fill out a continuum of expression states within a polyhedron. This occurs in intestinal progenitor cells, which fill out a tetrahedron in gene expression space. The four vertices of this tetrahedron are each enriched with genes for a specific task related to stemness and early differentiation. A polyhedral continuum of states is also found in spleen dendritic cells, known to perform multiple immune tasks: cells fill out a tetrahedron whose vertices correspond to key tasks related to maturation, pathogen sensing and communication with lymphocytes. A mixture of continuum-like distributions and discrete clusters is found in other cell types, including bone marrow and differentiated intestinal crypt cells. This approach can be used to understand the geometry and biological tasks of a wide range of single-cell datasets. The present results suggest that the concept of cell type may be expanded. In addition to discreet clusters in gene-expression space, we suggest a new possibility: a continuum of states within a polyhedron, in which the

  20. Geometry of the Gene Expression Space of Individual Cells.

    PubMed

    Korem, Yael; Szekely, Pablo; Hart, Yuval; Sheftel, Hila; Hausser, Jean; Mayo, Avi; Rothenberg, Michael E; Kalisky, Tomer; Alon, Uri

    2015-07-01

    There is a revolution in the ability to analyze gene expression of single cells in a tissue. To understand this data we must comprehend how cells are distributed in a high-dimensional gene expression space. One open question is whether cell types form discrete clusters or whether gene expression forms a continuum of states. If such a continuum exists, what is its geometry? Recent theory on evolutionary trade-offs suggests that cells that need to perform multiple tasks are arranged in a polygon or polyhedron (line, triangle, tetrahedron and so on, generally called polytopes) in gene expression space, whose vertices are the expression profiles optimal for each task. Here, we analyze single-cell data from human and mouse tissues profiled using a variety of single-cell technologies. We fit the data to shapes with different numbers of vertices, compute their statistical significance, and infer their tasks. We find cases in which single cells fill out a continuum of expression states within a polyhedron. This occurs in intestinal progenitor cells, which fill out a tetrahedron in gene expression space. The four vertices of this tetrahedron are each enriched with genes for a specific task related to stemness and early differentiation. A polyhedral continuum of states is also found in spleen dendritic cells, known to perform multiple immune tasks: cells fill out a tetrahedron whose vertices correspond to key tasks related to maturation, pathogen sensing and communication with lymphocytes. A mixture of continuum-like distributions and discrete clusters is found in other cell types, including bone marrow and differentiated intestinal crypt cells. This approach can be used to understand the geometry and biological tasks of a wide range of single-cell datasets. The present results suggest that the concept of cell type may be expanded. In addition to discreet clusters in gene-expression space, we suggest a new possibility: a continuum of states within a polyhedron, in which the

  1. Fragments of Target Cells are Internalized into Retroviral Envelope Protein-Expressing Cells during Cell-Cell Fusion by Endocytosis

    PubMed Central

    Izumida, Mai; Kamiyama, Haruka; Suematsu, Takashi; Honda, Eri; Koizumi, Yosuke; Yasui, Kiyoshi; Hayashi, Hideki; Ariyoshi, Koya; Kubo, Yoshinao

    2016-01-01

    Retroviruses enter into host cells by fusion between viral and host cell membranes. Retroviral envelope glycoprotein (Env) induces the membrane fusion, and also mediates cell-cell fusion. There are two types of cell-cell fusions induced by the Env protein. Fusion-from-within is induced by fusion between viral fusogenic Env protein-expressing cells and susceptible cells, and virions induce fusion-from-without by fusion between adjacent cells. Although entry of ecotropic murine leukemia virus (E-MLV) requires host cell endocytosis, the involvement of endocytosis in cell fusion is unclear. By fluorescent microscopic analysis of the fusion-from-within, we found that fragments of target cells are internalized into Env-expressing cells. Treatment of the Env-expressing cells with an endocytosis inhibitor more significantly inhibited the cell fusion than that of the target cells, indicating that endocytosis in Env-expressing cells is required for the cell fusion. The endocytosis inhibitor also attenuated the fusion-from-without. Electron microscopic analysis suggested that the membrane fusion resulting in fusion-from-within initiates in endocytic membrane dents. This study shows that two types of the viral cell fusion both require endocytosis, and provides the cascade of fusion-from-within. PMID:26834711

  2. Characteristics and EGFP expression of porcine mammary gland epithelial cells.

    PubMed

    Zheng, Yue-Mao; He, Xiao-Ying

    2010-12-01

    The aims of this study were to establish a porcine mammary gland epithelial (PMGE) cell line, and to determine if these PMGE cells could be maintained long-term in culture by continuous subculturing following transfection with a reporter gene, enhanced green fluorescence protein (EGFP). Primary culture of PMGE cells was achieved by outgrowth of migrating cells from the fragments of the mammary gland tissue of a lactating pig. The passage sixteen PMGE cells were transfected with EGFP gene using lipofection. The expression of Cell keratins of epithelial cells in PMGE cells was tested by immunofluorescence. Βeta-Casein gene mRNA was tested for PMGE cells by RT-PCR. The results showed that PMGE cells could form dome-like structure which looked like nipple, and the cells contained different cell types. The expression of Cell keratins demonstrated the property of epithelial cells, and the PMGE cells could express transcript encoding a Βeta-Casein protein. EGFP gene was successfully transferred into the PMGE cells, and the transfected cells could be maintained long-term in culture by continuous subculturing. In conclusion, we have established a EGFP gene transfected porcine mammary gland epithelial (ET-PMGE) cell line. PMID:20400167

  3. Glucagon increases hepatic oxygen supply-demand ratio in pigs

    SciTech Connect

    Gelman, S.; Dillard, E.; Parks, D.A.

    1987-05-01

    The present study was performed on eight young pigs to test the hypothesis that glucagon increases hepatic oxygen supply to a greater extent than hepatic oxygen uptake, providing a better hepatic oxygen supply-demand relationship. The experiments were performed under pentobarbital sodium anesthesia and controlled ventilation. Splanchnic blood flow was studied using radioactive microspheres. Glucagon was administered in doses of 1 and 5 ..mu..g x kg/sup -1/ x min/sup -1/. During glucagon infusion, hepatic arterial blood flow substantially increased, splenic and pancreatic blood flows increased moderately, while stomach and intestinal blood flows, as well as portal blood flow did not change significantly. Shunting of both 9- and 15-..mu..m spheres through preportal tissues did not change significantly. Oxygen content in arterial or portal venous blood did not change significantly, while it increased in hepatic venous blood by 30%. There were no differences in the effects between the doses of glucagon administered. There was no correlation found between changes in hepatic oxygen supply and cardiac output or blood pressure. The changes observed during glucagon administration resulted in an increase in oxygen delivery to the liver and hepatic oxygen supply-uptake ratio.

  4. HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins.

    PubMed

    Yadavalli, Raghavendra; Sam-Yellowe, Tobili

    2015-01-01

    Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is the challenge of identifying suitable vaccine candidates. Malarial proteins expressed using prokaryotic and eukaryotic cell based expression systems are poorly glycosylated, generally insoluble and undergo improper folding leading to reduced immunogenicity. The wheat germ, rabbit reticulocyte lysate and Escherichia coli lysate cell free expression systems are currently used for expression of malarial proteins. However, the length of expression time and improper glycosylation of proteins still remains a challenge. We demonstrate expression of Plasmodium proteins in vitro using HeLa based cell free expression systems, termed "in vitro human cell free expression systems". The 2 HeLa based cell free expression systems transcribe mRNA in 75 min and 3 µl of transcribed mRNA is sufficient to translate proteins in 90 min. The 1-step expression system is a transcription and translation coupled expression system; the transcription and co-translation occurs in 3 hr. The process can also be extended for 6 hr by providing additional energy. In the 2-step expression system, mRNA is first transcribed and then added to the translation mix for protein expression. We describe how to express malaria proteins; a hydrophobic PF3D7_0114100 Maurer's Cleft - 2 transmembrane (PfMC-2TM) protein, a hydrophilic PF3D7_0925900 protein and an armadillo repeats containing protein PF3D7_1361800, using the HeLa based cell free expression system. The proteins are expressed in micro volumes employing 2-step and 1-step expression strategies. An affinity purification method to purify 25 µl of proteins expressed using the in vitro human cell free expression system is also described. Protein yield is determined by Bradford's assay and the expressed and purified proteins can be confirmed by western blotting analysis. Expressed recombinant proteins can be

  5. Glucagon-Induced Vasospasm of Hepatic Artery Branches During Visceral Angiography

    SciTech Connect

    Dziedzic, T. Scott; Smith, Tony P.

    2008-07-15

    Glucagon is often used in radiology to decrease bowel motility for enhanced imaging, including visceral digital subtraction angiography. We present a case in which branch hepatic artery vasospasm followed the intravenous administration of glucagon during visceral angiography.

  6. Etoposide sensitizes neuroblastoma cells expressing caspase 8 to TRAIL.

    PubMed

    Kim, Hye Ryung; Lee, Myoung Woo; Kim, Dae Seong; Jo, Ha Yeong; Lee, Soo Hyun; Chueh, Hee Won; Jung, Hye Lim; Yoo, Keon Hee; Sung, Ki Woong; Koo, Hong Hoe

    2012-01-01

    TRAIL [TNF (tumour necrosis factor)-related apoptosis-inducing ligand] is a promising agent for clinical use since it kills a wide range of tumour cells without affecting normal cells. We provide evidence that pretreatment with etoposide significantly enhanced TRAIL-mediated apoptosis via up-regulation of DR5 (death receptor 5 or TRAIL-R2) expression in the caspase 8 expressing neuroblastoma cell line, SK-N-MC. In addition, sequential treatment with etoposide and TRAIL increased caspases 8, 9 and 3 activation, Mcl-1 cleavage and Bid truncation, which suggests that the ability of etoposide and TRAIL to induce apoptosis is mediated through activation of an intrinsic signalling pathway. Although TRAIL-R2 expression increased in IMR-32 cells in response to etoposide treatment, cell death was not increased by concurrent treatment with TRAIL compared with etoposide alone, because the cells lacked caspase 8 expression. Restoration of caspase 8 expression by exposure to IFNγ (interferon γ) sensitizes IMR-32 cells to TRAIL. Moreover, pretreatment with etoposide increased TRAIL-induced apoptosis in caspase 8 restored IMR-32 cells through activation of a caspase cascade that included caspases 8, 9 and 3. These results indicate that the etoposide-mediated sensitization of neuroblastoma cells to TRAIL is associated with an increase in TRAIL-R2 expression and requires caspase 8 expression. These observations support the potential use of a combination of etoposide and TRAIL in future clinical trials. PMID:23124518

  7. Effects of adenosine, adenosine triphosphate and structural analogues on glucagon secretion from the perfused pancreas of rat in vitro.

    PubMed Central

    Chapal, J.; Loubatières-Mariani, M. M.; Roye, M.; Zerbib, A.

    1984-01-01

    The effects of adenosine, adenosine triphosphate (ATP) and structural analogues have been studied on glucagon secretion from the isolated perfused pancreas of the rat in the presence of glucose (2.8 mM). Adenosine induced a transient increase of glucagon secretion. This effect was concentration-dependent in the range of 0.165 to 165 microM. ATP also induced an increase, but the effect was no greater at 165 microM than at 16.5 microM. 2-Chloroadenosine, an analogue more resistant to metabolism or uptake systems than adenosine, was more effective. Among the three structural analogues of ATP or ADP studied, beta, gamma-methylene ATP which can be hydrolyzed into AMP and adenosine had an effect similar to adenosine or ATP at the same concentrations (1.65 and 16.5 microM); in contrast alpha, beta-methylene ATP and alpha, beta-methylene ADP (resistant to hydrolysis into AMP and adenosine) were ineffective. Theophylline (50 microM) a specific blocker of the adenosine receptor, suppressed the glucagon peak induced by adenosine, 2-chloroadenosine, ATP and beta, gamma-methylene ATP (1.65 microM). An inhibitor of 5' nucleotidase, alpha, beta-methylene ADP (16.5 microM), reduced the glucagon increase induced by ATP and did not affect the response to adenosine (1.65 microM). These results support the hypothesis of adenosine receptors (P1-purinoceptors) on the pancreatic glucagon secretory cells and indicate that ATP acts after hydrolysis to adenosine. PMID:6097328

  8. Dibutyl Phthalate Exposure Disrupts Evolutionarily Conserved Insulin and Glucagon-Like Signaling in Drosophila Males.

    PubMed

    Williams, Michael J; Wiemerslage, Lyle; Gohel, Priya; Kheder, Sania; Kothegala, Lakshmi V; Schiöth, Helgi B

    2016-06-01

    Phthalate diesters are commonly used as industrial plasticisers, as well as in cosmetics and skin care products, as a result people are constantly exposed to these xenobiotics. Recent epidemiological studies have found a correlation between circulating phthalate levels and type 2 diabetes, whereas animal studies indicate that phthalates are capable of disrupting endocrine signaling. Nonetheless, how phthalates interfere with metabolic function is still unclear. Here, we show that feeding Drosophila males the xenobiotic dibutyl phthalate (DBP) affects conserved insulin- and glucagon-like signaling. We report that raising flies on food containing DBP leads to starvation resistance, increased lipid storage, hyperglycemia, and hyperphagia. We go on to show that the starvation-resistance phenotype can be rescued by overexpression of the glucagon analogue adipokinetic hormone (Akh). Furthermore, although acute DBP exposure in adult flies is able to affect insulin levels, only chronic feeding influences Akh expression. We establish that raising flies on DBP-containing food or feeding adults DBP food affects the expression of homologous genes involved in xenobiotic and lipid metabolism (AHR [Drosophila ss], NR1I2 [Hr96], ABCB1 [MDR50], ABCC3 [MRP], and CYP3A4 [Cyp9f2]). Finally, we determined that the expression of these genes is also influenced by Akh. Our results provide comprehensive evidence that DBP can disrupt metabolism in Drosophila males, by regulating genes involved in glucose, lipid, and xenobiotic metabolism. PMID:27100621

  9. Differential expression and function of CD27 in chronic lymphocytic leukemia cells expressing ZAP-70.

    PubMed

    Lafarge, Sandrine T; Hou, Sen; Pauls, Samantha D; Johnston, James B; Gibson, Spencer B; Marshall, Aaron J

    2015-07-01

    Chronic lymphocytic leukemia is a malignancy driven by abberant B cell signaling and survival. Leukemic B cells accumulate in the peripheral blood and the lymphoid organs where contact with stromal cells and T cells provide critical survival signals. Clinical severity of CLL is associated with several prognostic markers including expression of the kinase ZAP-70. ZAP-70 expression enhances signaling via the B cell antigen receptor and is associated with increased cell adhesion and migration capacity. Here we report that ZAP-70-positive CLL patients display significantly higher expression of the TNF superfamily receptor and memory marker CD27 than do ZAP-70 negative patients. CD27 expression by CLL was acutely elevated upon BCR cross-linking, or upon ectopic expression of ZAP-70. CD27 expression correlated with functional capacity to adhere to stromal cells and antibody blockade of CD27 impaired CLL binding to stroma. These results provide the first evidence for differential expression of CD27 among CLL prognostic groups, suggest a role for ZAP-70 dependent signaling in CD27 induction and implicate CD27 in cell-cell interactions with the lymphoid tissue microenvironment. PMID:26002513

  10. Chronic anemic hypoxemia increases plasma glucagon and hepatic PCK1 mRNA in late-gestation fetal sheep.

    PubMed

    Culpepper, Christine; Wesolowski, Stephanie R; Benjamin, Joshua; Bruce, Jennifer L; Brown, Laura D; Jonker, Sonnet S; Wilkening, Randall B; Hay, William W; Rozance, Paul J

    2016-07-01

    Hepatic glucose production (HGP) normally begins just prior to birth. Prolonged fetal hypoglycemia, intrauterine growth restriction, and acute hypoxemia produce an early activation of fetal HGP. To test the hypothesis that prolonged hypoxemia increases factors which regulate HGP, studies were performed in fetuses that were bled to anemic conditions (anemic: n = 11) for 8.9 ± 0.4 days and compared with control fetuses (n = 7). Fetal arterial hematocrit and oxygen content were 32% and 50% lower, respectively, in anemic vs. controls (P < 0.005). Arterial plasma glucose was 15% higher in the anemic group (P < 0.05). Hepatic mRNA expression of phosphonenolpyruvate carboxykinase (PCK1) was twofold higher in the anemic group (P < 0.05). Arterial plasma glucagon concentrations were 70% higher in anemic fetuses compared with controls (P < 0.05), and they were positively associated with hepatic PCK1 mRNA expression (P < 0.05). Arterial plasma cortisol concentrations increased 90% in the anemic fetuses (P < 0.05), but fetal cortisol concentrations were not correlated with hepatic PCK1 mRNA expression. Hepatic glycogen content was 30% lower in anemic vs. control fetuses (P < 0.05) and was inversely correlated with fetal arterial plasma glucagon concentrations. In isolated primary fetal sheep hepatocytes, incubation in low oxygen (3%) increased PCK1 mRNA threefold compared with incubation in normal oxygen (21%). Together, these results demonstrate that glucagon and PCK1 may potentiate fetal HGP during chronic fetal anemic hypoxemia. PMID:27170658

  11. Calreticulin: Roles in Cell-Surface Protein Expression

    PubMed Central

    Jiang, Yue; Dey, Sandeepa; Matsunami, Hiroaki

    2014-01-01

    In order to perform their designated functions, proteins require precise subcellular localizations. For cell-surface proteins, such as receptors and channels, they are able to transduce signals only when properly targeted to the cell membrane. Calreticulin is a multi-functional chaperone protein involved in protein folding, maturation, and trafficking. However, evidence has been accumulating that calreticulin can also negatively regulate the surface expression of certain receptors and channels. In these instances, depletion of calreticulin enhances cell-surface expression and function. In this review, we discuss the role of calreticulin with a focus on its negative effects on the expression of cell-surface proteins. PMID:25230046

  12. CD39 Expression Identifies Terminally Exhausted CD8+ T Cells

    PubMed Central

    Adland, Emily; Yates, Kathleen; Pauken, Kristen E.; Cosgrove, Cormac; Ledderose, Carola; Junger, Wolfgang G.; Robson, Simon C.; Wherry, E. John; Alter, Galit; Goulder, Philip J. R.; Klenerman, Paul; Sharpe, Arlene H.; Lauer, Georg M.; Haining, W. Nicholas

    2015-01-01

    Exhausted T cells express multiple co-inhibitory molecules that impair their function and limit immunity to chronic viral infection. Defining novel markers of exhaustion is important both for identifying and potentially reversing T cell exhaustion. Herein, we show that the ectonucleotidse CD39 is a marker of exhausted CD8+ T cells. CD8+ T cells specific for HCV or HIV express high levels of CD39, but those specific for EBV and CMV do not. CD39 expressed by CD8+ T cells in chronic infection is enzymatically active, co-expressed with PD-1, marks cells with a transcriptional signature of T cell exhaustion and correlates with viral load in HIV and HCV. In the mouse model of chronic Lymphocytic Choriomeningitis Virus infection, virus-specific CD8+ T cells contain a population of CD39high CD8+ T cells that is absent in functional memory cells elicited by acute infection. This CD39high CD8+ T cell population is enriched for cells with the phenotypic and functional profile of terminal exhaustion. These findings provide a new marker of T cell exhaustion, and implicate the purinergic pathway in the regulation of T cell exhaustion. PMID:26485519

  13. Studies in transgenic mice reveal potential relationships between secretin-producing cells and other endocrine cell types.

    PubMed

    Lopez, M J; Upchurch, B H; Rindi, G; Leiter, A B

    1995-01-13

    We have produced transgenic mice expressing fusion genes consisting of 1.6 kilobase pairs of the secretin gene 5' flanking region to direct the expression of human growth hormone (hGH) or simian virus 40 large T antigen to secretin-producing cells. Analysis of different mouse tissues for hGH transcripts revealed expression in each of the major secretin-producing tissues, namely the intestine and endocrine pancrease. Multiple label immunohistochemistry demonstrated that the transgene was correctly directed to secretin cells in the intestinal tract, including a previously unrecognized population of secretin cells in the colon of adult and developing mice. In the small intestine, subpopulations of hGH-containing cells frequently coexpressed substance P, serotonin, and cholecystokinin, whereas in the colon, cells expressing hGH frequently coexpressed glucagon, peptide YY, or neurotensin. Transgenic mice expressing large T antigen in secretin cells developed poorly differentiated neuroendocrine tumors of the small intestine, well differentiated colonic tumors containing glucagon-expressing cells, and insulin-producing tumors in pancreas. These studies indicate that the major cis-regulatory sequences necessary for secretin expression in enteroendocrine cells and fetal islets are localized with 1.6 kilobase pairs of the transcriptional start site. Coexpression of reporter transgenes with several gastrointestinal hormones suggests a potential relationships between secretin cells and other enteroendocrine cell types, as well as pancreatic beta cells. PMID:7822327

  14. Determining Physical Mechanisms of Gene Expression Regulation from Single Cell Gene Expression Data.

    PubMed

    Ezer, Daphne; Moignard, Victoria; Göttgens, Berthold; Adryan, Boris

    2016-08-01

    Many genes are expressed in bursts, which can contribute to cell-to-cell heterogeneity. It is now possible to measure this heterogeneity with high throughput single cell gene expression assays (single cell qPCR and RNA-seq). These experimental approaches generate gene expression distributions which can be used to estimate the kinetic parameters of gene expression bursting, namely the rate that genes turn on, the rate that genes turn off, and the rate of transcription. We construct a complete pipeline for the analysis of single cell qPCR data that uses the mathematics behind bursty expression to develop more accurate and robust algorithms for analyzing the origin of heterogeneity in experimental samples, specifically an algorithm for clustering cells by their bursting behavior (Simulated Annealing for Bursty Expression Clustering, SABEC) and a statistical tool for comparing the kinetic parameters of bursty expression across populations of cells (Estimation of Parameter changes in Kinetics, EPiK). We applied these methods to hematopoiesis, including a new single cell dataset in which transcription factors (TFs) involved in the earliest branchpoint of blood differentiation were individually up- and down-regulated. We could identify two unique sub-populations within a seemingly homogenous group of hematopoietic stem cells. In addition, we could predict regulatory mechanisms controlling the expression levels of eighteen key hematopoietic transcription factors throughout differentiation. Detailed information about gene regulatory mechanisms can therefore be obtained simply from high throughput single cell gene expression data, which should be widely applicable given the rapid expansion of single cell genomics. PMID:27551778

  15. Determining Physical Mechanisms of Gene Expression Regulation from Single Cell Gene Expression Data

    PubMed Central

    Moignard, Victoria; Göttgens, Berthold; Adryan, Boris

    2016-01-01

    Many genes are expressed in bursts, which can contribute to cell-to-cell heterogeneity. It is now possible to measure this heterogeneity with high throughput single cell gene expression assays (single cell qPCR and RNA-seq). These experimental approaches generate gene expression distributions which can be used to estimate the kinetic parameters of gene expression bursting, namely the rate that genes turn on, the rate that genes turn off, and the rate of transcription. We construct a complete pipeline for the analysis of single cell qPCR data that uses the mathematics behind bursty expression to develop more accurate and robust algorithms for analyzing the origin of heterogeneity in experimental samples, specifically an algorithm for clustering cells by their bursting behavior (Simulated Annealing for Bursty Expression Clustering, SABEC) and a statistical tool for comparing the kinetic parameters of bursty expression across populations of cells (Estimation of Parameter changes in Kinetics, EPiK). We applied these methods to hematopoiesis, including a new single cell dataset in which transcription factors (TFs) involved in the earliest branchpoint of blood differentiation were individually up- and down-regulated. We could identify two unique sub-populations within a seemingly homogenous group of hematopoietic stem cells. In addition, we could predict regulatory mechanisms controlling the expression levels of eighteen key hematopoietic transcription factors throughout differentiation. Detailed information about gene regulatory mechanisms can therefore be obtained simply from high throughput single cell gene expression data, which should be widely applicable given the rapid expansion of single cell genomics. PMID:27551778

  16. Stochasticity in gene expression in a cell-sized compartment.

    PubMed

    Nishimura, Kazuya; Tsuru, Saburo; Suzuki, Hiroaki; Yomo, Tetsuya

    2015-05-15

    The gene expression in a clonal cell population fluctuates significantly, and its relevance to various cellular functions is under intensive debate. A fundamental question is whether the fluctuation is a consequence of the complexity and redundancy in living cells or an inevitable attribute of the minute microreactor nature of cells. To answer this question, we constructed an artificial cell, which consists of only necessary components for the gene expression (in vitro transcription and translation system) and its boundary as a microreactor (cell-sized lipid vesicle), and investigated the gene expression noise. The variation in the expression of two fluorescent proteins was decomposed into the components that were correlated and uncorrelated between the two proteins using a method similar to the one used by Elowitz and co-workers to analyze the expression noise in E. coli. The observed fluctuation was compared with a theoretical model that expresses the amplitude of noise as a function of the average number of intermediate molecules and products. With the assumption that the transcripts are partly active, the theoretical model was able to well describe the noise in the artificial system. Furthermore, the same measurement for E. coli cells harboring an identical plasmid revealed that the E. coli exhibited a similar level of expression noise. Our results demonstrated that the level of fluctuation found in bacterial cells is mostly an intrinsic property that arises even in a primitive form of the cell. PMID:25280237

  17. CARD14 Expression in Dermal Endothelial Cells in Psoriasis

    PubMed Central

    Harden, Jamie L.; Lewis, Steven M.; Pierson, Katherine C.; Suárez-Fariñas, Mayte; Lentini, Tim; Ortenzio, Francesca S.; Zaba, Lisa C.; Goldbach-Mansky, Raphaela; Bowcock, Anne M.; Lowes, Michelle A.

    2014-01-01

    Mutations in the caspase recruitment domain, family member 14 (CARD14) gene have recently been described in psoriasis patients, and explain the psoriasis susceptibility locus 2 (PSORS2). CARD14 is a scaffolding protein that regulates NF-κB activation, and psoriasis-associated CARD14 mutations lead to enhanced NF-κB signaling. CARD14 is expressed mainly in epidermal keratinocytes, but also in unidentified dermal cells. In this manuscript, the identity of the dermal cell types expressing CARD14, as well the potential functional consequence of overactive CARD14 in these dermal cell types, was determined. Using two-color immunofluorescence, dermal CARD14 did not co-localize with T-cells, dendritic cells, or macrophages. However, dermal CARD14 did highly co-localize with CD31+ endothelial cells (ECs). CARD14 was also expressed non-dermal endothelial cells, such as aortic endothelial cells, which may indicate a role of CARD14+ECs in the systemic inflammation and cardiovascular comorbidities associated with psoriasis. Additionally, phosphorylated NF-κB was found in psoriatic CARD14+ CD31+ ECs, demonstrating this pathway is active in dermal ECs in psoriasis. Transfection of dermal ECs with psoriasis-associated CARD14 mutations resulted in increased expression of several chemokines, including CXCL10, IL-8, and CCL2. These results provide preliminary evidence that CARD14 expression in ECs may contribute to psoriasis through increased expression of chemokines and facilitating recruitment of immune cells into skin. PMID:25369198

  18. CARD14 expression in dermal endothelial cells in psoriasis.

    PubMed

    Harden, Jamie L; Lewis, Steven M; Pierson, Katherine C; Suárez-Fariñas, Mayte; Lentini, Tim; Ortenzio, Francesca S; Zaba, Lisa C; Goldbach-Mansky, Raphaela; Bowcock, Anne M; Lowes, Michelle A

    2014-01-01

    Mutations in the caspase recruitment domain, family member 14 (CARD14) gene have recently been described in psoriasis patients, and explain the psoriasis susceptibility locus 2 (PSORS2). CARD14 is a scaffolding protein that regulates NF-κB activation, and psoriasis-associated CARD14 mutations lead to enhanced NF-κB signaling. CARD14 is expressed mainly in epidermal keratinocytes, but also in unidentified dermal cells. In this manuscript, the identity of the dermal cell types expressing CARD14, as well the potential functional consequence of overactive CARD14 in these dermal cell types, was determined. Using two-color immunofluorescence, dermal CARD14 did not co-localize with T-cells, dendritic cells, or macrophages. However, dermal CARD14 did highly co-localize with CD31(+) endothelial cells (ECs). CARD14 was also expressed non-dermal endothelial cells, such as aortic endothelial cells, which may indicate a role of CARD14(+)ECs in the systemic inflammation and cardiovascular comorbidities associated with psoriasis. Additionally, phosphorylated NF-κB was found in psoriatic CARD14(+) CD31(+) ECs, demonstrating this pathway is active in dermal ECs in psoriasis. Transfection of dermal ECs with psoriasis-associated CARD14 mutations resulted in increased expression of several chemokines, including CXCL10, IL-8, and CCL2. These results provide preliminary evidence that CARD14 expression in ECs may contribute to psoriasis through increased expression of chemokines and facilitating recruitment of immune cells into skin. PMID:25369198

  19. CD40 expression in Wehi-164 cell line

    PubMed Central

    Ebadi, Padideh; Pourfathollah, Ali Akbar; Soheili, Zahra Soheila; Moazzeni, Seyed Mohammad

    2010-01-01

    CD40-CD154 interaction is an important process for cellular and humoral immunity regulation and can be effective in the body’s defense against tumors. In the present study, we evaluated the expression of CD40 in Wehi-164 cell line. CD40 expressions on the cell surface and in the cytoplasm were assessed by flow cytometry and intracellular staining assay, respectively. Also, the mRNA expression was identified by real time-PCR. The obtained results showed the high mRNA and cytoplasmic protein expression of CD40 but no surface expression. These results suggest that the Wehi-164 cell line down regulates expression of CD40 on the surface for evasion of immune system. PMID:20496113

  20. Glucagon-like peptide-1 regulates calcium homeostasis and electrophysiological activities of HL-1 cardiomyocytes.

    PubMed

    Huang, Jen-Hung; Chen, Yao-Chang; Lee, Ting-I; Kao, Yu-Hsun; Chazo, Tze-Fan; Chen, Shih-Ann; Chen, Yi-Jen

    2016-04-01

    Glucagon like-peptide-1 (GLP-1) is an incretin hormone with antidiabetic effects through stimulating insulin secretion, β cell neogenesis, satiety sensation, and inhibiting glucagon secretion. Administration of GLP-1 provides cardioprotective effects through attenuating cardiac inflammation and insulin resistance. GLP-1 also modulates the heart rate and systolic pressure, which suggests that GLP-1 may have cardiac electrical effects. Therefore, the purposes of this study were to evaluate whether GLP-1 has direct cardiac effects and identify the underlying mechanisms. Patch clamp, confocal microscopy with Fluo-3 fluorescence, and Western blot analyses were used to evaluate the electrophysiological characteristics, calcium homeostasis, and calcium regulatory proteins in HL-1 atrial myocytes with and without GLP-1 (1 and 10nM) incubation for 24h. GLP-1 (1 and 10nM) and control cells had similar action potential durations. However, GLP-1 at 10nM significantly increased calcium transients and sarcoplasmic reticular Ca(2+) contents. Compared to the control, GLP-1 (10nM)-treated cells significantly decreased phosphorylation of the ryanodine receptor at S2814 and total phospholamban, but there were similar protein levels of sarcoplasmic reticular Ca(2+)-ATPase and the sodium-calcium exchanger. Moreover, exendin (9-39) amide (a GLP-1 receptor antagonist, 10nM) attenuated GLP-1-mediated effects on total SR content and phosphorylated ryanodine receptor S2814. This study demonstrates GLP-1 may regulate HL-1 cell arrhythmogenesis through modulating calcium handling proteins. PMID:26930508

  1. Molecular Background of Estrogen Receptor Gene Expression in Endometriotic Cells.

    PubMed

    Izawa, Masao; Taniguchi, Fuminori; Harada, Tasuku

    2016-07-01

    The molecular background of estrogen receptor (ER) expression is important to understand the pathophysiology of the high estrogen environment in endometriosis. However, the molecular details have not been fully understood. The objective of this study is to evaluate the molecular background of ERα and ERβ messenger RNA (mRNA) expression in endometriotic cells. The following summarizes our observations: (1) ERα mRNA expression in endometriotic cells was estimated to be approximately one-tenth of that in endometrial cells. (2) Three mRNAs, which include 3 different 5'-untranslated exons tagged to an open reading frame of wild-type ERα, were detected. (3) Expression of ERβ mRNA depends mostly on 0N promoter and includes 2 open reading frames: one for a wild-type ERβ1 and another for a splice variant ERβ2. (4) Expression of ERβ1 mRNA was approximately 40-fold higher than that in endometrial cells. (5) Expression of ERβ2 mRNA was almost at a comparable level of the ERβ1. 9 (6) ERα and ERβ mRNAs are equivalently expressed in endometriotic cells. These observations show the molecular background of ER mRNA expression in endometriotic cells and provide a clue to further understanding the estrogen-dependent pathophysiology leading to clinical application in endometriosis. PMID:26704524

  2. Cyclin Dl expression in B-cell non Hodgkin lymphoma.

    PubMed

    Aref, Salah; Mossad, Y; El-Khodary, T; Awad, M; El-Shahat, E

    2006-10-01

    Disorders of the cell cycle regulatory machinery play a key role in the pathogenesis of cancer. Over-expression of cyclin D1 protein has been reported in several solid tumors and certain lymphoid malignancies, but little is known about the effect of its expression on clinical behavior and outcome in B-cell Non-Hodgkin lymphoma (NHL). In this study, we investigated the expression of cyclin Dl in group of patients with NHL and correlated the results with the clinical and laboratory data. The degree of expression of cyclin Dl protein was evaluated by flow cytometry in a group of NHL patients (n = 46) and in normal control group (n = 10). Cyclin Dl over expression was detected in 10 out of 46 (21.7%) patients; they were 5/5-mantle cell lymphoma (MCL) (100%) and 5/28 large B-cell lymphoma (17.8%). All other NHL subtypes showed normal cyclin D1 expression. The clinical signs (hepatomegaly, splenomegaly and B-symptoms, clinical staging) and laboratory data (hemoglobin, white cell count (WBCs), platelet count, and bone marrow infiltration) were not significantly different between NHL subgroup with cyclin Dl over expression and that with normal cyclin Dl expression. Serum lactic dehydrogenase (LDH) levels and lymphadenopathy were significantly higher in NHL group with cyclin D1 over expression as compared to those without. Also, cyclin D1 over expression is associated with poor outcome of NHL patients. Cyclin Dl over expression was evident among all cases of MCL and few cases of large B-cell lymphoma. Cyclin Dl over expression might be used as adjuvant tool for diagnosis of MCL; has role in NHL biology and is bad prognostic index in NHL. PMID:17607588

  3. Expression of MIF and CD74 in leukemic cell lines: correlation to DR expression destiny.

    PubMed

    Georgouli, Mirella; Papadimitriou, Lina; Glymenaki, Maria; Patsaki, Valia; Athanassakis, Irene

    2016-06-01

    Invariant chain (Ii) or CD74 is a non-polymorphic glycoprotein, which apart from its role as a chaperone dedicated to MHCII molecules, is known to be a high-affinity receptor for macrophage migration inhibitory factor (MIF). The present study aimed to define the roles of CD74 and MIF in the immune surveillance escape process. Towards this direction, the cell lines HL-60, Raji, K562 and primary pre-B leukemic cells were examined for expression and secretion of MIF. Flow cytometry analysis detected high levels of MIF and intracellular/membrane CD74 expression in all leukemic cells tested, while MIF secretion was shown to be inversely proportional to intracellular HLA-DR (DR) expression. In the MHCII-negative cells, IFN-γ increased MIF expression and induced its secretion in HL-60 and K562 cells, respectively. In K562 cells, CD74 (Iip33Iip35) was shown to co-precipitate with HLA-DOβ (DOβ), inhibiting thus MIF or DR binding. Induced expression of DOα in K562 (DOα-DOβ+) cells in different transfection combinations decreased MIF expression and secretion, while increasing surface DR expression. Thus, MIF could indeed be part of the antigen presentation process. PMID:26866879

  4. Glucagon-like peptide-2 and short-chain fatty acids: a new twist to an old story.

    PubMed

    Tappenden, Kelly A; Albin, David M; Bartholome, Anne L; Mangian, Heather Fottler

    2003-11-01

    The nutritional regulation of intestinal adaptation extends beyond the route of nutrient administration as specific nutrients are known to mediate the adaptive response. Dietary carbohydrates are known to enhance intestinal adaptation in patients with short-bowel syndrome. This review discusses SCFA-induced adaptation in intestinal structure and function in adult rat and neonatal piglet models. Potential mechanisms relate to the salvage of energy as SCFA in the colon, direct mediation of intestinal adaptation by SCFA and stimulated release of glucagon-like peptide-2 (GLP-2) from enteroendocrine L cells by SCFA. Among the produced SCFA, butyrate appears to be responsible for increasing plasma GLP-2 concentration, in addition to the enterotrophic effects. Emerging evidence reveals that physiological concentrations of butyrate acutely upregulate the expression of key enterocyte-associated nutrient transporters. Focused experiments are needed to carefully identify the critical components of intestinal adaptation and yield conclusions regarding the relative contributions of SCFA and GLP-2 during the various phases of this process. PMID:14608102

  5. Glucagon regulates gluconeogenesis through KAT2B- and WDR5-mediated epigenetic effects

    PubMed Central

    Ravnskjaer, Kim; Hogan, Meghan F.; Lackey, Denise; Tora, Laszlo; Dent, Sharon Y.R.; Olefsky, Jerrold; Montminy, Marc

    2013-01-01

    Circulating pancreatic glucagon is increased during fasting and maintains glucose balance by stimulating hepatic gluconeogenesis. Glucagon triggering of the cAMP pathway upregulates the gluconeogenic program through the phosphorylation of cAMP response element–binding protein (CREB) and the dephosphorylation of the CREB coactivator CRTC2. Hormonal and nutrient signals are also thought to modulate gluconeogenic gene expression by promoting epigenetic changes that facilitate assembly of the transcriptional machinery. However, the nature of these modifications is unclear. Using mouse models and in vitro assays, we show that histone H3 acetylation at Lys 9 (H3K9Ac) was elevated over gluconeogenic genes and contributed to increased hepatic glucose production during fasting and in diabetes. Dephosphorylation of CRTC2 promoted increased H3K9Ac through recruitment of the lysine acetyltransferase 2B (KAT2B) and WD repeat–containing protein 5 (WDR5), a core subunit of histone methyltransferase (HMT) complexes. KAT2B and WDR5 stimulated the gluconeogenic program through a self-reinforcing cycle, whereby increases in H3K9Ac further potentiated CRTC2 occupancy at CREB binding sites. Depletion of KAT2B or WDR5 decreased gluconeogenic gene expression, consequently breaking the cycle. Administration of a small-molecule KAT2B antagonist lowered circulating blood glucose concentrations in insulin resistance, suggesting that this enzyme may be a useful target for diabetes treatment. PMID:24051374

  6. Biased Allelic Expression in Human Primary Fibroblast Single Cells

    PubMed Central

    Borel, Christelle; Ferreira, Pedro G.; Santoni, Federico; Delaneau, Olivier; Fort, Alexandre; Popadin, Konstantin Y.; Garieri, Marco; Falconnet, Emilie; Ribaux, Pascale; Guipponi, Michel; Padioleau, Ismael; Carninci, Piero; Dermitzakis, Emmanouil T.; Antonarakis, Stylianos E.

    2015-01-01

    The study of gene expression in mammalian single cells via genomic technologies now provides the possibility to investigate the patterns of allelic gene expression. We used single-cell RNA sequencing to detect the allele-specific mRNA level in 203 single human primary fibroblasts over 133,633 unique heterozygous single-nucleotide variants (hetSNVs). We observed that at the snapshot of analyses, each cell contained mostly transcripts from one allele from the majority of genes; indeed, 76.4% of the hetSNVs displayed stochastic monoallelic expression in single cells. Remarkably, adjacent hetSNVs exhibited a haplotype-consistent allelic ratio; in contrast, distant sites located in two different genes were independent of the haplotype structure. Moreover, the allele-specific expression in single cells correlated with the abundance of the cellular transcript. We observed that genes expressing both alleles in the majority of the single cells at a given time point were rare and enriched with highly expressed genes. The relative abundance of each allele in a cell was controlled by some regulatory mechanisms given that we observed related single-cell allelic profiles according to genes. Overall, these results have direct implications in cellular phenotypic variability. PMID:25557783

  7. Optimizing transient recombinant protein expression in mammalian cells.

    PubMed

    Hopkins, Ralph F; Wall, Vanessa E; Esposito, Dominic

    2012-01-01

    Transient gene expression (TGE) in mammalian cells has become a routine process for expressing recombinant proteins in cell lines such as human embryonic kidney 293 and Chinese hamster ovary cells. The rapidly increasing need for recombinant proteins requires further improvements in TGE technology. While a great deal of focus has been directed toward optimizing the secretion of antibodies and other naturally secreted targets, much less work has been done on ways to improve cytoplasmic expression in mammalian cells. The benefits to protein production in mammalian cells, particularly for eukaryotic proteins, should be very significant - glycosylation and other posttranslational modifications will likely be native or near-native, solubility and protein folding would likely improve overexpression in heterologous hosts, and expression of proteins in their proper intracellular compartments is much more likely to occur. Improvements in this area have been slow, however, due to limited development of the cell culture processes needed for low-cost, higher-throughput expression in mammalian cells, and the relatively low diversity of DNA vectors for protein production in these systems. Here, we describe how the use of recombinational cloning, coupled with improvements in transfection protocols which increase speed and lower cost, can be combined to make mammalian cells much more amenable for routine recombinant protein expression. PMID:21987258

  8. The subcellular location, maturation and response to increased plasma glucagon of Ruthenium Red-insensitive calcium-ion transport in rat liver

    PubMed Central

    Bygrave, Fyfe L.; Tranter, Charmaine J.

    1978-01-01

    1. The subcellular distribution and maturation of Ruthenium Red-insensitive Ca2+ transport activity were determined in livers of rats ranging in age from 3 days pre-term to 10 weeks of adult life and compared with those of glucose 6-phosphatase, 5′-nucleotidase and Ruthenium Red-sensitive Ca2+ transport. Initial rates of Ruthenium Red-insensitive Ca2+ transport were highest in those fractions enriched in glucose 6-phosphatase, i.e. the microsomal fraction; this fraction was devoid of Ruthenium Red-sensitive Ca2+ transport activity. Although the heaviest fraction (nuclear) contained significant amounts of 5′-nucleotidase activity it was devoid of Ruthenium Red-insensitive Ca2+ transport activity. 2. Foetal rat liver contain minimal amounts of Ruthenium Red-insensitive Ca2+ transport activity, glucose 6-phosphatase and 5′-nucleotidase activities. These begin to be expressed concomitantly soon after birth; Ruthenium Red-insensitive Ca2+ transport is maximal by 3 to 4 days and remains so for up to at least 10 weeks of adult life. Glucose 6-phosphatase also reaches a peak at 3–4 days, but then rapidly decreases to approach adult values. Maximal activity of 5′-nucleotidase in the microsomal and nuclear fractions is seen about 4–6 days after birth; this enzyme activity remains increased for up to about 10 days and then falls, but not as rapidly as glucose 6-phosphatase. It is tentatively suggested that the bulk of the Ruthenium Red-insensitive Ca2+ transport is attributable to the system derived from the endoplasmic reticulum. 3. Administration of glucagon to adult rats enhances by 2–3-fold the initial rate of Ruthenium Red-insensitive Ca2+ transport in the intermediate but not the microsomal fraction. The hormone-induced effect is fully suppressed by co-administration of puromycin, is dose-dependent with half-maximal response at approx. 1μg of glucagon/100g body wt. and time-dependent exhibiting a half-maximal response about 1h after administration of the

  9. Evidence of tricellulin expression by immune cells, particularly microglia.

    PubMed

    Mariano, Cibelle; Silva, Sandra Leitão; Pereira, Pedro; Fernandes, Adelaide; Brites, Dora; Brito, Maria A

    2011-06-17

    Tight junctions (TJs) are elaborate structures located on the apical region of epithelial cells that limit paracellular permeability. Tricellulin is a recently discovered TJ protein, which is concentrated at the structurally specialized tricellular TJs but also present at bicellular contacts between epithelial cells, namely in the stomach. Interestingly, several TJ proteins have been found in other than epithelial cells, as astrocytes, and tricellulin mRNA expression was reported in mature dendritic cells. These findings prompted us to look for tricellulin expression in both epithelial and immune cells in the stomach, as well as in microglia, the brain resident immunocompetent cells. Immunohistochemical analysis of human stomach tissue sections revealed peroxidase staining at three-corner contact sites, as well as at the contact between two adjacent epithelial cells, thus evidencing the expression of tricellulin not only at tricellullar but at bicellular junctions as well. Such analysis, further revealed tricellulin immunostaining in cells of the monocyte/macrophage lineage, scattered throughout the lamina propria. Cultured rat microglia exhibited a notorious tricellulin staining, consistent with an extensive expression of the protein along the cell, which was not absolutely coincident with the lysosomal marker CD68. Detection of mRNA expression by real-time PCR provided supportive evidence for the expression of the TJ protein in microglia. These data demonstrate for the first time that microglia express a TJ protein. Moreover, the expression of tricellulin both in microglia and in the stomach immune cells point to a possible role of this new TJ protein in the immune system. PMID:21624353

  10. Regulated expression of erythropoietin by two human hepatoma cell lines

    SciTech Connect

    Goldberg, M.A.; Glass, G.A.; Cunningham, J.M.; Bunn, H.F.

    1987-11-01

    The development of a cell culture system that produces erythropoietin (Epo) in a regulated manner has been the focus of much effort. The authors have screened multiple renal and hepatic cell lines for either constitutive or regulated expression of Epo. Only the human hepatoma cell lines, Hep3B and HepG2, made significant amounts of Epo as measured both by radioimmunoassay and in vitro bioassay (as much as 330 milliunits per 10/sup 6/ cells in 24 hr). The constitutive production of Epo increased dramatically as a function of cell density in both cell lines. At cell densities < 3.3 x 10/sup 5/ cells per cm/sup 2/, there was little constitutive release of Epo in the medium. With Hep3B cells grown at low cell densities, a mean 18-fold increase in Epo expression was seen in response to hypoxia and a 6-fold increase was observed in response to incubation in medium containing 50 ..mu..M cobalt(II) chloride. At similar low cell densities, Epo production in HepG2 cells could be enhanced an average of about 3-fold by stimulation with either hypoxia or cobalt(II) chloride. Upon such stimulation, both cell lines demonstrated markedly elevated levels of Epo mRNA. Hence, both Hep3B and HepG2 cell lines provide an excellent in vitro system in which to study the physiological regulation of Epo expression.

  11. Regulation of global gene expression and cell proliferation by APP.

    PubMed

    Wu, Yili; Zhang, Si; Xu, Qin; Zou, Haiyan; Zhou, Weihui; Cai, Fang; Li, Tingyu; Song, Weihong

    2016-01-01

    Down syndrome (DS), caused by trisomy of chromosome 21, is one of the most common genetic disorders. Patients with DS display growth retardation and inevitably develop characteristic Alzheimer's disease (AD) neuropathology, including neurofibrillary tangles and neuritic plaques. The expression of amyloid precursor protein (APP) is increased in both DS and AD patients. To reveal the function of APP and elucidate the pathogenic role of increased APP expression in DS and AD, we performed gene expression profiling using microarray method in human cells overexpressing APP. A set of genes are significantly altered, which are involved in cell cycle, cell proliferation and p53 signaling. We found that overexpression of APP inhibits cell proliferation. Furthermore, we confirmed that the downregulation of two validated genes, PSMA5 and PSMB7, inhibits cell proliferation, suggesting that the downregulation of PSMA5 and PSMB7 is involved in APP-induced cell proliferation impairment. Taken together, this study suggests that APP regulates global gene expression and increased APP expression inhibits cell proliferation. Our study provides a novel insight that APP overexpression may contribute to the growth impairment in DS patients and promote AD pathogenesis by inhibiting cell proliferation including neural stem cell proliferation and neurogenesis. PMID:26936520

  12. Regulation of global gene expression and cell proliferation by APP

    PubMed Central

    Wu, Yili; Zhang, Si; Xu, Qin; Zou, Haiyan; Zhou, Weihui; Cai, Fang; Li, Tingyu; Song, Weihong

    2016-01-01

    Down syndrome (DS), caused by trisomy of chromosome 21, is one of the most common genetic disorders. Patients with DS display growth retardation and inevitably develop characteristic Alzheimer’s disease (AD) neuropathology, including neurofibrillary tangles and neuritic plaques. The expression of amyloid precursor protein (APP) is increased in both DS and AD patients. To reveal the function of APP and elucidate the pathogenic role of increased APP expression in DS and AD, we performed gene expression profiling using microarray method in human cells overexpressing APP. A set of genes are significantly altered, which are involved in cell cycle, cell proliferation and p53 signaling. We found that overexpression of APP inhibits cell proliferation. Furthermore, we confirmed that the downregulation of two validated genes, PSMA5 and PSMB7, inhibits cell proliferation, suggesting that the downregulation of PSMA5 and PSMB7 is involved in APP-induced cell proliferation impairment. Taken together, this study suggests that APP regulates global gene expression and increased APP expression inhibits cell proliferation. Our study provides a novel insight that APP overexpression may contribute to the growth impairment in DS patients and promote AD pathogenesis by inhibiting cell proliferation including neural stem cell proliferation and neurogenesis. PMID:26936520

  13. Expression Profiling of Developing Zebrafish Retinal Cells.

    PubMed

    Mullally, Madelyn; Albrecht, Caitlin; Horton, Mary; Laboissonniere, Lauren A; Goetz, Jillian J; Chowdhury, Rebecca; Manning, Alicia; Wester, Andrea K; Bose, Quinton; Trimarchi, Jeffrey M

    2016-08-01

    During retinal development, a variety of different types of neurons are produced. Understanding how each of these types of retinal nerve cells is generated is important from a developmental biology perspective. It is equally important if one is interested in how to regenerate cells after an injury or a disease. To gain more insight into how retinal neurons develop in the zebrafish, we performed single-cell mRNA profiling and in situ hybridizations (ISHs) on retinal sections and whole-mount zebrafish. Through the series of ISHs, designed and performed solely by undergraduate students in the laboratory, we were able to retrospectively identify our single-cell mRNA profiles as most likely coming from developing amacrine cells. Further analysis of these profiles will reveal genes that can be mutated using genome editing techniques. Together these studies increase our knowledge of the genes driving development of different cell types in the zebrafish retina. PMID:26982811

  14. ALDH isozymes downregulation affects cell growth, cell motility and gene expression in lung cancer cells

    PubMed Central

    Moreb, Jan S; Baker, Henry V; Chang, Lung-Ji; Amaya, Maria; Lopez, M Cecilia; Ostmark, Blanca; Chou, Wayne

    2008-01-01

    Background Aldehyde dehydrogenase isozymes ALDH1A1 and ALDH3A1 are highly expressed in non small cell lung cancer. Neither the mechanisms nor the biologic significance for such over expression have been studied. Methods We have employed oligonucleotide microarrays to analyze changes in gene profiles in A549 lung cancer cell line in which ALDH activity was reduced by up to 95% using lentiviral mediated expression of siRNA against both isozymes (Lenti 1+3). Stringent analysis methods were used to identify gene expression patterns that are specific to the knock down of ALDH activity and significantly different in comparison to wild type A549 cells (WT) or cells similarly transduced with green fluorescent protein (GFP) siRNA. Results We confirmed significant and specific down regulation of ALDH1A1 and ALDH3A1 in Lenti 1+3 cells and in comparison to 12 other ALDH genes detected. The results of the microarray analysis were validated by real time RT-PCR on RNA obtained from Lenti 1+3 or WT cells treated with ALDH activity inhibitors. Detailed functional analysis was performed on 101 genes that were significantly different (P < 0.001) and their expression changed by ≥ 2 folds in the Lenti 1+3 group versus the control groups. There were 75 down regulated and 26 up regulated genes. Protein binding, organ development, signal transduction, transcription, lipid metabolism, and cell migration and adhesion were among the most affected pathways. Conclusion These molecular effects of the ALDH knock-down are associated with in vitro functional changes in the proliferation and motility of these cells and demonstrate the significance of ALDH enzymes in cell homeostasis with a potentially significant impact on the treatment of lung cancer. PMID:19025616

  15. 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. PMID:25535828

  16. Engineering Cells to Improve Protein Expression

    PubMed Central

    Xiao, Su; Shiloach, Joseph; Betenbaugh, Michael J.

    2014-01-01

    Cellular engineering of bacteria, fungi, insect cells and mammalian cells is a promising methodology to improve recombinant protein production for structural, biochemical, and commercial applications. Increased understanding of the host organism biology has suggested engineering strategies targeting bottlenecks in transcription, translation, protein processing and secretory pathways, as well as cell growth and survival. A combination of metabolic engineering and synthetic biology has been used to improve the properties of cells for protein production, which has resulted in enhanced yields of multiple protein classes. PMID:24704806

  17. Endocrine Taste Cells

    PubMed Central

    Kokrashvili, Zaza; Yee, Karen K.; Ilegems, Erwin; Iwatsuki, Ken; Li, Yan; Mosinger, Bedrich; Margolskee, Robert F.

    2014-01-01

    In taste cells, taste receptors, their coupled G proteins, and downstream signaling elements mediate detection and transduction of sweet, bitter and umami compounds. In some intestinal endocrine cells, taste receptors and gustducin contribute to the release of glucagon-like peptide-1 (GLP-1) and other gut hormones in response to glucose and non-caloric sweeteners. Conversely, taste cells have been found to express multiple hormones typically found in intestinal endocrine cells, e.g. GLP-1, glucagon, somatostatin and ghrelin. By immunohistochemistry multiple subsets of taste cells were found to express GLP-1. The release of GLP-1 from “endocrine taste cells” into the bloodstream was examined. In wild-type mice, even after esophagealectomy and vagotomy, application of glucose to the tongue induced an elevation of GLP-1 in the bloodstream within 10 minutes of stimulation of the taste buds with glucose. Glucose stimulation of taste cell explants from wild-type mice led to release of GLP-1 into the medium. Knocking out the T1R3 gene did not eliminate glucose-stimulated GLP-1 release from taste cells in vivo. Our results indicate that a portion of the cephalic phase rise in circulating GLP-1 is mediated by direct release of GLP-1 from taste cells into the bloodstream. PMID:24382120

  18. Over-expression of secreted proteins from mammalian cell lines

    PubMed Central

    Dalton, Annamarie C; Barton, William A

    2014-01-01

    Secreted mammalian proteins require the development of robust protein over-expression systems for crystallographic and biophysical studies of protein function. Due to complex disulfide bonds and distinct glycosylation patterns preventing folding and expression in prokaryotic expression hosts, many secreted proteins necessitate production in more complex eukaryotic expression systems. Here, we elaborate on the methods used to obtain high yields of purified secreted proteins from transiently or stably transfected mammalian cell lines. Among the issues discussed are the selection of appropriate expression vectors, choice of signal sequences for protein secretion, availability of fusion tags for enhancing protein stability and purification, choice of cell line, and the large-scale growth of cells in a variety of formats. PMID:24510886

  19. Analysis of variation of amplitudes in cell cycle gene expression

    PubMed Central

    Liu, Delong; Gaido, Kevin W; Wolfinger, Russ

    2005-01-01

    Background Variation in gene expression among cells in a population is often considered as noise produced from gene transcription and post-transcription processes and experimental artifacts. Most studies on noise in gene expression have emphasized a few well-characterized genes and proteins. We investigated whether different cell-arresting methods have impacts on the maximum expression levels (amplitudes) of a cell cycle related gene. Results By introducing random noise, modeled by a von Mises distribution, to the phase angle in a sinusoidal model in a cell population, we derived a relationship between amplitude and the distribution of noise in maximum transcription time (phase). We applied our analysis to Whitfield's HeLa cell cycle data. Our analysis suggests that among 47 cell cycle related genes common to the 2nd experiment (thymidine-thymidine method) and the 4th experiment (thymidine-nocodazole method): (i) the amplitudes of CDC6 and PCNA, which are expressed during G1/S phase, are smaller in the 2nd experiment than in the 4th, while the amplitude of CDC20, which is expressed during G2/M phase, is smaller in the 4th experiment; and (ii) the two cell-arresting methods had little impact on the amplitudes of the other 43 genes in the 2nd and 4th experiments. Conclusion Our analysis suggests that procedures that arrest cells in different stages of the cell cycle differentially affect expression of some cell cycle related genes once the cells are released from arrest. The impact of the cell-arresting method on expression of a cell cycle related gene can be quantitatively estimated from the ratio of two estimated amplitudes in two experiments. The ratio can be used to gauge the variation in the phase/peak expression time distribution involved in stochastic transcription and post-transcriptional processes for the gene. Further investigations are needed using normal, unperturbed and synchronized HeLa cells as a reference to compare how many cell cycle related genes

  20. Langerhans' cell expression of the selectin ligand, sialyl Lewis x.

    PubMed Central

    Ross, E L; Barker, J N; Allen, M H; Chu, A C; Groves, R W; MacDonald, D M

    1994-01-01

    Cellular adhesion molecules play a central role in leucocyte migration through peripheral blood and tissues. A crucial stage in these events in selectin-mediated adhesion involving E-selectin expressed on activated endothelium interacting with a range of carbohydrate ligands expressed by specific subpopulations of leucocytes. As such mechanisms may be relevant to bone marrow-derived dendritic epidermal Langerhans' cell (LC) migration, expression of these carbohydrate ligands was assessed immunocytochemically in whole skin biopsies and in epidermal cell suspensions obtained from adult humans. Double-labelling experiments revealed that sialyl Lewis x, recognized by the monoclonal antibody CSLEX1, was expressed on epidermal LC (n = 9). Furthermore, expression was enhanced at 24 hr following epicutaneous application of antigen and in the inflammatory disorder psoriasis (n = 10). E-selectin was concomitantly strongly expressed on dermal endothelium in psoriasis and allergic contact dermatitis. Intradermal injection of the T-cell-derived cytokine interferon-gamma (IFN-gamma) led to increased LC expression of sialyl Lewis x. In epidermal cell suspensions, in contrast to keratinocytes, CD1a+ cells expressed sialyl Lewis x, intensity of which was enhanced after 4 days in culture. CSLEX1 staining could be abolished and CD15 (non-sialated Lewis x) expression induced by saponification and treatment with neuraminidase. Expression of other selectin ligands was also examined. While the cutaneous lymphocyte antigen defined by the monoclonal antibody HECA-452 reacted with a small minority of LC, sialyl Lewis a and sulphatide were not expressed under any experimental conditions. These studies indicate that E-selectin-sialyl Lewis x interactions are potentially important in LC migration, both into and out of skin. Images Figure 2 Figure 3 Figure 5 Figure 6 PMID:7512530

  1. Glucagon-like peptide-2 intracellularly stimulates eNOS phosphorylation and specifically induces submucosal arteriole vasodilation via a sheer stress-independent, local neural mechanism

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Glucagon-like peptide-2 (GLP-2) is a nutrient-responsive neuropeptide that exerts diverse actions in the gastrointestinal tract, including enhancing mucosal cell survival and proliferation, mucosal blood flow, luminal nutrient uptake, and suppressing gastric motility and secretion. We have shown th...

  2. Ectopic ERK Expression Induces Phenotypic Conversion of C10 Cells and Alters DNA Methyltransferase Expression

    SciTech Connect

    Sontag, Ryan L.; Weber, Thomas J.

    2012-05-04

    In some model systems constitutive extracellular signal regulated kinase (ERK) activation is sufficient to promote an oncogenic phenotype. Here we investigate whether constitutive ERK expression influences phenotypic conversion in murine C10 type II alveolar epithelial cells. C10 cells were stably transduced with an ERK1-green fluorescent protein (ERK1-GFP) chimera or empty vector and ectopic ERK expression was associated with the acquisition of soft agar focus-forming potential in late passage, but not early passage cells. Late passage ERK1-GFP cells exhibited a significant increase in the expression of DNA methyl transferases (DNMT1 and 3b) and a marked increase in sensitivity to 5-azacytidine (5-azaC)-mediated toxicity, relative to early passage ERK1-GFP cells and vector controls. The expression of xeroderma pigmentosum complementation group A (XPA) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) were significantly increased in late passage cells, suggesting enhanced DNA damage recognition and repair activity which we interpret as a reflection of genomic instability. Phospho-ERK levels were dramatically decreased in late passage ERK1-GFP cells, relative to early passage and vector controls, and phospho-ERK levels were restored by treatment with sodium orthovanadate, indicating a role for phosphatase activity in this response. Collectively these observations suggest that ectopic ERK expression promotes phenotypic conversion of C10 cells that is associated with latent effects on epigenetic programming and phosphatase activities.

  3. 21 CFR 862.1335 - Glucagon test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Glucagon test system. 862.1335 Section 862.1335 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems §...

  4. 21 CFR 862.1335 - Glucagon test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Glucagon test system. 862.1335 Section 862.1335 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems §...

  5. 21 CFR 862.1335 - Glucagon test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Glucagon test system. 862.1335 Section 862.1335 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems §...

  6. 21 CFR 862.1335 - Glucagon test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Glucagon test system. 862.1335 Section 862.1335 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED... and treatment of patients with various disorders of carbohydrate metabolism, including...

  7. 21 CFR 862.1335 - Glucagon test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Glucagon test system. 862.1335 Section 862.1335 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED... and treatment of patients with various disorders of carbohydrate metabolism, including...

  8. Metal-Catalyzed Oxidation and Photo-oxidation of Glucagon.

    PubMed

    Zhang, Jian

    2016-08-01

    The oxidation of glucagon by the H2O2/Cu(2+) system and by simulated sunlight was studied using HPLC-MS methodologies. It was found that copper ion-catalyzed oxidation is much faster in the residue 1-12 region than in photo-oxidation, but it is slower than photo-oxidation in the residue 18-29 region. This difference is due to the unique feature of the primary sequence of glucagon. The residue 1-12 region contains His-1 and Asp-9 that can bind to Cu(2+) ions and catalyze the oxidation of His-1 and Tyr-10, while the residue 18-29 region lacks these charged residues near the liable Met-27 and Trp-25 and hence no catalysis by the neighboring groups occurs. Fragment (residue 13-17) was more stable than the other regions of the peptide toward photo-oxidation because it contains only one oxidizable residue, Tyr-13. These findings may help explain the mechanism of action of glucagon and provide some hints for the development of effective anti-diabetic drug molecules and stable glucagon formulations. PMID:27435200

  9. An Exercise to Estimate Differential Gene Expression in Human Cells

    ERIC Educational Resources Information Center

    Chaudhry, M. Ahmad

    2006-01-01

    The expression of genes in cells of various tissue types varies considerably and is correlated with the function of a particular organ. The pattern of gene expression changes in diseased tissues, in response to therapy or infection and exposure to environmental mutagens, chemicals, ultraviolet light, and ionizing radiation. To better understand…

  10. Behavioral experience induces zif268 expression in mature granule cells but suppresses its expression in immature granule cells

    PubMed Central

    Huckleberry, Kylie A.; Kane, Gary A.; Mathis, Rita J.; Cook, Sarah G.; Clutton, Jonathan E.; Drew, Michael R.

    2015-01-01

    Thousands of neurons are born each day in the dentate gyrus (DG), but many of these cells die before reaching maturity. Both death and survival of adult-born neurons are regulated by neuronal activity in the DG. The immediate-early gene (IEG) zif268 appears to be an important mediator of these effects, as its expression can be induced by neural activity and knockout of zif268 impairs survival of adult-born neurons (Richardson et al., 1992; Veyrac et al., 2013). Despite the apparent importance of zif268 for adult neurogenesis, its behavior-induced expression has not been fully characterized in adult-born neurons. Here we characterize behavior-evoked expression of zif268 in mature and newborn dentate granule cells (DGCs). We first quantified zif268 expression in doublecortin-positive (DCX+) immature neurons and in the general granule cell population after brief exposure to a novel environment (NE). In the general granule cell population, zif268 expression peaked 1 h after NE exposure and returned to baseline by 8 h post-exposure. However, in the DCX+ cells, zif268 expression was suppressed relative to home cage for at least 8 h post-exposure. We next asked whether suppression of zif268 in DCX+ immature cells occurs in other behavioral paradigms that recruit the hippocampus. Exposure to Morris water maze (MWM) training, an enriched environment, or a NE caused approximately equal suppression of zif268 expression in DCX+ cells and approximately equal activation of zif268 expression among the general granule cell population. The same behavioral procedures activated zif268 expression in 6-week-old BrdU-labeled adult-born neurons, indicating that zif268 suppression is specific to immature neurons. Finally, we asked whether zif268 suppression varied as a function of age within the DCX+ population, which ranges in age from 0 to approximately 4 weeks. NE exposure had no significant effect on zif268 expression in 2- or 4-week-old BrdU-labeled neurons, but it significantly

  11. Neurogenin 3 Expressing Cells in the Human Exocrine Pancreas Have the Capacity for Endocrine Cell Fate

    PubMed Central

    Gomez, Danielle L.; O’Driscoll, Marci; Sheets, Timothy P.; Hruban, Ralph H.; Oberholzer, Jose; McGarrigle, James J.; Shamblott, Michael J.

    2015-01-01

    Neurogenin 3 (NGN3) is necessary and sufficient for endocrine differentiation during pancreatic development and is expressed by a population of progenitor cells that give rise exclusively to hormone-secreting cells within islets. NGN3 protein can be detected in the adult rodent pancreas only following certain types of injury, when it is transiently expressed by exocrine cells undergoing reprogramming to an endocrine cell fate. Here, NGN3 protein can be detected in 2% of acinar and duct cells in living biopsies of histologically normal adult human pancreata and 10% in cadaveric biopsies of organ donor pancreata. The percentage and total number of NGN3+ cells increase during culture without evidence of proliferation or selective cell death. Isolation of highly purified and viable NGN3+ cell populations can be achieved based on coexpression of the cell surface glycoprotein CD133. Transcriptome and targeted expression analyses of isolated CD133+ / NGN3+ cells indicate that they are distinct from surrounding exocrine tissue with respect to expression phenotype and Notch signaling activity, but retain high level mRNA expression of genes indicative of acinar and duct cell function. NGN3+ cells have an mRNA expression profile that resembles that of mouse early endocrine progenitor cells. During in vitro differentiation, NGN3+ cells express genes in a pattern characteristic of endocrine development and result in cells that resemble beta cells on the basis of coexpression of insulin C-peptide, chromogranin A and pancreatic and duodenal homeobox 1. NGN3 expression in the adult human exocrine pancreas marks a dedifferentiating cell population with the capacity to take on an endocrine cell fate. These cells represent a potential source for the treatment of diabetes either through ex vivo manipulation, or in vivo by targeting mechanisms controlling their population size and endocrine cell fate commitment. PMID:26288179

  12. Shifts in dietary carbohydrate-lipid exposure regulate expression of the non-alcoholic fatty liver disease-associated gene PNPLA3/adiponutrin in mouse liver and HepG2 human liver cells

    PubMed Central

    Hao, Lei; Ito, Kyoko; Huang, Kuan-Hsun; Sae-tan, Sudathip; Lambert, Joshua D.; Ross, A. Catharine

    2014-01-01

    Objective Patatin-like phospholipase domain containing 3 (PNPLA3, adiponutrin) has been identified as a modifier of lipid metabolism. To better understand the physiological role of PNPLA3/adiponutrin, we have investigated its regulation in intact mice and human hepatocytes under various nutritional/metabolic conditions. Material/Methods PNPLA3 gene expression was determined by real-time PCR in liver of C57BL/6 mice after dietary treatments and in HepG2 cells exposed to various nutritional/metabolic stimuli. Intracellular lipid content was determined in HepG2 cells after siRNA-mediated knockdown of PNPLA3. Results In vivo, mice fed a high-carbohydrate (HC) liquid diet had elevated hepatic lipid content, and PNPLA3 mRNA and protein expression, compared to chow-fed mice. Elevated expression was completely abrogated by addition of unsaturated lipid emulsion to the HC diet. By contrast, in mice with high-fat diet-induced steatosis, Pnpla3 expression did not differ compared to low-fat fed mice. In HepG2 cells, Pnpla3 expression was reversibly suppressed by glucose depletion and increased by glucose refeeding, but unchanged by addition of insulin and glucagon. Several unsaturated fatty acids each significantly decreased Pnpla3 mRNA, similar to lipid emulsion in vivo. However, Pnpla3 knockdown in HepG2 cells did not alter total lipid content in high glucose- or oleic acid-treated cells. Conclusions Our results provide evidence that PNPLA3 expression is an early signal/signature of carbohydrate-induced lipogenesis, but its expression is not associated with steatosis per se. Under lipogenic conditions due to high-carbohydrate feeding, certain unsaturated fatty acids can effectively suppress both lipogenesis and PNPLA3 expression, both in vivo and in a hepatocyte cell line. PMID:25060692

  13. Recombinant cells that highly express chromosomally-integrated heterologous genes

    DOEpatents

    Ingram, L.O.; Ohta, Kazuyoshi; Wood, B.E.

    1998-10-13

    Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol. 13 figs.

  14. Recombinant cells that highly express chromosomally-integrated heterologous gene

    DOEpatents

    Ingram, Lonnie O.; Ohta, Kazuyoshi; Wood, Brent E.

    2007-03-20

    Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol.

  15. Recombinant cells that highly express chromosomally-integrated heterologous genes

    DOEpatents

    Ingram, Lonnie O.; Ohta, Kazuyoshi; Wood, Brent E.

    1998-01-01

    Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol.

  16. Recombinant cells that highly express chromosomally-integrated heterologous genes

    DOEpatents

    Ingram, Lonnie O.; Ohta, Kazuyoshi; Wood, Brent E.

    2000-08-22

    Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol.

  17. Abnormally high expression of proteasomes in human leukemic cells.

    PubMed Central

    Kumatori, A; Tanaka, K; Inamura, N; Sone, S; Ogura, T; Matsumoto, T; Tachikawa, T; Shin, S; Ichihara, A

    1990-01-01

    Proteasomes are eukaryotic ring-shaped or cylindrical particles with multicatalytic protease activities. To clarify the involvement of proteasomes in tumorigenesis of human blood cells, we compared their expression in human hematopoietic malignant tumor cells with that in normal peripheral blood mononuclear cells. Immunohistochemical staining showed considerably increased concentrations of proteasomes in leukemic cells from the bone marrow of patients with various types of leukemia and the predominant localization of these proteasomes in the nuclei. Moreover, enzyme immunoassay and Northern blot analysis indicated that the concentrations of proteasomes and their mRNA levels were consistently much higher in a variety of malignant human hematopoietic cell lines than in resting peripheral lymphocytes and monocytes from healthy adults. Proteasome expression was also greatly increased in normal blood mononuclear cells during blastogenic transformation induced by phytohemagglutinin; their expression increased in parallel with induction of DNA synthesis and returned to the basal level with progress of the cell cycle. Thus, abnormally high expression of proteasomes may play an important role in transformation and proliferation of blood cells and in specific functions of hematopoietic tumor cells. Images PMID:2205851

  18. Amino Acids Regulate Transgene Expression in MDCK Cells

    PubMed Central

    Torrente, Marta; Guetg, Adriano; Sass, Jörn Oliver; Arps, Lisa; Ruckstuhl, Lisa; Camargo, Simone M. R.; Verrey, François

    2014-01-01

    Gene expression and cell growth rely on the intracellular concentration of amino acids, which in metazoans depends on extracellular amino acid availability and transmembrane transport. To investigate the impact of extracellular amino acid concentrations on the expression of a concentrative amino acid transporter, we overexpressed the main kidney proximal tubule luminal neutral amino acid transporter B0AT1-collectrin (SLC6A19-TMEM27) in MDCK cell epithelia. Exogenously expressed proteins co-localized at the luminal membrane and mediated neutral amino acid uptake. However, the transgenes were lost over few cell culture passages. In contrast, the expression of a control transgene remained stable. To test whether this loss was due to inappropriately high amino acid uptake, freshly transduced MDCK cell lines were cultivated either with physiological amounts of amino acids or with the high concentration found in standard cell culture media. Expression of exogenous transporters was unaffected by physiological amino acid concentration in the media. Interestingly, mycoplasma infection resulted in a significant increase in transgene expression and correlated with the rapid metabolism of L-arginine. However, L-arginine metabolites were shown to play no role in transgene expression. In contrast, activation of the GCN2 pathway revealed by an increase in eIF2α phosphorylation may trigger transgene derepression. Taken together, high extracellular amino acid concentration provided by cell culture media appears to inhibit the constitutive expression of concentrative amino acid transporters whereas L-arginine depletion by mycoplasma induces the expression of transgenes possibly via stimulation of the GCN2 pathway. PMID:24797296

  19. UV-induced changes in cell cycle and gene expression within rabbit lens epithelial cells

    SciTech Connect

    Sidjanin, D.; Grdina, D.; Woloschak, G.E.

    1994-11-01

    Damage to lens epithelial cells is a probable initiation process in cataract formation induced by ultraviolet radiation. These experiments investigated the ability of 254 nm radiation on cell cycle progression and gene expression in rabbit lens epithelial cell line N/N1003A. No changes in expression of c-fos, c-jun, alpha- tubulin, or vimentin was observed following UV exposure. Using flow cytometry, an accumulation of cells in G1/S phase of the cell cycle 1 hr following exposure. The observed changes in gene expression, especially the decreased histone transcripts reported here may play a role in UV induced inhibition of cell cycle progression.

  20. Expression and purification of recombinant nattokinase in Spodoptera frugiperda cells.

    PubMed

    Li, Xiaoxiang; Wang, Xiaoli; Xiong, Shaoling; Zhang, Jing; Cai, Litao; Yang, Yanyan

    2007-10-01

    A recombinant baculovirus, rv-egfp-NK, containing a reporter gene encoding the enhanced green fluorescent protein (EGFP), was used to express nattokinase (NK), a fibrinolytic enzyme, in Spodoptera frugiperda (SF-9) cells. The recombinant protein also included a histidine tag for purification using Ni(2+) resins. The recombinant NK, approximately 30 kDa, retained fibrinolytic activity (60 U/ml). The integration of the EGFP expression cassette in the Bac-to-Bac system is thus an effective method for the expression and purification of recombinant NK protein in Spodoptera frugiperda insect cells. PMID:17581705

  1. Quantitative analysis of intermediary metabolism in hepatocytes incubated in the presence and absence of glucagon with a substrate mixture containing glucose, ribose, fructose, alanine and acetate.

    PubMed Central

    Rabkin, M; Blum, J J

    1985-01-01

    Hepatocytes were isolated from the livers of fed rats and incubated, in the presence and absence of 100 nM-glucagon, with a substrate mixture containing glucose (10 mM), fructose (4 mM), alanine (3.5 mM), acetate (1.25 mM), and ribose (1 mM). In any given incubation one substrate was labelled with 14C. Incorporation of 14C into glucose, glycogen, CO2, lactate, alanine, glutamate, lipid glycerol and fatty acids was measured after 20 and 40 min of incubation under quasi-steady-state conditions [Borowitz, Stein & Blum (1977) J. Biol. Chem. 252, 1589-1605]. These data and the measured O2 consumption were analysed with the aid of a structural metabolic model incorporating all reactions of the glycolytic, gluconeogenic, and pentose phosphate pathways, and associated mitochondrial and cytosolic reactions. A considerable excess of experimental measurements over independent flux parameters and a number of independent measurements of changes in metabolite concentrations allowed for a stringent test of the model. A satisfactory fit to the data was obtained for each condition. Significant findings included: control cells were glycogenic and glucagon-treated cells glycogenolytic during the second interval; an ordered (last in, first out) model of glycogen degradation [Devos & Hers (1979) Eur. J. Biochem. 99, 161-167] was required in order to fit the experimental data; the pentose shunt contributed approx. 15% of the carbon for gluconeogenesis in both control and glucagon-treated cells; net flux through the lower Embden-Meyerhof pathway was in the glycolytic direction except during the 20-40 min interval in glucagon-treated cells; the increased gluconeogenesis in response to glucagon was correlated with a decreased pyruvate kinase flux and lactate output; fluxes through pyruvate kinase, pyruvate carboxylase, and phosphoenolpyruvate carboxykinase were not coordinately controlled; Krebs cycle activity did not change with glucagon treatment; flux through the malic enzyme was

  2. Human rhabdomyosarcoma cells express functional erythropoietin receptor: Potential therapeutic implications

    PubMed Central

    PONIEWIERSKA-BARAN, AGATA; SUSZYNSKA, MALWINA; SUN, WENYUE; ABDELBASET-ISMAIL, AHMED; SCHNEIDER, GABRIELA; BARR, FREDERIC G.; RATAJCZAK, MARIUSZ Z.

    2015-01-01

    The erythropoietin receptor (EpoR) is expressed by cells from the erythroid lineage; however, evidence has accumulated that it is also expressed by some solid tumors. This is an important observation, because recombinant erythropoietin (EPO) is employed in cancer patients to treat anemia related to chemo/radiotherapy. In our studies we employed eight rhabdomyosarcoma (RMS) cell lines (three alveolar-type RMS cell lines and five embrional-type RMS cell lines), and mRNA samples obtained from positive, PAX7-FOXO1-positive, and fusion-negative RMS patient samples. Expression of EpoR was evaluated by RT-PCR, gene array and FACS. The functionality of EpoR in RMS cell lines was evaluated by chemotaxis, adhesion, and direct cell proliferation assays. In some of the experiments, RMS cells were exposed to vincristine (VCR) in the presence or absence of EPO to test whether EPO may impair the therapeutic effect of VCR. We report for a first time that functional EpoR is expressed in human RMS cell lines as well as by primary tumors from RMS patients. Furthermore, EpoR is detectably expressed in both embryonal and alveolar RMS subtypes. At the functional level, several human RMS cell lines responded to EPO stimulation by enhanced proliferation, chemotaxis, cell adhesion, and phosphorylation of MAPKp42/44 and AKT. Moreover, RMS cells became more resistant to VCR treatment in the presence of EPO. Our findings have important potential clinical implications, indicating that EPO supplementation in RMS patients may have the unwanted side effect of tumor progression. PMID:26412593

  3. Human rhabdomyosarcoma cells express functional erythropoietin receptor: Potential therapeutic implications.

    PubMed

    Poniewierska-Baran, Agata; Suszynska, Malwina; Sun, Wenyue; Abdelbaset-Ismail, Ahmed; Schneider, Gabriela; Barr, Frederic G; Ratajczak, Mariusz Z

    2015-11-01

    The erythropoietin receptor (EpoR) is expressed by cells from the erythroid lineage; however, evidence has accumulated that it is also expressed by some solid tumors. This is an important observation, because recombinant erythropoietin (EPO) is employed in cancer patients to treat anemia related to chemo/radiotherapy. In our studies we employed eight rhabdomyosarcoma (RMS) cell lines (three alveolar-type RMS cell lines and five embrional-type RMS cell lines), and mRNA samples obtained from positive, PAX7-FOXO1-positive, and fusion-negative RMS patient samples. Expression of EpoR was evaluated by RT-PCR, gene array and FACS. The functionality of EpoR in RMS cell lines was evaluated by chemotaxis, adhesion, and direct cell proliferation assays. In some of the experiments, RMS cells were exposed to vincristine (VCR) in the presence or absence of EPO to test whether EPO may impair the therapeutic effect of VCR. We report for a first time that functional EpoR is expressed in human RMS cell lines as well as by primary tumors from RMS patients. Furthermore, EpoR is detectably expressed in both embryonal and alveolar RMS subtypes. At the functional level, several human RMS cell lines responded to EPO stimulation by enhanced proliferation, chemotaxis, cell adhesion, and phosphorylation of MAPKp42/44 and AKT. Moreover, RMS cells became more resistant to VCR treatment in the presence of EPO. Our findings have important potential clinical implications, indicating that EPO supplementation in RMS patients may have the unwanted side effect of tumor progression. PMID:26412593

  4. Magnetic field-controlled gene expression in encapsulated cells

    PubMed Central

    Ortner, Viktoria; Kaspar, Cornelius; Halter, Christian; Töllner, Lars; Mykhaylyk, Olga; Walzer, Johann; Günzburg, Walter H.; Dangerfield, John A.; Hohenadl, Christine; Czerny, Thomas

    2012-01-01

    Cell and gene therapies have an enormous range of potential applications, but as for most other therapies, dosing is a critical issue, which makes regulated gene expression a prerequisite for advanced strategies. Several inducible expression systems have been established, which mainly rely on small molecules as inducers, such as hormones or antibiotics. The application of these inducers is difficult to control and the effects on gene regulation are slow. Here we describe a novel system for induction of gene expression in encapsulated cells. This involves the modification of cells to express potential therapeutic genes under the control of a heat inducible promoter and the co-encapsulation of these cells with magnetic nanoparticles. These nanoparticles produce heat when subjected to an alternating magnetic field; the elevated temperatures in the capsules then induce gene expression. In the present study we define the parameters of such systems and provide proof-of-principle using reporter gene constructs. The fine-tuned heating of nanoparticles in the magnetic field allows regulation of gene expression from the outside over a broad range and within short time. Such a system has great potential for advancement of cell and gene therapy approaches. PMID:22197778

  5. Leukomogenic factors downregulate heparanase expression in acute myeloid leukemia cells

    SciTech Connect

    Eshel, Rinat; Ben-Zaken, Olga; Vainas, Oded; Nadir, Yona; Minucci, Saverio; Polliack, Aaron; Naparstek, Ella; Vlodavsky, Israel; Katz, Ben-Zion; E-mail: bkatz@tasmc.healt.gov.il

    2005-10-07

    Heparanase is a heparan sulfate-degrading endoglycosidase expressed by mature monocytes and myeloid cells, but not by immature hematopoietic progenitors. Heparanase gene expression is upregulated during differentiation of immature myeloid cells. PML-RAR{alpha} and PLZF-RAR{alpha} fusion gene products associated with acute promyelocytic leukemia abrogate myeloid differentiation and heparanase expression. AML-Eto, a translocation product associated with AML FAB M2, also downregulates heparanase gene expression. The common mechanism that underlines the activity of these three fusion gene products involves the recruitment of histone deacetylase complexes to specific locations within the DNA. We found that retinoic acid that dissociates PML-RAR{alpha} from the DNA, and which is used to treat acute promyelocytic leukemia patients, restores heparanase expression to normal levels in an acute promyelocytic leukemia cell line. The retinoic acid effects were also observed in primary acute promyelocytic leukemia cells and in a retinoic acid-treated acute promyelocytic leukemia patient. Histone deacetylase inhibitor reverses the downregulation of heparanase expression induced by the AML-Eto fusion gene product in M2 type AML. In summary, we have characterized a link between leukomogenic factors and the downregulation of heparanase in myeloid leukemic cells.

  6. Inducible regulation of GDNF expression in human neural stem cells.

    PubMed

    Wang, ShuYan; Ren, Ping; Guan, YunQian; Zou, ChunLin; Fu, LinLin; Zhang, Yu

    2013-01-01

    Glial cell derived neurotrophic factor (GDNF) holds promises for treating neurodegenerative diseases such as Parkinson's disease. Human neural stem cells (hNSCs) have proved to be a suitable cell delivery vehicle for the safe and efficient introduction of GDNF into the brain. In this study, we used hNSCs-infected with a lentivirus encoding GDNF and the hygromycin resistance gene as such vehicles. A modified tetracycline operator 7 (tetO7) was inserted into a region upstream of the EF1-α promoter to drive GDNF expression. After hygromycin selection, hNSCs were infected with a lentivirus encoding a KRAB-tetracycline repressor fusion protein (TTS). TTS bound to tetO7 and suppressed the expression of GDNF in hNSCs. Upon administration of doxycycline (Dox) the TTS-tetO7 complex separated and the expression of GDNF resumed. The hNSCs infected with GDNF expressed the neural stem cell specific markers, nestin and sox2, and exhibited no significant change in proliferation rate. However, the rate of apoptosis in hNSCs expressing GDNF was lower compared with normal NSCs in response to actinomycin treatment. Furthermore, a higher percentage of Tuj-1 positive cells were obtained from GDNF-producing NSCs under conditions that induced differentiation compared to control NSCs. The inducible expression of GDNF in hNSCs may provide a system for the controllable delivery of GDNF in patients with neurodegenerative diseases. PMID:23269553

  7. All-optical regulation of gene expression in targeted cells

    NASA Astrophysics Data System (ADS)

    Wang, Yisen; He, Hao; Li, Shiyang; Liu, Dayong; Lan, Bei; Hu, Minglie; Cao, Youjia; Wang, Chingyue

    2014-06-01

    Controllable gene expression is always a challenge and of great significance to biomedical research and clinical applications. Recently, various approaches based on extra-engineered light-sensitive proteins have been developed to provide optogenetic actuators for gene expression. Complicated biomedical techniques including exogenous genes engineering, transfection, and material delivery are needed. Here we present an all-optical method to regulate gene expression in targeted cells. Intrinsic or exogenous genes can be activated by a Ca2+-sensitive transcription factor nuclear factor of activated T cells (NFAT) driven by a short flash of femtosecond-laser irradiation. When applied to mesenchymal stem cells, expression of a differentiation regulator Osterix can be activated by this method to potentially induce differentiation of them. A laser-induced ``Ca2+-comb'' (LiCCo) by multi-time laser exposure is further developed to enhance gene expression efficiency. This noninvasive method hence provides an encouraging advance of gene expression regulation, with promising potential of applying in cell biology and stem-cell science.

  8. Estradiol regulates MICA expression in human endometrial cells

    PubMed Central

    Basu, Satarupa; Pioli, Patricia A.; Conejo-Garcia, Jose; Wira, Charles R.; Sentman, Charles L.

    2008-01-01

    The human endometrium undergoes cyclical changes regulated by sex hormones. Evidence suggests sex hormones regulate NK cell recruitment into the uterus in large numbers. NKG2D is an activating receptor expressed on human NK cells, γδ and CD8 T cells. NKG2D ligands are known to be sensors of cellular “stress”. In this study, we investigated whether sex hormones directly regulate expression of NKG2D ligands in the human uterus. Estradiol increased MICA expression on uterine epithelial cells; regulation was estrogen receptor-dependent. Real-time PCR analysis showed that NKG2D ligands MICA and MICB were expressed in the human endometrium. MICA protein was detected primarily on epithelial cells, and greater expression was observed in immunohistochemical analysis of tissues from patients in the secretory phase of the menstrual cycle. Thus, estrogens regulate expression of MICA. These data suggest hormonal regulation of innate immunity and NKG2D-mediated recognition in other tissues and diseases where estrogen may be involved. PMID:18728002

  9. The expression of ADAMTS13 in human microvascular endothelial cells.

    PubMed

    Wang, Anyou; Duan, Qiaohong; Wu, Jingsheng; Liu, Xin; Sun, Zimin

    2016-06-01

    ADAMTS13, as a specific von Willebrand factor (VWF)-cleaving protease, prevents microvascular thrombosis of VWF/platelet thrombi. It has been reported that human vascular endothelial cells could also synthesize and secrete ADAMTS13, and these reports were focused in human umbilical vascular endothelial cells. Considering the particularity of its huge quantity and structure of human microvascular endothelial cells (HMECs) in the body, whether ADAMTS13 is expressed in HMECs also needs to be confirmed. To investigate whether ADAMTS13 is expressed in HMECs. Real-time PCR (RT-PCR) amplification detected ADAMTS13 mRNA in HMEC-1 cell line. The expression and distribution of ADAMTS13 protein and VWF were detected by fluorescence immunoassay and western blot. We observed the expression and distribution of ADAMTS13 in HMECs. We confirmed the expression of ADAMTS13 mRNA in HMEC-1, and found that there were some partly common distributions of ADAMTS13 protein and VWF. This study provides the evidence that HMECs also express ADAMTS13. HMECs might also be a primary source for human plasma ADAMTS13. The overlap region for the distribution of ADAMTS13 and VWF suggests that ADAMTS13 might have a potential regulation role for VWF inside cells. PMID:26366828

  10. Expression of the somatostatin gene in human astrocytoma cell lines.

    PubMed Central

    Mercure, L; Tannenbaum, G S; Schipper, H M; Phaneuf, D; Wainberg, M A

    1996-01-01

    Somatostatin (somatotropin release-inhibiting hormone; SRIH) has been demonstrated in neurons of the central nervous system (CNS) as well as in endocrine cells of the pancreas and gastrointestinal tract and can suppress various immune functions including lymphocyte proliferation, immunoglobulin synthesis, and cytokine production. Since astrocytes possess antigen-presenting activity and can secrete a wide array of immunoregulatory and inflammatory cytokines, we studied SRIH gene expression in both astrocyte cell lines and mitogen-stimulated peripheral blood mononuclear leukocytes from healthy donors. We now report by means of a complementary DNA-based reverse transcription PCR that differential levels of SRIH mRNA were expressed in 9 of 11 human astrocytoma cell lines tested but were undetectable in activated peripheral blood mononuclear leukocytes as well as in a variety of human lymphocyte and monocyte cell lines. The synthesis and secretion of SRIH protein by astrocytoma cells that expressed SRIH transcripts were confirmed by specific radioimmunoassay of cell culture fluids. These findings support the notion that SRIH gene expression occurs in human astrocytoma cells but not in mature lymphoid cells of the immune system. PMID:8991628

  11. Structural Determinants of Binding the Seven-transmembrane Domain of the Glucagon-like Peptide-1 Receptor (GLP-1R).

    PubMed

    Yang, Dehua; de Graaf, Chris; Yang, Linlin; Song, Gaojie; Dai, Antao; Cai, Xiaoqing; Feng, Yang; Reedtz-Runge, Steffen; Hanson, Michael A; Yang, Huaiyu; Jiang, Hualiang; Stevens, Raymond C; Wang, Ming-Wei

    2016-06-17

    The glucagon-like peptide-1 receptor (GLP-1R) belongs to the secretin-like (class B) family of G protein-coupled receptors. Members of the class B family are distinguished by their large extracellular domain, which works cooperatively with the canonical seven-transmembrane (7TM) helical domain to signal in response to binding of various peptide hormones. We have combined structure-based site-specific mutational studies with molecular dynamics simulations of a full-length model of GLP-1R bound to multiple peptide ligand variants. Despite the high sequence similarity between GLP-1R and its closest structural homologue, the glucagon receptor (GCGR), nearly half of the 62 stably expressed mutants affected GLP-1R in a different manner than the corresponding mutants in GCGR. The molecular dynamics simulations of wild-type and mutant GLP-1R·ligand complexes provided molecular insights into GLP-1R-specific recognition mechanisms for the N terminus of GLP-1 by residues in the 7TM pocket and explained how glucagon-mimicking GLP-1 mutants restored binding affinity for (GCGR-mimicking) GLP-1R mutants. Structural analysis of the simulations suggested that peptide ligand binding mode variations in the 7TM binding pocket are facilitated by movement of the extracellular domain relative to the 7TM bundle. These differences in binding modes may account for the pharmacological differences between GLP-1 peptide variants. PMID:27059958

  12. Dual elimination of the glucagon and GLP-1 receptors in mice reveals plasticity in the incretin axis.

    PubMed

    Ali, Safina; Lamont, Benjamin J; Charron, Maureen J; Drucker, Daniel J

    2011-05-01

    Disordered glucagon secretion contributes to the symptoms of diabetes, and reduced glucagon action is known to improve glucose homeostasis. In mice, genetic deletion of the glucagon receptor (Gcgr) results in increased levels of the insulinotropic hormone glucagon-like peptide 1 (GLP-1), which may contribute to the alterations in glucose homeostasis observed in Gcgr-/- mice. Here, we assessed the contribution of GLP-1 receptor (GLP-1R) signaling to the phenotype of Gcgr-/- mice by generating Gcgr-/-Glp1r-/- mice. Although insulin sensitivity was similar in all genotypes, fasting glucose was increased in Gcgr-/-Glp1r-/- mice. Elimination of the Glp1r normalized gastric emptying and impaired intraperitoneal glucose tolerance in Gcgr-/- mice. Unexpectedly, deletion of Glp1r in Gcgr-/- mice did not alter the improved oral glucose tolerance and increased insulin secretion characteristic of that genotype. Although Gcgr-/-Glp1r-/- islets exhibited increased sensitivity to the incretin glucose-dependent insulinotropic polypeptide (GIP), mice lacking both Glp1r and the GIP receptor (Gipr) maintained preservation of the enteroinsular axis following reduction of Gcgr signaling. Moreover, Gcgr-/-Glp1r-/- islets expressed increased levels of the cholecystokinin A receptor (Cckar) and G protein-coupled receptor 119 (Gpr119) mRNA transcripts, and Gcgr-/-Glp1r-/- mice exhibited increased sensitivity to exogenous CCK and the GPR119 agonist AR231453. Our data reveal extensive functional plasticity in the enteroinsular axis via induction of compensatory mechanisms that control nutrient-dependent regulation of insulin secretion. PMID:21540554

  13. PAX8 Expression in Ovarian Surface Epithelial Cells

    PubMed Central

    Adler, Emily; Mhawech-Fauceglia, Paulette; Gayther, Simon A; Lawrenson, Kate

    2015-01-01

    High-grade serous ovarian carcinoma (HGSOC) is usually diagnosed at a late stage and is associated with poor prognosis. Understanding early stage disease biology is essential in developing clinical biomarkers to detect HGSOC earlier. While recent studies indicate that HGSOCs arise from fallopian tube secretory epithelial cells (FTSECs), a considerable body of evidence also suggests that HGSOC can also arise from ovarian surface epithelial cells (OSECs). PAX8 is overexpressed in HGSOCs and expressed in FTSECs, but there are conflicting reports about PAX8 expression in OSECs. The purpose of this study was to comprehensively characterize PAX8 expression in a large series of OSECs, and to investigate the role of PAX8 in early HGSOC development. PAX8 protein expression was analyzed in the OSECs of 27 normal ovaries and 7 primary OSEC cultures using immunohistochemistry and immunofluorescent cytochemistry. PAX8 mRNA expression was quantified in 66 primary OSEC cultures. Cellular transformation was evaluated in OSECs expressing a PAX8 construct. PAX8 was expressed by 44-71% of OSECs. Calretinin and E-cadherin were frequently co-expressed with PAX8. Expression of PAX8 in OSECs decreased cellular migration (P=0.028), but had no other effects on cellular transformation. In addition, PAX8 expression was significantly increased (P=0.003) in an in vitro stepwise model of neoplastic transformation. In conclusion, PAX8 is frequently expressed by OSECs and endogenous levels of PAX8 expression are non-transforming. These data indicate that in OSECs PAX8 expression may represent a normal state and that OSECs may represent an origin of HGSOCs. PMID:26079312

  14. HLA antigen expression in enteropathy associated T cell lymphoma.

    PubMed Central

    Ashton-Key, M; Singh, N; Pan, L X; Smith, M E

    1996-01-01

    AIMS: To investigate the occurrence of abnormal patterns of HLA-ABC and HLA-DR expression in enteropathy associated T cell lymphoma and to relate such abnormalities to the Epstein Barr virus (EBV) status of the tumours. METHODS: Eleven enteropathy associated T cell lymphomas were immunostained with HC10 (HLA-ABC heavy chain) and TAL 1B5 (HLA-DR alpha chain) monoclonal antibodies and polyclonal anti-beta 2 microglobulin (beta 2m, the HLA-ABC light chain) antibodies. In situ hybridisation for EBV using EBER probes was performed on all cases. RESULTS: Tumour cells of two of 11 patients were EBER positive. One of these showed partial, and the other, complete loss of beta 2m. HLA-DR expression was undetectable in both patients. Of the remaining nine EBER negative tumours, two were HLA-ABC heavy chain negative or showed only occasional positive cells and five of nine showed partial or complete loss of the HLA-ABC light chain, beta 2m. Seven of the nine cases were either negative for HLA-DR or showed weak expression in a proportion of tumour cells. CONCLUSIONS: These data show that low or absent HLA-ABC and HLA-DR antigen expression occurs commonly in enteropathy associated T cell lymphoma. These abnormal patterns of HLA expression may be associated with escape from immune attack which, in a minority of patients, could be directed against EBV antigens. Images PMID:8813950

  15. Cell-Free Expression of G Protein-Coupled Receptors.

    PubMed

    Segers, Kenneth; Masure, Stefan

    2015-01-01

    The large-scale production of recombinant G protein-coupled receptors (GPCRs) is one of the major bottlenecks that hamper functional and structural studies of this important class of integral membrane proteins. Heterologous overexpression of GPCRs often results in low yields of active protein, usually due to a combination of several factors, such as low expression levels, protein insolubility, host cell toxicity, and the need to use harsh and often denaturing detergents (e.g., SDS, LDAO, OG, and DDM, among others) to extract the recombinant receptor from the host cell membrane. Many of these problematic issues are inherently linked to cell-based expression systems and can therefore be circumvented by the use of cell-free systems. In this unit, we provide a range of protocols for the production of GPCRs in a cell-free expression system. Using this system, we typically obtain GPCR expression levels of ∼1 mg per ml of reaction mixture in the continuous-exchange configuration. Although the protocols in this unit have been optimized for the cell-free expression of GPCRs, they should provide a good starting point for the production of other classes of membrane proteins, such as ion channels, aquaporins, carrier proteins, membrane-bound enzymes, and even large molecular complexes. PMID:26237676

  16. Positive Allosteric Modulation of the Glucagon-like Peptide-1 Receptor by Diverse Electrophiles.

    PubMed

    Bueno, Ana B; Showalter, Aaron D; Wainscott, David B; Stutsman, Cynthia; Marín, Aranzazu; Ficorilli, James; Cabrera, Over; Willard, Francis S; Sloop, Kyle W

    2016-05-13

    Therapeutic intervention to activate the glucagon-like peptide-1 receptor (GLP-1R) enhances glucose-dependent insulin secretion and improves energy balance in patients with type 2 diabetes mellitus. Studies investigating mechanisms whereby peptide ligands activate GLP-1R have utilized mutagenesis, receptor chimeras, photo-affinity labeling, hydrogen-deuterium exchange, and crystallography of the ligand-binding ectodomain to establish receptor homology models. However, this has not enabled the design or discovery of drug-like non-peptide GLP-1R activators. Recently, studies investigating 4-(3-benzyloxyphenyl)-2-ethylsulfinyl-6-(trifluoromethyl)pyrimidine (BETP), a GLP-1R-positive allosteric modulator, determined that Cys-347 in the GLP-1R is required for positive allosteric modulator activity via covalent modification. To advance small molecule activation of the GLP-1R, we characterized the insulinotropic mechanism of BETP. In guanosine 5'-3-O-(thio)triphosphate binding and INS1 832-3 insulinoma cell cAMP assays, BETP enhanced GLP-1(9-36)-NH2-stimulated cAMP signaling. Using isolated pancreatic islets, BETP potentiated insulin secretion in a glucose-dependent manner that requires both the peptide ligand and GLP-1R. In studies of the covalent mechanism, PAGE fluorography showed labeling of GLP-1R in immunoprecipitation experiments from GLP-1R-expressing cells incubated with [(3)H]BETP. Furthermore, we investigated whether other reported GLP-1R activators and compounds identified from screening campaigns modulate GLP-1R by covalent modification. Similar to BETP, several molecules were found to enhance GLP-1R signaling in a Cys-347-dependent manner. These chemotypes are electrophiles that react with GSH, and LC/MS determined the cysteine adducts formed upon conjugation. Together, our results suggest covalent modification may be used to stabilize the GLP-1R in an active conformation. Moreover, the findings provide pharmacological guidance for the discovery and

  17. Positive Allosteric Modulation of the Glucagon-like Peptide-1 Receptor by Diverse Electrophiles*

    PubMed Central

    Showalter, Aaron D.; Wainscott, David B.; Stutsman, Cynthia; Marín, Aranzazu; Ficorilli, James; Cabrera, Over

    2016-01-01

    Therapeutic intervention to activate the glucagon-like peptide-1 receptor (GLP-1R) enhances glucose-dependent insulin secretion and improves energy balance in patients with type 2 diabetes mellitus. Studies investigating mechanisms whereby peptide ligands activate GLP-1R have utilized mutagenesis, receptor chimeras, photo-affinity labeling, hydrogen-deuterium exchange, and crystallography of the ligand-binding ectodomain to establish receptor homology models. However, this has not enabled the design or discovery of drug-like non-peptide GLP-1R activators. Recently, studies investigating 4-(3-benzyloxyphenyl)-2-ethylsulfinyl-6-(trifluoromethyl)pyrimidine (BETP), a GLP-1R-positive allosteric modulator, determined that Cys-347 in the GLP-1R is required for positive allosteric modulator activity via covalent modification. To advance small molecule activation of the GLP-1R, we characterized the insulinotropic mechanism of BETP. In guanosine 5′-3-O-(thio)triphosphate binding and INS1 832-3 insulinoma cell cAMP assays, BETP enhanced GLP-1(9–36)-NH2-stimulated cAMP signaling. Using isolated pancreatic islets, BETP potentiated insulin secretion in a glucose-dependent manner that requires both the peptide ligand and GLP-1R. In studies of the covalent mechanism, PAGE fluorography showed labeling of GLP-1R in immunoprecipitation experiments from GLP-1R-expressing cells incubated with [3H]BETP. Furthermore, we investigated whether other reported GLP-1R activators and compounds identified from screening campaigns modulate GLP-1R by covalent modification. Similar to BETP, several molecules were found to enhance GLP-1R signaling in a Cys-347-dependent manner. These chemotypes are electrophiles that react with GSH, and LC/MS determined the cysteine adducts formed upon conjugation. Together, our results suggest covalent modification may be used to stabilize the GLP-1R in an active conformation. Moreover, the findings provide pharmacological guidance for the discovery and

  18. Expression profiling. Combinatorial labeling of single cells for gene expression cytometry.

    PubMed

    Fan, H Christina; Fu, Glenn K; Fodor, Stephen P A

    2015-02-01

    We present a technically simple approach for gene expression cytometry combining next-generation sequencing with stochastic barcoding of single cells. A combinatorial library of beads bearing cell- and molecular-barcoding capture probes is used to uniquely label transcripts and reconstruct the digital gene expression profile of thousands of individual cells in a single experiment without the need for robotics or automation. We applied the technology to dissect the human hematopoietic system and to characterize heterogeneous response to in vitro stimulation. High sensitivity is demonstrated by detection of low-abundance transcripts and rare cells. Under current implementation, the technique can analyze a few thousand cells simultaneously and can readily scale to 10,000s or 100,000s of cells. PMID:25657253

  19. Significance of Parafibromin Expression in Laryngeal Squamous Cell Carcinomas

    PubMed Central

    Cho, Inju; Lee, Mija; Lim, Sharon; Hong, Ran

    2016-01-01

    Background: Parafibromin is a product of the tumor suppressor gene that has been studied as a potential indicator of tumor aggressiveness in the parathyroid, breast, colorectum, and stomach. However, the clinical significance and potential function of parafibromin expression in head and neck squamous cell carcinomas remain largely unknown. The aim of this study was to evaluate the expression of parafibromin in laryngeal squamous cell carcinoma (LSCC) and to verify its potential as a biomarker of tumor behavior. Methods: Parafibromin expression was evaluated in 30 cases of LSCC using immunohistochemistry. The correlations between parafibromin expression and clinicopathologic parameters were investigated. Results: Parafibromin expression was positive in 15 cases (50%) and negative in 15 cases (50%). Tumor size and T stage showed a statistically significant inverse relationship with parafibromin expression (p=.028 and p<.001, respectively). Parafibromin expression was not associated with age, sex, lymph node metastasis, tumor differentiation, or tumor location. There was no statistically significant relationship between parafibromin expression and progression-free survival in the patients (p>.05). Conclusions: Our results indicate that the downregulation or loss of parafibromin expression can be employed as a novel marker of tumor progression or aggressiveness in LSCC. PMID:27334641

  20. B cell priming for extrafollicular antibody responses requires Bcl-6 expression by T cells.

    PubMed

    Lee, Sau K; Rigby, Robert J; Zotos, Dimitra; Tsai, Louis M; Kawamoto, Shimpei; Marshall, Jennifer L; Ramiscal, Roybel R; Chan, Tyani D; Gatto, Dominique; Brink, Robert; Yu, Di; Fagarasan, Sidonia; Tarlinton, David M; Cunningham, Adam F; Vinuesa, Carola G

    2011-07-01

    T follicular helper cells (Tfh cells) localize to follicles where they provide growth and selection signals to mutated germinal center (GC) B cells, thus promoting their differentiation into high affinity long-lived plasma cells and memory B cells. T-dependent B cell differentiation also occurs extrafollicularly, giving rise to unmutated plasma cells that are important for early protection against microbial infections. Bcl-6 expression in T cells has been shown to be essential for the formation of Tfh cells and GC B cells, but little is known about its requirement in physiological extrafollicular antibody responses. We use several mouse models in which extrafollicular plasma cells can be unequivocally distinguished from those of GC origin, combined with antigen-specific T and B cells, to show that the absence of T cell-expressed Bcl-6 significantly reduces T-dependent extrafollicular antibody responses. Bcl-6(+) T cells appear at the T-B border soon after T cell priming and before GC formation, and these cells express low amounts of PD-1. Their appearance precedes that of Bcl-6(+) PD-1(hi) T cells, which are found within the GC. IL-21 acts early to promote both follicular and extrafollicular antibody responses. In conclusion, Bcl-6(+) T cells are necessary at B cell priming to form extrafollicular antibody responses, and these pre-GC Tfh cells can be distinguished phenotypically from GC Tfh cells. PMID:21708925

  1. SPARC expression induces cell cycle arrest via STAT3 signaling pathway in medulloblastoma cells

    SciTech Connect

    Chetty, Chandramu; Dontula, Ranadheer; Gujrati, Meena; Lakka, Sajani S.

    2012-01-13

    Highlights: Black-Right-Pointing-Pointer Ectopic expression of SPARC impaired cell proliferation in medulloblastoma cells. Black-Right-Pointing-Pointer SPARC expression induces STAT3 mediated cell cycle arrest in medulloblastoma cells. Black-Right-Pointing-Pointer SPARC expression significantly inhibited pre-established tumor growth in nude-mice. -- Abstract: Dynamic cell interaction with ECM components has profound influence in cancer progression. SPARC is a component of the ECM, impairs the proliferation of different cell types and modulates tumor cell aggressive features. We previously reported that SPARC expression significantly impairs medulloblastoma tumor growth in vivo. In this study, we demonstrate that expression of SPARC inhibits medulloblastoma cell proliferation. MTT assay indicated a dose-dependent reduction in tumor cell proliferation in adenoviral mediated expression of SPARC full length cDNA (Ad-DsRed-SP) in D425 and UW228 cells. Flow cytometric analysis showed that Ad-DsRed-SP-infected cells accumulate in the G2/M phase of cell cycle. Further, immunoblot and immunoprecipitation analyses revealed that SPARC induced G2/M cell cycle arrest was mediated through inhibition of the Cyclin-B-regulated signaling pathway involving p21 and Cdc2 expression. Additionally, expression of SPARC decreased STAT3 phosphorylation at Tyr-705; constitutively active STAT3 expression reversed SPARC induced G2/M arrest. Ad-DsRed-SP significantly inhibited the pre-established orthotopic tumor growth and tumor volume in nude-mice. Immunohistochemical analysis of tumor sections from mice treated with Ad-DsRed-SP showed decreased immunoreactivity for pSTAT3 and increased immunoreactivity for p21 compared to tumor section from mice treated with mock and Ad-DsRed. Taken together our studies further reveal that STAT3 plays a key role in SPARC induced G2/M arrest in medulloblastoma cells. These new findings provide a molecular basis for the mechanistic understanding of the

  2. 293 cells express both epithelial as well as mesenchymal cell adhesion molecules

    PubMed Central

    INADA, MASAKAZU; IZAWA, GENYA; KOBAYASHI, WAKAKO; OZAWA, MASAYUKI

    2016-01-01

    The 293 cell line, used extensively in various types of studies due to the ease with which these cells can be transfected, was thought to be derived by the transformation of primary cultures of human embryonic kidney cells with sheared adenovirus type 5 DNA. Although the 293 cells were assumed to originate from epithelial cells, the exact origin of these cells remains unknown. Previous attempts to characterize these cells combined immunostaining, immunoblot analysis and microarray analysis to demonstrate that 293 cells express neurofilament subunits, α-internexin, and several other proteins typically found in neurons. These findings raised the possibility that the 293 cell line may have originated from human neuronal lineage cells. Contrary to this suggestion, in this study, we found that the 293 cells expressed N-cadherin and vimentin, which are marker proteins expressed in mesenchymal cells. Furthermore, the 293 cells also expressed E-cadherin, cytokeratins 5/8 and desmoglein 2, which are epithelial cell markers. When the cells, primarily cultured from the kidneys of Clawn miniature swine and passaged 10–15 generations [termed porcine kidney epithelial (PKE) cells] were examined, they were found to be positive for the expression of both mesenchymal and epithelial markers. Thus, transformation by adenovirus was not necessary for the cells to express N-cadherin. Occludin and zonula occludens (ZO)-1, two components of tight junctions in epithelial and endothelial cells, were detected in the 293 and the PKE cells. Thus, the findings of the present study demonstrate that 293 cells retain several characteristics of epithelial cells. PMID:27121032

  3. Reduced Ang2 expression in aging endothelial cells.

    PubMed

    Hohensinner, P J; Ebenbauer, B; Kaun, C; Maurer, G; Huber, K; Wojta, J

    2016-06-01

    Aging endothelial cells are characterized by increased cell size, reduced telomere length and increased expression of proinflammatory cytokines. In addition, we describe here that aging reduces the migratory distance of endothelial cells. Furthermore, we observe an increase of the quiescence protein Ang1 and a decrease of the endothelial activation protein Ang2 upon aging. Supplementing Ang2 to aged endothelial cells restored their migratory capacity. We conclude that aging shifts the balance of the Ang1/Ang2 network favouring a quiescent state. Activation of endothelial cells in aging might be necessary to enhance wound healing capacities. PMID:27137842

  4. Immunoglobulin Expression in Non-Lymphoid Lineage and Neoplastic Cells

    PubMed Central

    Chen, Zhengshan; Qiu, Xiaoyan; Gu, Jiang

    2009-01-01

    It has traditionally been believed that the production of immunoglobulin (Ig) molecules is restricted to B lineage cells. However, immunoglobulin genes and proteins have been recently found in a variety of types of cancer cells, as well as some proliferating epithelial cells and neurons. The immunoglobulin molecules expressed by these cells consist predominantly of IgG, IgM, and IgA, and the light chains expressed are mainly kappa chains. Recombination activating genes 1 and 2, which are required for V(D)J recombination, are also expressed in these cells. Knowledge about the function of these non-lymphoid cell-derived immunoglobulins is limited. Preliminary data suggests that Ig secreted by epithelial cancer cells has some unidentified capacity to promote the growth and survival of tumor cells. As immunoglobulins are known to have a wide spectrum of important functions, the discovery of non-lymphoid cells and cancers that produce immunoglobulin calls for in-depth investigation of the functional and pathological significance of this previously unrecognized phenomenon. PMID:19246641

  5. Survivin expression is associated with lens epithelial cell proliferation and fiber cell differentiation

    PubMed Central

    Mansergh, Fiona C.; Boulton, Michael E.; Gunhaga, Lena

    2012-01-01

    Purpose Survivin (Birc5) is the smallest member of the inhibitor of apoptosis (IAP) protein family, which regulates the cell cycle/apoptosis balance. The purpose of this study was to examine Survivin expression in the embryonic chick lens, in chick lens epithelial cell cultures, and in the postnatal mouse lens. Methods Survivin expression was examined using a combination of quantitative real-time polymerase chain reaction, western blotting, and immunocytochemistry. To correlate Survivin expression with the timing of proliferation, we determined the profile of cell proliferation in the developing lens using the cell cycle marker proliferating cell nuclear antigen (PCNA) in quantitative western blotting and immunocytochemistry studies. We also examined the expression of PCNA and the extent of denucleation using terminal deoxynucleotidyl transferase (TdT)-mediated biotin-dUTP nick-end labeling (TUNEL) of lentoids (lens fiber-like cells) during chick lens epithelial cell differentiation in vitro. Results At embryonic day (ED) 4, Survivin immunostaining was present in two pools in lens epithelial cells and fiber cells: cytoplasmic and nuclear. The nuclear staining became more pronounced as the lens epithelial cells differentiated into lens fiber cells. At ED12, Survivin staining was observed in lens fiber cell nuclei containing marginalized chromatin, indicative of early denucleation events. Using western blotting, Survivin expression peaked at ED6, diminishing thereafter. This profile of expression correlated with the events in chick lens epithelial cell cultures: i) increased Survivin expression was associated with an increase in PCNA staining up to day 6 of culture and ii) downregulation of Survivin expression at day 8 of culture was coincident with a dramatic decrease in PCNA staining and an increase in TdT-mediated biotin-dUTP nick-end labeling in lentoids. In early postnatal mouse lenses, Survivin and PCNA were highly expressed and decreased thereafter during

  6. Effect of glucagon on intracellular pH regulation in isolated rat hepatocyte couplets.

    PubMed Central

    Alvaro, D; Della Guardia, P; Bini, A; Gigliozzi, A; Furfaro, S; La Rosa, T; Piat, C; Capocaccia, L

    1995-01-01

    To elucidate mechanisms of glucagon-induced bicarbonate-rich choleresis, we investigated the effect of glucagon on ion transport processes involved in the regulation of intracellular pH (pHi) in isolated rat hepatocyte couplets. It was found that glucagon (200 nM), without influencing resting pHi, significantly stimulates the Cl-/HCO3- exchange activity. The effect of glucagon was associated with a sevenfold increase in cAMP levels in rat hepatocytes. The activity of the Cl-/HCO3- exchanger was also stimulated by DBcAMP + forskolin. The effect of glucagon on the Cl-/HCO3- exchange was individually blocked by two specific and selective inhibitors of protein kinase A, Rp-cAMPs (10 microM) and H-89 (30 microM), the latter having no influence on the glucagon-induced cAMP accumulation in isolated rat hepatocytes. The Cl- channel blocker, NPPB (10 microM), showed no effect on either the basal or the glucagon-stimulated Cl-/HCO3 exchange. In contrast, the protein kinase C agonist, PMA (10 microM), completely blocked the glucagon stimulation of the Cl-/HCO3- exchange; however, this effect was achieved through a significant inhibition of the glucagon-stimulated cAMP accumulation in rat hepatocytes. Colchicine pretreatment inhibited the basal as well as the glucagon-stimulated Cl-/HCO3- exchange activity. The Na+/H+ exchanger was unaffected by glucagon either at basal pHi or at acid pHi values. In contrast, glucagon, at basal pHi, stimulated the Na(+)-HCO3- symport. The main findings of this study indicate that glucagon, through the cAMP-dependent protein kinase A pathway, stimulates the activity of the Cl-/HCO3- exchanger in isolated rat hepatocyte couplets, a mechanism which could account for the in vivo induced bicarbonate-rich choleresis. Images PMID:7635959

  7. Glucagon clearance is regulated by nutritional state: evidence from experimental studies in mice

    PubMed Central

    Zhou, Alyssa; Pacini, Giovanni; Ahrén, Bo; D’Argenio, David Z.

    2014-01-01

    Aims/hypothesis Given the importance of glucagon in the development of type 2 diabetes and as a potential therapeutic agent, the aim of this study was to characterise glucagon kinetics in mice and its regulation by the nutritional state. Methods Anaesthetised C57BL/6 mice fed normal or high-fat diets, or fasted, were injected intravenously with glucagon (0.1, 0.3, 1.0, 10.0 or 20 μg/kg); blood samples were withdrawn before injection and 1, 3, 5, 10, 20 min thereafter for glucagon assay by RIA. Glucagon kinetics were described by two-compartment models using a population analysis. Results The population mean and between-animal SD of glucagon clearance in the fed mice was 6.03 ± 2.58 ml/min, with a rapid elimination half-life of 2.92 ± 1.21 min. Fasted mice showed a slower glucagon clearance. The kinetics of glucagon in the fed and fasted group was linear across this large dose range. The mice fed a high-fat diet, however, showed non-linear kinetics with a faster terminal clearance of 20.4 ± 5.45 ml/min (p < 0.001) and a shorter elimination half-life of 1.59 ± 0.606 (p < 0.001) min relative to normal mice. Conclusions/interpretation This first systematic dose-ranging study of glucagon kinetics produced several findings: (1) a linear two-compartment model describes glucagon in normal C57BL/6 mice; (2) fasting reduces the clearance of glucagon and (3) high-fat diet enhances the clearance of glucagon. These results may direct future studies on glucagon physiology and indicate that there are other mechanisms, not included in the current model, needed to fully explain glucagon’s kinetics. PMID:24370975

  8. Enhanced expression of codon optimized interferon gamma in CHO cells.

    PubMed

    Chung, Bevan Kai-Sheng; Yusufi, Faraaz N K; Mariati; Yang, Yuansheng; Lee, Dong-Yup

    2013-09-10

    The human interferon-gamma (IFN-γ) is a potential drug candidate for treating various diseases due to its immunomodulatory properties. The efficient production of this protein can be achieved through a popular industrial host, Chinese hamster ovary (CHO) cells. However, recombinant expression of foreign proteins is typically suboptimal possibly due to the usage of non-native codon patterns within the coding sequence. Therefore, we demonstrated the application of a recently developed codon optimization approach to design synthetic IFN-γ coding sequences for enhanced heterologous expression in CHO cells. For codon optimization, earlier studies suggested to establish the target usage distribution pattern in terms of selected design parameters such as individual codon usage (ICU) and codon context (CC), mainly based on the host's highly expressed genes. However, our RNA-Seq based transcriptome profiling indicated that the ICU and CC distribution patterns of different gene expression classes in CHO cell are relatively similar, unlike other microbial expression hosts, Escherichia coli and Saccharomyces cerevisiae. This finding was further corroborated through the in vivo expression of various ICU and CC optimized IFN-γ in CHO cells. Interestingly, the CC-optimized genes exhibited at least 13-fold increase in expression level compared to the wild-type IFN-γ while a maximum of 10-fold increase was observed for the ICU-optimized genes. Although design criteria based on individual codons, such as ICU, have been widely used for gene optimization, our experimental results suggested that codon context is relatively more effective parameter for improving recombinant IFN-γ expression in CHO cells. PMID:23876479

  9. Glucagon-like peptide-1 protects cardiomyocytes from advanced oxidation protein product-induced apoptosis via the PI3K/Akt/Bad signaling pathway

    PubMed Central

    ZHANG, HUA; XIONG, ZHOUYI; WANG, JIAO; ZHANG, SHUANGSHUANG; LEI, LEI; YANG, LI; ZHANG, ZHEN

    2016-01-01

    Cardiomyocyte apoptosis is a major event in the pathogenesis of diabetic cardiomyopathy. Currently, no single effective treatment for diabetic cardiomyopathy exists. The present study investigated whether advanced oxidative protein products (AOPPs) have a detrimental role in the survival of cardiomyocytes and if glucagon-like peptide-1 (GLP-1) exerts a cardioprotective effect under these circumstances. The present study also aimed to determine the underlying mechanisms. H9c2 cells were exposed to increasing concentrations of AOPPs in the presence or absence of GLP-1, and the viability and apoptotic rate were detected using a cell counting kit-8 assay and flow cytometry, respectively. In addition, a phosphatidylino-sitol-4,5-bisphosphate 3-kinase (PI3K) inhibitor, LY294002, was employed to illustrate the mechanism of the antiapoptotic effect of GLP-1. The expression levels of the apoptotic-associated proteins, Akt, B-cell lymphoma (Bcl)-2, Bcl-2-associated death promoter (Bad), Bcl-2-associated X protein (Bax) and caspase-3 were measured by western blotting. It was revealed that GLP-1 significantly attenuated AOPP-induced cell toxicity and apoptosis. AOPPs inactivated the phosphorylation of Akt, reduced the phosphorylation of Bad, decreased the expression of Bcl-2, increased the expression of Bax and the activation of caspase-3 in H9c2 cells. GLP-1 reversed the above changes induced by AOPPs and the protective effects of GLP-1 were abolished by the PI3K inhibitor, LY294002. In conclusion, the present data suggested that GLP-1 protected cardiomyocytes against AOPP-induced apoptosis, predominantly via the PI3K/Akt/Bad pathway. These results provided a conceivable mechanism for the development of diabetic cardiomyopathy and rendered a novel application of GLP-1 exerting favorable cardiac effects for the treatment of diabetic cardiomyopathy. PMID:26717963

  10. Expression and stabilization of bacterial luciferase in mammalian cells

    NASA Astrophysics Data System (ADS)

    Patterson, Stacey S.; Dionisi, Hebe M.; Gupta, Rakesh K.; Sayler, Gary S.

    2004-06-01

    Current mammalian bioreporters using either firefly luciferase (luc) or GFP constructs require lysis and/or exogenous excitation to evoke a measurable response. Consequently, these cells cannot serve as continuous, on-line monitoring devices for in vivo imaging. Bacterial luciferase, lux, produces a photonic reaction that is cyclic, resulting in autonomous signal generation without the requirement for exogenous substrates or external activation. Therefore, lux-based bioluminescent bioreporters are the only truly autonomous light-generating sensors in existence. Unfortunately, the bacterial lux system has not yet been efficiently expressed in mammalian cells. In this research, three approaches for optimal expression of the a and b subunits of the bacterial luciferase protein were compared and reporter signal stability was evaluated from stably transfected human embryonic kidney cells. Maximum light levels were obtained from cells expressing the luciferase subunits linked with an internal ribosomal entry site (IRES). Cells harboring this construct produced bioluminescence equaling 2.6 X 106 photons/sec compared to 7.2 X 104 photons/sec obtained from cells expressing the luciferase from a dual promoter vector and 3.5 X 104 photons/sec from a Lux fusion protein. Furthermore, the bioluminescence levels remained stable for more than forty cell passages (5 months) in the absence of antibiotic selection. After this time, bioluminescence signals dropped at a rate of approximately 5% per cell passage. These data indicate that mammalian cell lines can be engineered to efficiently express the bacterial lux system, thus lending themselves to possible long-term continuous monitoring or imaging applications in vivo.

  11. Expression of apoptotic regulatory molecules in renal cell carcinoma: elevated expression of Fas ligand.

    PubMed

    Olive, C; Cheung, C; Nicol, D; Falk, M C

    1999-02-01

    Renal cell carcinoma (RCC) is the most common renal neoplasm. Despite being infiltrated by tumour infiltrating lymphocytes (TIL), these TIL are unable to control tumour growth in vivo, suggesting that the cytotoxic capacity of TIL against RCC is impaired, or that the tumour cells are resistant to killing and therefore escape detection by the immune system. It is postulated that the expression of apoptotic regulatory molecules in RCC favours tumour cell survival. The present study has therefore determined the expression of Fas (APO-1/CD95), Fas ligand (Fas L) and bcl-2 in these tumours. The expression of Fas, Fas L and bcl-2 mRNA transcripts was determined in RCC, normal kidney and peripheral blood by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR), following RNA extraction and cDNA synthesis from tissues and cell samples. Transcript levels were measured by densitometry after Southern blot hybridization of PCR products with internal radio-labelled oligonucleotide probes; a densitometry score was assigned to each hybridizing DNA band and expressed as a ratio of the glyceraldehyde-3-phosphate dehydrogenase content. In peripheral blood, the expression of Fas L and bcl-2 transcripts was similar between patients and normal healthy individuals; however, Fas transcript expression was significantly down-regulated in the patients' versus normal peripheral blood (P = 0.026). Most interestingly, significantly up-regulated Fas L expression was observed in RCC compared to normal kidney (P = 0.041). In contrast, bcl-2 transcripts were well represented in normal kidney but markedly decreased in RCC (P = 0.021). The expression of Fas transcripts in normal kidney and RCC was variable. These data demonstrate elevated expression of Fas L transcripts in RCC, but the functional relevance of this remains to be investigated. PMID:10101681

  12. Effect of pure zinc deficiency on glucose tolerance and insulin and glucagon levels

    SciTech Connect

    Park, J.H.Y.; Grandjean, C.J.; Hart, M.H.; Erdman, S.H.; Pour, P.; Vanderhoof, J.A.

    1986-03-05

    The effect of zinc deficiency on glucose tolerance was investigated using intragastric force-feeding to obviate decreased food intake and altered eating patterns. Three groups of weanling male Sprague-Dawley rats were fed a purified powdered zinc-deficient diet: zinc-deficient ad libitum fed animals (ZDA); zinc-replete gavage force-fed controls (ZRF) fed the zinc-deficient diet in water with zinc (25 ppm); zinc-deficient gavage force-fed animals (ZDF) fed the zinc-deficient diet in distilled water. A fourth group of zinc-supplemented rats fed the diet ad libitum was included to determine caloric intake for ZDF and ZRF gavage fed groups. After 8 days of feeding, the zinc concentration in the serum and pancreas were lower in both zinc-deficient groups, but the difference was much greater in the ZDF rats than in the ZDA. The ZDF group had impaired glucose tolerance curves, yet blood insulin and glucagon levels were normal. The ZDA group had normal glucose tolerance with low insulin levels compared to the ZRF group. The islet cell morphology among the three dietary groups were similar. These results suggest that the glucose intolerance observed in ZDF rats is not due to altered blood insulin and glucagon levels but rather to peripheral resistance to insulin action.

  13. Transcriptional Regulation of Tlr11 Gene Expression in Epithelial Cells*

    PubMed Central

    Cai, Zhenyu; Shi, Zhongcheng; Sanchez, Amir; Zhang, Tingting; Liu, Mingyao; Yang, Jianghua; Wang, Fen; Zhang, Dekai

    2009-01-01

    As sensors of invading microorganisms, Toll-like receptors (TLRs) are expressed not only on macrophages and dendritic cells (DCs) but also on epithelial cells. In the TLR family, Tlr11 appears to have the unique feature in that it is expressed primarily on epithelial cells, although it is also expressed on DCs and macrophages. Here, we demonstrate that transcription of the Tlr11 gene is regulated through two cis-acting elements, one Ets-binding site and one interferon regulatory factor (IRF)-binding site. The Ets element interacts with the epithelium-specific transcription factors, ESE-1 and ESE-3, and the IRF motif interacts with IRF-8. Thus, Tlr11 expression on epithelial cells is regulated by the transcription factors that are presumably distinct from transcription factors that regulate the expression of TLRs in innate immune cells such as macrophages and DCs. Our results imply that the distinctive transcription regulatory machinery for TLRs on epithelium may represent a promising new avenue for the development of epithelia-specific therapeutic interventions. PMID:19801549

  14. Regulation of somatic cell reprogramming through inducible mir-302 expression.

    PubMed

    Lin, Shi-Lung; Chang, Donald C; Lin, Chun-Hung; Ying, Shao-Yao; Leu, Davey; Wu, David T S

    2011-02-01

    Global demethylation is required for early zygote development to establish stem cell pluripotency, yet our findings reiterate this epigenetic reprogramming event in somatic cells through ectopic introduction of mir-302 function. Here, we report that induced mir-302 expression beyond 1.3-fold of the concentration in human embryonic stem (hES) H1 and H9 cells led to reprogramming of human hair follicle cells (hHFCs) to induced pluripotent stem (iPS) cells. This reprogramming mechanism functioned through mir-302-targeted co-suppression of four epigenetic regulators, AOF2 (also known as KDM1 or LSD1), AOF1, MECP1-p66 and MECP2. Silencing AOF2 also caused DNMT1 deficiency and further enhanced global demethylation during somatic cell reprogramming (SCR) of hHFCs. Re-supplementing AOF2 in iPS cells disrupted such global demethylation and induced cell differentiation. Given that both hES and iPS cells highly express mir-302, our findings suggest a novel link between zygotic reprogramming and SCR, providing a regulatory mechanism responsible for global demethylation in both events. As the mechanism of conventional iPS cell induction methods remains largely unknown, understanding this microRNA (miRNA)-mediated SCR mechanism may shed light on the improvements of iPS cell generation. PMID:20870751

  15. Evaluating cell-surface expression and measuring activation of mammalian odorant receptors in heterologous cells

    PubMed Central

    Zhuang, Hanyi; Matsunami, Hiroaki

    2009-01-01

    A fundamental question in olfaction is which odorant receptors (ORs) are activated by a given odorant. A major roadblock to investigate odorant-OR relationship in mammals has been an inability to express ORs in heterologous cells suitable for screening active ligands for ORs. The discovery of the receptor-transporting protein (RTP) family has facilitated the effective cell-surface expression of ORs in heterologous cells. The establishment of a robust heterologous expression system for mammalian ORs facilitates the high-throughput “deorphanization” of these receptors by matching them to their cognate ligands. This protocol details the method used for evaluating the cell-surface expression and measuring the functional activation of ORs of transiently-expressed mammalian odorant receptors in HEK293T cells. The stages of odorant receptor cell-surface expression include cell culture preparation, transfer of cells, transfection, and immunocytochemistry/flow cytometry, odorant stimulation, and luciferase assay. This protocol can be completed in a period of 3 days from transfer of cells to cell-surface expression detection and/or measurement of functional activation. PMID:18772867

  16. Effects of glucagon-like peptide-1 receptor agonists on non-alcoholic fatty liver disease and inflammation.

    PubMed

    Wang, Xing-Chun; Gusdon, Aaron M; Liu, Huan; Qu, Shen

    2014-10-28

    Glucagon-like peptide1 (GLP-1) is secreted from Langerhans cells in response to oral nutrient intake. Glucagon-like peptide-1 receptor agonists (GLP-1RAs) are a new class of incretin-based anti-diabetic drugs. They function to stimulate insulin secretion while suppressing glucagon secretion. GLP-1-based therapies are now well established in the management of type 2 diabetes mellitus (T2DM), and recent literature has suggested potential applications of these drugs in the treatment of obesity and for protection against cardiovascular and neurological diseases. As we know, along with change in lifestyles, the prevalence of non-alcoholic fatty liver disease (NAFLD) in China is rising more than that of viral hepatitis and alcoholic fatty liver disease, and NAFLD has become the most common chronic liver disease in recent years. Recent studies further suggest that GLP-1RAs can reduce transaminase levels to improve NAFLD by improving blood lipid levels, cutting down the fat content to promote fat redistribution, directly decreasing fatty degeneration of the liver, reducing the degree of liver fibrosis and improving inflammation. This review shows the NAFLD-associated effects of GLP-1RAs in animal models and in patients with T2DM or obesity who are participants in clinical trials. PMID:25356042

  17. c-myc protooncogene expression in mouse erythroleukemia cells.

    PubMed Central

    Lachman, H M

    1989-01-01

    Murine erythroleukemia (MEL) cells are erythroid progenitors whose programs of erythroid differentiation has been interrupted by transformation with the Friend virus complex. As a result of the ability of certain chemicals such as dimethylsulfoxide (DMSO) to induce terminal erythroid differentiation, the cells have been used as a model for understanding the molecular basis of cellular differentiation. Recent work on MEL cells as well as other differentiating systems indicates that expression of cellular protooncogenes is implicated in chemically mediated differentiation. In MEL cells the expression of the c-myc protooncogene undergoes unusual biphasic changes following inducer treatment. Levels of c-myc mRNA decrease 10- to 20-fold between 1 and 2 hr and are then reexpressed between 12 and 24 hr. These changes occur as a result of complex transcriptional and posttranscriptional regulatory events. Recent DNA transfection experiments, in which MEL cells were transfected with myc expression vectors, indicate that both the early decrease in c-myc expression and its subsequent reexpression are important events in the differentiation pathway. The work on MEL cells, as well as on other models of differentiation, is directed at understanding the molecular basis of leukemogenic transformation and cellular differentiation. The ability of c-myc, as well as other protooncogenes, to influence both of these events indicates that cellular protooncogenes play a central role in their regulation. Images FIGURE 1. FIGURE 2. FIGURE 3. FIGURE 4. FIGURE 5. PMID:2647476

  18. Expression Profiling of Single Mammalian Cells – Small is Beautiful

    PubMed Central

    2000-01-01

    Increasingly mRNA expression patterns established using a variety of molecular technologies such as cDNA microarrays, SAGE and cDNA display are being used to identify potential regulatory genes and as a means of providing valuable insights into the biological status of the starting sample. Until recently, the application of these techniques has been limited to mRNA isolated from millions or, at very best, several thousand cells thereby restricting the study of small samples and complex tissues. To overcome this limitation a variety of amplification approaches have been developed which are capable of broadly evaluating mRNA expression patterns in single cells. This review will describe approaches that have been employed to examine global gene expression patterns either in small numbers of cells or, wherever possible, in actual isolated single cells. The first half of the review will summarize the technical aspects of methods developed for single-cell analysis and the latter half of the review will describe the areas of biological research that have benefited from single-cell expression analysis. PMID:11025531

  19. Sp3 regulates fas expression in lung epithelial cells.

    PubMed Central

    Pang, H; Miranda, K; Fine, A

    1998-01-01

    By transducing an apoptotic signal in immune effector cells, Fas has been directly implicated in the control of immunological activity. Expression and functional results, however, have also suggested a role for Fas in regulating cell turnover in specific epithelial populations. To characterize factors responsible for Fas expression in epithelial cells, approximately 3 kb of the 5' flanking region of the mouse Fas gene was isolated. By rapid amplification of cDNA ends and primer extension, transcriptional start sites were identified within 50 bp upstream of the translation start site. Transient transfection of promoter-luciferase constructs in a mouse lung epithelial cell line, MLE-15, localized promoter activity to the first 77 bp of upstream sequence. By using a 60 bp DNA probe (-18 to -77) in electrophoretic mobility-shift assays, three shifted complexes were found. Incubation with excess cold Sp1 oligonucleotide or an anti-Sp3 antibody inhibited complex formation. Site-directed mutagenesis of the Sp1 site resulted in 60-70% loss of promoter activity. In Drosophila SL-2 cells, promoter activity was markedly increased by co-transfection of an Sp3 expression construct. These results show that the Sp3 protein is involved in regulating Fas gene expression in lung epithelial cells. PMID:9639581

  20. SNAP25 Expression in Mammalian Retinal Horizontal Cells

    PubMed Central

    Hirano, Arlene A.; Brandstätter, Johann Helmut; Morgans, Catherine W.; Brecha, Nicholas C.

    2014-01-01

    Horizontal cells mediate inhibitory feedforward and feedback lateral interactions in the outer retina at photoreceptor terminals and bipolar cell dendrites; however, the mechanisms that underlie synaptic transmission from mammalian horizontal cells are poorly understood. The localization of a vesicular γ-aminobutyric acid (GABA) transporter (VGAT) to horizontal cell processes in primate and rodent retinae suggested that mammalian horizontal cells release transmitter in a vesicular manner. Toward determining whether the molecular machinery for vesicular transmitter release is present in horizontal cells, we investigated the expression of SNAP25 (synaptosomal-associated protein of 25 kDa), a key SNARE protein, by immunocytochemistry with cell type-specific markers in the retinae of mouse, rat, rabbit, and monkey. Different commercial antibodies to SNAP25 were tested on vertical sections of retina. We report the robust expression of SNAP25 in both plexiform layers. Double labeling with SNAP25 and calbindin antibodies demonstrated that horizontal cell processes and their endings in photoreceptor triad synapses were strongly labeled for both proteins in mouse, rat, rabbit, and monkey retinae. Double labeling with parvalbumin antibodies in monkey retina verified SNAP25 immunoreactivity in all horizontal cells. Pre-embedding immunoelectron microscopy in rabbit retina confirmed expression of SNAP25 in lateral elements within photoreceptor triad synapses. The SNAP25 immunoreactivity in the plexiform layers and outer nuclear layer fell into at least three patterns depending on the antibody, suggesting a differential distribution of SNAP25 isoforms. The presence of SNAP25a and SNAP25b isoforms in mouse retina was established by reverse transcriptase-polymerase chain reaction. SNAP25 expression in mammalian horizontal cells along with other SNARE proteins is consistent with vesicular exocytosis. PMID:21280047

  1. Regulation of cell-to-cell variability in divergent gene expression

    NASA Astrophysics Data System (ADS)

    Yan, Chao; Wu, Shuyang; Pocetti, Christopher; Bai, Lu

    2016-03-01

    Cell-to-cell variability (noise) is an important feature of gene expression that impacts cell fitness and development. The regulatory mechanism of this variability is not fully understood. Here we investigate the effect on gene expression noise in divergent gene pairs (DGPs). We generated reporters driven by divergent promoters, rearranged their gene order, and probed their expressions using time-lapse fluorescence microscopy and single-molecule fluorescence in situ hybridization (smFISH). We show that two genes in a co-regulated DGP have higher expression covariance compared with the separate, tandem and convergent configurations, and this higher covariance is caused by more synchronized firing of the divergent transcriptions. For differentially regulated DGPs, the regulatory signal of one gene can stochastically `leak' to the other, causing increased gene expression noise. We propose that the DGPs' function in limiting or promoting gene expression noise may enhance or compromise cell fitness, providing an explanation for the conservation pattern of DGPs.

  2. Relation of CD30 expression to survival and morphology in large cell B cell lymphomas.

    PubMed Central

    Noorduyn, L A; de Bruin, P C; van Heerde, P; van de Sandt, M M; Ossenkoppele, G J; Meijer, C J

    1994-01-01

    AIMS--To investigate whether CD30 expression is correlated with anaplastic morphology, and whether this correlated with a better survival in large cell B cell lymphomas, as has been described for T cell lymphomas. METHODS--CD30 expression was investigated using frozen sections in a series of 146 large cell B cell lymphomas. Clinical data and follow up information were collected from 25 lymphomas with strong CD30 expression, 30 lymphomas with partial CD30 expression, and a control group of 25 lymphomas which did not express CD30. RESULTS--Morphological distinction between anaplastic and non-anaplastic tumours was difficult. Of the cases with an anaplastic morphology, 50% were CD30 positive, as were 24% of the polymorphic centroblastic B cell lymphomas. Only 65% of the morphologically non-anaplastic tumours were completely CD30 negative. There was no difference in survival among patients with lymphomas expressing CD30 and those that did not. Patients with morphologically anaplastic B cell lymphomas did not differ in their survivals from those with other high grade B cell lymphomas. Clinical stage at presentation was the only variable that was significantly associated with survival. CONCLUSIONS--CD30 expression occurs frequently in large cell B cell lymphomas and is poorly related to anaplastic morphology. Morphological distinction between anaplastic and non-anaplastic tumours is difficult. In contrast to T cell lymphomas, CD30 positive B cell lymphomas do not show a relatively favourable clinical course. The results presented here raise serious doubts as to whether large cell B cell lymphoma, based on the expression of CD30 or anaplastic morphology, can really be termed a separate entity. Images PMID:8132806

  3. Targeting telomerase-expressing cancer cells

    PubMed Central

    Ouellette, Michel M; Wright, Woodring E; Shay, Jerry W

    2011-01-01

    Abstract The role of telomeres and telomerase as a target for cancer therapeutics is an area of continuing interest. This review is intended to provide an update on the field, pointing to areas in which our knowledge remains deficient and exploring the details of the most promising areas being advanced into clinical trials. Topics that will be covered include the role of dysfunctional telomeres in cellular aging and how replicative senescence provides an initial barrier to the emergence of immortalized cells, a hallmark of cancer. As an important translational theme, this review will consider possibilities for selectively targeting telomeres and telomerase to enhance cancer therapy. The role of telomerase as an immunotherapy, as a gene therapy approach using telomerase promoter driven oncolytic viruses and as a small oligonucleotide targeted therapy (Imetelstat) will be discussed. PMID:21332640

  4. Targeting telomerase-expressing cancer cells.

    PubMed

    Ouellette, Michel M; Wright, Woodring E; Shay, Jerry W

    2011-07-01

    The role of telomeres and telomerase as a target for cancer therapeutics is an area of continuing interest. This review is intended to provide an update on the field, pointing to areas in which our knowledge remains deficient and exploring the details of the most promising areas being advanced into clinical trials. Topics that will be covered include the role of dysfunctional telomeres in cellular aging and how replicative senescence provides an initial barrier to the emergence of immortalized cells, a hallmark of cancer. As an important translational theme, this review will consider possibilities for selectively targeting telomeres and telomerase to enhance cancer therapy. The role of telomerase as an immunotherapy, as a gene therapy approach using telomerase promoter driven oncolytic viruses and as a small oligonucleotide targeted therapy (Imetelstat) will be discussed. PMID:21332640

  5. Human Neural Cells Transiently Express Reelin during Olfactory Placode Development

    PubMed Central

    Antal, M. Cristina; Samama, Brigitte; Ghandour, M. Said; Boehm, Nelly

    2015-01-01

    Reelin, an extracellular glycoprotein is essential for migration and correct positioning of neurons during development. Since the olfactory system is known as a source of various migrating neuronal cells, we studied Reelin expression in the two chemosensory olfactory systems, main and accessory, during early developmental stages of human foetuses/embryos from Carnegie Stage (CS) 15 to gestational week (GW) 14. From CS 15 to CS 18, but not at later stages, a transient expression of Reelin was detected first in the presumptive olfactory and then in the presumptive vomeronasal epithelium. During the same period, Reelin-positive cells detach from the olfactory/vomeronasal epithelium and migrate through the mesenchyme beneath the telencephalon. Dab 1, an adaptor protein of the Reelin pathway, was simultaneously expressed in the migratory mass from CS16 to CS17 and, at later stages, in the presumptive olfactory ensheathing cells. Possible involvements of Reelin and Dab 1 in the peripheral migrating stream are discussed. PMID:26270645

  6. Protein expression analyses at the single cell level.

    PubMed

    Ohno, Masae; Karagiannis, Peter; Taniguchi, Yuichi

    2014-01-01

    The central dogma of molecular biology explains how genetic information is converted into its end product, proteins, which are responsible for the phenotypic state of the cell. Along with the protein type, the phenotypic state depends on the protein copy number. Therefore, quantification of the protein expression in a single cell is critical for quantitative characterization of the phenotypic states. Protein expression is typically a dynamic and stochastic phenomenon that cannot be well described by standard experimental methods. As an alternative, fluorescence imaging is being explored for the study of protein expression, because of its high sensitivity and high throughput. Here we review key recent progresses in fluorescence imaging-based methods and discuss their application to proteome analysis at the single cell level. PMID:25197931

  7. Human Neural Cells Transiently Express Reelin during Olfactory Placode Development.

    PubMed

    Antal, M Cristina; Samama, Brigitte; Ghandour, M Said; Boehm, Nelly

    2015-01-01

    Reelin, an extracellular glycoprotein is essential for migration and correct positioning of neurons during development. Since the olfactory system is known as a source of various migrating neuronal cells, we studied Reelin expression in the two chemosensory olfactory systems, main and accessory, during early developmental stages of human foetuses/embryos from Carnegie Stage (CS) 15 to gestational week (GW) 14. From CS 15 to CS 18, but not at later stages, a transient expression of Reelin was detected first in the presumptive olfactory and then in the presumptive vomeronasal epithelium. During the same period, Reelin-positive cells detach from the olfactory/vomeronasal epithelium and migrate through the mesenchyme beneath the telencephalon. Dab 1, an adaptor protein of the Reelin pathway, was simultaneously expressed in the migratory mass from CS16 to CS17 and, at later stages, in the presumptive olfactory ensheathing cells. Possible involvements of Reelin and Dab 1 in the peripheral migrating stream are discussed. PMID:26270645

  8. Glucagon like peptide-1 attenuates bleomycin-induced pulmonary fibrosis, involving the inactivation of NF-κB in mice.

    PubMed

    Gou, Si; Zhu, Tao; Wang, Wei; Xiao, Min; Wang, Xi-chen; Chen, Zhong-hua

    2014-10-01

    Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with high mortality and poor prognosis. Previous studies confirmed that NF-κB plays a critical role in the pathogenesis of pulmonary fibrosis and glucagon like peptide-1 (GLP-1) has a property of anti-inflammation by inactivation of NF-κB. Furthermore, the GLP-1 receptor was detected in the lung tissues. Our aim was to investigate the potential value and mechanisms of GLP-1 on BLM-induced pulmonary fibrosis in mice. Mice with BLM-induced pulmonary fibrosis were treated with or without GLP-1 administration. 28 days after BLM infusion, the number of total cells, macrophages, neutrophils, lymphocytes, and the content of TGF-β1 in BALF were measured. Hematoxylin-eosin (HE) staining and Masson's trichrome (MT) staining were performed. The Ashcroft score and hydroxyproline content were analyzed. RT-qPCR and western blot were used to evaluate the expression of α-SMA and VCAM-1. The phosphorylation of NF-κB p65 was also assessed by western blot. DNA binding of NF-κB p65 was measured through Trans(AM) p65 transcription factor ELISA kit. GLP-1 reduced inflammatory cell infiltration and the content of TGF-β1 in BLAF in mice with BLM injection. The Ashcroft score and hydroxyproline content were decreased by GLP-1 administration. Meanwhile, BLM-induced overexpression of α-SMA and VCAM-1 were blocked by GLP-1 treatment in mice. GLP-1 also reduced the ratio of phosphor-NF-κB p65/total-NF-κB p65 and NF-κB p65 DNA binding activity in BLM-induced pulmonary fibrosis in mice. Our data found that BLM-induced lung inflammation and pulmonary fibrosis were significantly alleviated by GLP-1 treatment in mice, possibly through inactivation of NF-κB. PMID:25111852

  9. Glucagon-like peptide 1: a potent glycogenic hormone.

    PubMed

    Valverde, I; Morales, M; Clemente, F; López-Delgado, M I; Delgado, E; Perea, A; Villanueva-Peñacarrillo, M L

    1994-08-01

    GLP-1(7-36)amide is an insulinotropic peptide derived from the intestinal post-translational proglucagon process, the release of which is increased mainly after a carbohydrate meal; also, its anti-diabetogenic effect in normal and diabetic states has been reported. In this study, GLP-1(7-36)amide stimulates the formation of glycogen from glucose in isolated rat hepatocytes, such a glycogenic effect being achieved with physiological concentrations of the peptide. The GLP-1(7-36)amide-induced glycogenesis is abolished by glucagon, and it is accompanied by stimulation of the glycogen synthase alpha activity and by a decrease in the basal and glucagon-stimulated cyclic AMP content. These findings could explain, at least in part, the GLP-1(7-36)amide insulin-independent plasma glucose lowering effect. PMID:8050588

  10. Regulation of gene expression in ovarian cancer cells by luteinizing hormone receptor expression and activation

    PubMed Central

    2011-01-01

    Background Since a substantial percentage of ovarian cancers express gonadotropin receptors and are responsive to the relatively high concentrations of pituitary gonadotropins during the postmenopausal years, it has been suggested that receptor activation may contribute to the etiology and/or progression of the neoplasm. The goal of the present study was to develop a cell model to determine the impact of luteinizing hormone (LH) receptor (LHR) expression and LH-mediated LHR activation on gene expression and thus obtain insights into the mechanism of gonadotropin action on ovarian surface epithelial (OSE) carcinoma cells. Methods The human ovarian cancer cell line, SKOV-3, was stably transfected to express functional LHR and incubated with LH for various periods of time (0-20 hours). Transcriptomic profiling was performed on these cells to identify LHR expression/activation-dependent changes in gene expression levels and pathways by microarray and qRT-PCR analyses. Results Through comparative analysis on the LHR-transfected SKOV-3 cells exposed to LH, we observed the differential expression of 1,783 genes in response to LH treatment, among which five significant families were enriched, including those of growth factors, translation regulators, transporters, G-protein coupled receptors, and ligand-dependent nuclear receptors. The most highly induced early and intermediate responses were found to occupy a network impacting transcriptional regulation, cell growth, apoptosis, and multiple signaling transductions, giving indications of LH-induced apoptosis and cell growth inhibition through the significant changes in, for example, tumor necrosis factor, Jun and many others, supportive of the observed cell growth reduction in in vitro assays. However, other observations, e.g. the substantial up-regulation of the genes encoding the endothelin-1 subtype A receptor, stromal cell-derived factor 1, and insulin-like growth factor II, all of which are potential therapeutic

  11. Oxytocin increases extrapancreatic glucagon secretion and glucose production in pancreatectomized dogs

    SciTech Connect

    Altszuler, N.; Puma, F.; Winkler, B.; Fontan, N.; Saudek, C.D.

    1986-05-01

    Infusion of oxytocin into normal dogs increases plasma levels of insulin and glucagon and glucose production and uptake. To determine whether infused oxytocin also increases glucagon secretion from extrapancreatic sites, pancreatectomized dogs, off insulin of 18 hr, were infused with oxytocin and plasma glucagon, and glucose production and uptake were measured using the (6-/sup 3/H)glucose primer-infusion technique. The diabetic dogs, in the control period, had elevated plasma glucose and glucagon levels, an increased rate of glucose production, and a relative decrease in glucose uptake (decreased clearance). Infusion of oxytocin (500 ..mu..U/kg/min) caused a rise in plasma glucagon and glucose levels, increased glucose production, and further decreased glucose clearance. it is concluded that oxytocin can stimulate secretion of extrapancreatic glucagon, which contributes to the increased glucose production.

  12. Tolerance Associated Gene Expression following Allogeneic Hematopoietic Cell Transplantation

    PubMed Central

    Pidala, Joseph; Bloom, Gregory C.; Eschrich, Steven; Sarwal, Minnie; Enkemann, Steve; Betts, Brian C.; Beato, Francisca; Yoder, Sean; Anasetti, Claudio

    2015-01-01

    Biologic markers of immune tolerance may facilitate tailoring of immune suppression duration after allogeneic hematopoietic cell transplantation (HCT). In a cross-sectional study, peripheral blood samples were obtained from tolerant (n = 15, median 38.5 months post-HCT) and non-tolerant (n = 17, median 39.5 post-HCT) HCT recipients and healthy control subjects (n = 10) for analysis of immune cell subsets and differential gene expression. There were no significant differences in immune subsets across groups. We identified 281 probe sets unique to the tolerant (TOL) group and 122 for non-tolerant (non-TOL). These were enriched for process networks including NK cell cytotoxicity, antigen presentation, lymphocyte proliferation, and cell cycle and apoptosis. Differential gene expression was enriched for CD56, CD66, and CD14 human lineage-specific gene expression. Differential expression of 20 probe sets between groups was sufficient to develop a classifier with > 90% accuracy, correctly classifying 14/15 TOL cases and 15/17 non-TOL cases. These data suggest that differential gene expression can be utilized to accurately classify tolerant patients following HCT. Prospective investigation of immune tolerance biologic markers is warranted. PMID:25774806

  13. Glucagon-Like Peptide-2 Requires a Full Complement of Bmi-1 for Its Proliferative Effects in the Murine Small Intestine.

    PubMed

    Smither, Bradley R; Pang, Hilary Y M; Brubaker, Patricia L

    2016-07-01

    The intestinal hormone, glucagon-like peptide-2 (GLP-2), stimulates growth, survival, and function of the intestinal epithelium through increased crypt cell proliferation, and a long-acting analog has recently been approved to enhance intestinal capacity in patients with short bowel syndrome. The goal of the present study was to determine whether GLP-2-induced crypt cell proliferation requires a full complement of B-cell lymphoma Moloney murine leukemia virus insertion region-1 homolog (Bmi-1), using the Bmi-1(eGFP/+) mouse model in comparison with age- and sex-matched Bmi-1(+/+) littermates. Bmi-1 is a member of the polycomb-repressive complex family that promotes stem cell proliferation and self-renewal and is expressed by both stem cells and transit-amplifying (TA) cells in the crypt. The acute (6 h) and chronic (11 d) proliferative responses to long-acting human (Gly(2))GLP-2 in the crypt TA zone, but not in the active or reserve stem cell zones, were both impaired by Bmi-1 haploinsufficiency. Similarly, GLP-2-induced crypt regeneration after 10-Gy irradiation was reduced in the Bmi-1(eGFP/+) animals. Despite these findings, chronic GLP-2 treatment enhanced overall intestinal growth in the Bmi-1(eGFP/+) mice, as demonstrated by increases in small intestinal weight per body weight and in the length of the crypt-villus axis, in association with decreased apoptosis and an adaptive increase in crypt epithelial cell migration rate. The results of these studies therefore demonstrate that a full complement of Bmi-1 is required for the intestinal proliferative effects of GLP-2 in both the physiological and pathological setting, and mediates, at least in part, the proliferation kinetics of cells in the TA zone. PMID:27187177

  14. Conserved Expression Signatures between Medaka and Human Pigment Cell Tumors

    PubMed Central

    Schartl, Manfred; Kneitz, Susanne; Wilde, Brigitta; Wagner, Toni; Henkel, Christiaan V.; Spaink, Herman P.; Meierjohann, Svenja

    2012-01-01

    Aberrations in gene expression are a hallmark of cancer cells. Differential tumor-specific transcript levels of single genes or whole sets of genes may be critical for the neoplastic phenotype and important for therapeutic considerations or useful as biomarkers. As an approach to filter out such relevant expression differences from the plethora of changes noted in global expression profiling studies, we searched for changes of gene expression levels that are conserved. Transcriptomes from massive parallel sequencing of different types of melanoma from medaka were generated and compared to microarray datasets from zebrafish and human melanoma. This revealed molecular conservation at various levels between fish models and human tumors providing a useful strategy for identifying expression signatures strongly associated with disease phenotypes and uncovering new melanoma molecules. PMID:22693581

  15. Reproductive fitness advantage of BCR-ABL expressing leukemia cells.

    PubMed

    Traulsen, Arne; Pacheco, Jorge M; Dingli, David

    2010-08-01

    Mutations in oncogenes and tumor suppressor genes confer a fitness advantage to cells that can lead to cancer. The tumor phenotype normally results from the interaction of many mutant genes making it difficult to estimate the fitness advantage provided by any oncogene, except when tumors depend on one oncogene only. We utilize a model of chronic myeloid leukemia (CML), to quantitate the fitness advantage conferred by expression of BCR-ABL in hematopoietic cells from in vivo patient data. We show that BCR-ABL expression provides a high fitness advantage, which explains why this single mutation drives the chronic phase of CML. PMID:20153920

  16. Do CD8 effector cells need IL-7R expression to become resting memory cells?

    PubMed

    Buentke, Eva; Mathiot, Anne; Tolaini, Mauro; Di Santo, James; Zamoyska, Rose; Seddon, Benedict

    2006-09-15

    The role for IL-7R expression in the differentiation of effector T cells into resting memory remains controversial. Here, using a conditional IL-7R transgenic model, we were able to test directly whether CD8 effector T cells require IL-7R expression for their differentiation into resting memory cells. In the absence of IL-7R expression, effector cells transferred into "full" hosts underwent a protracted and unremitting contraction compared with IL-7R-expressing control cells and were unable to develop into long-term resting memory cells. Surprisingly, when the same effector cells were transferred into empty T-cell-deficient hosts, they could generate long-lived fully functional resting memory cells independently of IL-7R expression. Formation of these latter cells was found to be dependent on IL-15, because the same IL-7R-deficient effector cells were rapidly lost from IL-15-deficient hosts, having a half-life of less than 40 hours. Therefore, our data suggest that, under physiological conditions, both IL-7 and IL-15 synergize to promote the formation of memory cells directly by limiting the contraction of effectors that occurs following an immune response and that reexpression of IL-7R is a key checkpoint in the regulation of this process. PMID:16705084

  17. Expression of gamma-glutamyl transpeptidase protects ramos B cells from oxidation-induced cell death.

    PubMed

    Karp, D R; Shimooku, K; Lipsky, P E

    2001-02-01

    The ectoenzyme, gamma-glutamyl transpeptidase (GGT, EC ) cleaves glutathione (GSH) to facilitate the recapture of cysteine for synthesis of intracellular GSH. The impact of GGT expression on cell survival during oxidative stress was investigated using the human B cell lymphoblastoid cell line, Ramos. Ramos cells did not express surface GGT and exhibited no GGT enzyme activity. In contrast, Ramos cells stably transfected with the human GGT cDNA expressed high levels of surface GGT and enzymatic activity. GGT-transfected Ramos cells were protected from apoptosis when cultured in cyst(e)ine-deficient medium. The GGT-expressing cells also had lower levels of intracellular reactive oxygen species (ROS). Homocysteic acid and alanine, inhibitors of cystine and cysteine uptake, respectively, caused increased ROS content and diminished viability of GGT expressing cells. Exogenous GSH increased the viability of the GGT-transfected cells more effectively than that of control cells, whereas the products of GSH metabolism prevented death of both the control and GGT-transfected cells comparably. These data indicate that GGT cleavage of GSH and the subsequent recapture of cysteine and cystine allow cells to maintain low levels of cellular ROS and thereby avoid apoptosis induced by oxidative stress. PMID:11080500

  18. Cell-by-Cell Dissection of Gene Expression and Chromosomal Interactions Reveals Consequences of Nuclear Reorganization

    PubMed Central

    2005-01-01

    The functional consequences of long-range nuclear reorganization were studied in a cell-by-cell analysis of gene expression and long-range chromosomal interactions in the Drosophila eye and eye imaginal disk. Position-effect variegation was used to stochastically perturb gene expression and probe nuclear reorganization. Variegating genes on rearrangements of Chromosomes X, 2, and 3 were probed for long-range interactions with heterochromatin. Studies were conducted only in tissues known to express the variegating genes. Nuclear structure was revealed by fluorescence in situ hybridization with probes to the variegating gene and heterochromatin. Gene expression was determined alternately by immunofluorescence against specific proteins and by eye pigment autofluorescence. This allowed cell-by-cell comparisons of nuclear architecture between cells in which the variegating gene was either expressed or silenced. Very strong correlations between heterochromatic association and silencing were found. Expressing cells showed a broad distribution of distances between variegating genes and their own centromeric heterochromatin, while silenced cells showed a very tight distribution centered around very short distances, consistent with interaction between the silenced genes and heterochromatin. Spatial and temporal analysis of interactions with heterochromatin indicated that variegating genes primarily associate with heterochromatin in cells that have exited the cell cycle. Differentiation was not a requirement for association, and no differences in association were observed between cell types. Thus, long-range interactions between distal chromosome regions and their own heterochromatin have functional consequences for the organism. PMID:15737020

  19. Glucagon-like Peptide-1 (GLP-1) Analogs: Recent Advances, New Possibilities, and Therapeutic Implications

    PubMed Central

    2015-01-01

    Glucagon-like peptide-1 (GLP-1) is an incretin that plays important physiological roles in glucose homeostasis. Produced from intestine upon food intake, it stimulates insulin secretion and keeps pancreatic β-cells healthy and proliferating. Because of these beneficial effects, it has attracted a great deal of attention in the past decade, and an entirely new line of diabetic therapeutics has emerged based on the peptide. In addition to the therapeutic applications, GLP-1 analogs have demonstrated a potential in molecular imaging of pancreatic β-cells; this may be useful in early detection of the disease and evaluation of therapeutic interventions, including islet transplantation. In this Perspective, we focus on GLP-1 analogs for their studies on improvement of biological activities, enhancement of metabolic stability, investigation of receptor interaction, and visualization of the pancreatic islets. PMID:25349901

  20. Glucagon-Like Peptide 1 Analogs and their Effects on Pancreatic Islets.

    PubMed

    Tudurí, Eva; López, Miguel; Diéguez, Carlos; Nadal, Angel; Nogueiras, Rubén

    2016-05-01

    Glucagon-like peptide 1 (GLP-1) exerts many actions that improve glycemic control. GLP-1 stimulates glucose-stimulated insulin secretion and protects β cells, while its extrapancreatic effects include cardioprotection, reduction of hepatic glucose production, and regulation of satiety. Although an appealing antidiabetic drug candidate, the rapid degradation of GLP-1 by dipeptidyl peptidase 4 (DPP-4) means that its therapeutic use is unfeasible, and this prompted the development of two main GLP-1 therapies: long-acting GLP-1 analogs and DPP-4 inhibitors. In this review, we focus on the pancreatic effects exerted by current GLP-1 derivatives used to treat diabetes. Based on the results from in vitro and in vivo studies in humans and animal models, we describe the specific actions of GLP-1 analogs on the synthesis, processing, and secretion of insulin, islet morphology, and β cell proliferation and apoptosis. PMID:27062006

  1. From single-cell to cell-pool transcriptomes: stochasticity in gene expression and RNA splicing.

    PubMed

    Marinov, Georgi K; Williams, Brian A; McCue, Ken; Schroth, Gary P; Gertz, Jason; Myers, Richard M; Wold, Barbara J

    2014-03-01

    Single-cell RNA-seq mammalian transcriptome studies are at an early stage in uncovering cell-to-cell variation in gene expression, transcript processing and editing, and regulatory module activity. Despite great progress recently, substantial challenges remain, including discriminating biological variation from technical noise. Here we apply the SMART-seq single-cell RNA-seq protocol to study the reference lymphoblastoid cell line GM12878. By using spike-in quantification standards, we estimate the absolute number of RNA molecules per cell for each gene and find significant variation in total mRNA content: between 50,000 and 300,000 transcripts per cell. We directly measure technical stochasticity by a pool/split design and find that there are significant differences in expression between individual cells, over and above technical variation. Specific gene coexpression modules were preferentially expressed in subsets of individual cells, including one enriched for mRNA processing and splicing factors. We assess cell-to-cell variation in alternative splicing and allelic bias and report evidence of significant differences in splice site usage that exceed splice variation in the pool/split comparison. Finally, we show that transcriptomes from small pools of 30-100 cells approach the information content and reproducibility of contemporary RNA-seq from large amounts of input material. Together, our results define an experimental and computational path forward for analyzing gene expression in rare cell types and cell states. PMID:24299736

  2. Neuropilin 1 expression correlates with differentiation status of epidermal cells and cutaneous squamous cell carcinomas.

    PubMed

    Shahrabi-Farahani, Shokoufeh; Wang, Lili; Zwaans, Bernadette M M; Santana, Jeans M; Shimizu, Akio; Takashima, Seiji; Kreuter, Michael; Coultas, Leigh; D'Amore, Patricia A; Arbeit, Jeffrey M; Akslen, Lars A; Bielenberg, Diane R

    2014-07-01

    Neuropilins (NRPs) are cell surface receptors for vascular endothelial growth factor (VEGF) and SEMA3 (class 3 semaphorin) family members. The role of NRPs in neurons and endothelial cells has been investigated, but the expression and role of NRPs in epithelial cells is much less clear. Herein, the expression and localization of NRP1 was investigated in human and mouse skin and squamous cell carcinomas (SCCs). Results indicated that NRP1 mRNA and protein was expressed in the suprabasal epithelial layers of the skin sections. NRP1 staining did not overlap with that of keratin 14 (K14) or proliferating cell nuclear antigen, but did co-localize with staining for keratin 1, indicating that differentiated keratinocytes express NRP1. Similar to the expression of NRP1, VEGF-A was expressed in suprabasal epithelial cells, whereas Nrp2 and VEGFR2 were not detectable in the epidermis. The expression of NRP1 correlated with a high degree of differentiation in human SCC specimens, human SCC xenografts, and mouse K14-HPV16 transgenic SCC. UVB irradiation of mouse skin induced Nrp1 upregulation. In vitro, Nrp1 was upregulated in primary keratinocytes in response to differentiating media or epidermal growth factor-family growth factors. In conclusion, the expression of NRP1 is regulated in the skin and is selectively produced in differentiated epithelial cells. NRP1 may function as a reservoir to sequester VEGF ligand within the epithelial compartment, thereby modulating its bioactivity. PMID:24791743

  3. Polyclonal T-Cells Express CD1a in Langerhans Cell Histiocytosis (LCH) Lesions

    PubMed Central

    West, Jennifer A.; Olsen, Sharon L.; Mitchell, Jenée M.; Priddle, Ross E.; Luke, Jennifer M.; Åkefeldt, Selma Olsson; Henter, Jan-Inge; Turville, Christopher; Kannourakis, George

    2014-01-01

    Langerhans cell histiocytosis (LCH) is a complex and poorly understood disorder that has characteristics of both inflammatory and neoplastic disease. By using eight-colour flow cytometry, we have identified a previously unreported population of CD1a+/CD3+ T-cells in LCH lesions. The expression of CD1a is regarded as a hallmark of this disease; however, it has always been presumed that it was only expressed by pathogenic Langerhans cells (LCs). We have now detected CD1a expression by a range of T-cell subsets within all of the LCH lesions that were examined, establishing that CD1a expression in these lesions is no longer restricted to pathogenic LCs. The presence of CD1a+ T-cells in all of the LCH lesions that we have studied to date warrants further investigation into their biological function to determine whether these cells are important in the pathogenesis of LCH. PMID:25343480

  4. A second glucagon in the pancreatic islets of the daddy sculpin Cottus scorpius.

    PubMed

    Cutfield, S M; Cutfield, J F

    1993-09-01

    The peptide hormone glucagon has been isolated from the islet tissue (Brockmann bodies) of the teleost Cottus scorpius (daddy sculpin) and sequenced. The sequence is HSEGTSNDYSKYLEDRKAQDFVQWLMNN differing at four positions from the glucagon found earlier in the same species by Conlon and coworkers (1987b, Eur. J. Biochem, 164, 117-122). Thus sculpin, in common with anglerfish, possesses two distinct glucagons. Comparative sequence data are presented as a phylogenetic tree. PMID:8224771

  5. Glucagon release from rat pancreatic islets. A combined morphological and functional approach.

    PubMed Central

    Carpentier, J L; Malaisse-Lagae, F; Müller, W A

    1977-01-01

    The release of glucagon induced in isolated rat islets by arginine or by calcium deprivation has been subjected to combined functional and morphological quantifications. Arginine-stimulated glucagon release was associated with a significant increase of morphological events linked to exocytosis. By contrast, the paradoxical events linked to exocytosis. By contrast, the paradoxical release of glucagon provoked by calcium deprivation, although accompanied by a significant loss of granule stores, was not associated with an increase of morphologically detectable exocytosis. Images PMID:409734

  6. Single-cell PCR profiling of gene expression in hematopoiesis.

    PubMed

    Teles, José; Enver, Tariq; Pina, Cristina

    2014-01-01

    Single-cell analysis of gene expression offers the possibility of exploring cellular and molecular heterogeneity in stem and developmental cell systems, including cancer, to infer routes of cellular specification and their respective gene regulatory modules. PCR-based technologies, although limited to the analysis of a predefined set of genes, afford a cost-effective balance of throughput and biological information and have become a method of choice in stem cell laboratories. Here we describe an experimental and analytical protocol based on the Fluidigm microfluidics platform for the simultaneous expression analysis of 48 or 96 genes in multiples of 48 or 96 cells. We detail wet laboratory procedures and describe clustering, principal component analysis, correlation, and classification tools for the inference of cellular pathways and gene networks. PMID:25062620

  7. Spatial reconstruction of single-cell gene expression

    PubMed Central

    Satija, Rahul; Farrell, Jeffrey A.; Gennert, David; Schier, Alexander F.; Regev, Aviv

    2015-01-01

    Spatial localization is a key determinant of cellular fate and behavior, but spatial RNA assays traditionally rely on staining for a limited number of RNA species. In contrast, single-cell RNA-seq allows for deep profiling of cellular gene expression, but established methods separate cells from their native spatial context. Here we present Seurat, a computational strategy to infer cellular localization by integrating single-cell RNA-seq data with in situ RNA patterns. We applied Seurat to spatially map 851 single cells from dissociated zebrafish (Danio rerio) embryos, inferring a transcriptome-wide map of spatial patterning. We confirmed Seurat’s accuracy using several experimental approaches, and used it to identify a set of archetypal expression patterns and spatial markers. Additionally, Seurat correctly localizes rare subpopulations, accurately mapping both spatially restricted and scattered groups. Seurat will be applicable to mapping cellular localization within complex patterned tissues in diverse systems. PMID:25867923

  8. Syndecan-4-expressing muscle progenitor cells in the SP engraft as satellite cells during muscle regeneration.

    PubMed

    Tanaka, Kathleen Kelly; Hall, John K; Troy, Andrew A; Cornelison, D D W; Majka, Susan M; Olwin, Bradley B

    2009-03-01

    Skeletal muscle satellite cells, located between the basal lamina and plasma membrane of myofibers, are required for skeletal muscle regeneration. The capacity of satellite cells as well as other cell lineages including mesoangioblasts, mesenchymal stem cells, and side population (SP) cells to contribute to muscle regeneration has complicated the identification of a satellite stem cell. We have characterized a rare subset of the muscle SP that efficiently engrafts into the host satellite cell niche when transplanted into regenerating muscle, providing 75% of the satellite cell population and 30% of the myonuclear population, respectively. These cells are found in the satellite cell position, adhere to isolated myofibers, and spontaneously undergo myogenesis in culture. We propose that this subset of SP cells (satellite-SP cells), characterized by ABCG2, Syndecan-4, and Pax7 expression, constitutes a self-renewing muscle stem cell capable of generating both satellite cells and their myonuclear progeny in vivo. PMID:19265661

  9. Immunohistological recognition of cyclin D1 expression by non-lymphoid cells among lymphoid neoplastic cells.

    PubMed

    Abdulla, Zainalabideen; Turley, Helen; Gatter, Kevin; Pezzella, Francesco

    2014-03-01

    Cyclin D1 immunostaining of non-neoplastic cells has been a source of diagnostic confusion especially in lymphoproliferative lesions. This study has reviewed these in two hundred and thirty-one haematopathological samples stained for cyclin D1. Most cases were formalin-fixed except for a few bone marrow trephines, which were B-5 fixed, and EDTA decalcified. Overall, 94% (216/231) of cases showed one or more types of non-neoplastic cells expressing Cyclin D1 of variable intensity. Endothelial cells and histiocytes were the most commonly identified Cyclin D1 positive cells being positive in 92% (214/231) of cases. Other normal cell types identified included fat cells, stromal fibroblasts, glial cells, spermatocytes, smooth muscle cells, osteoblasts and where present epithelial cells. Many normal cell types can express cyclinD1. Knowledge of these is useful to prevent misinterpretation of cyclin D1 positive tumours. PMID:23758159

  10. Robust enumeration of cell subsets from tissue expression profiles

    PubMed Central

    Newman, Aaron M.; Liu, Chih Long; Green, Michael R.; Gentles, Andrew J.; Feng, Weiguo; Xu, Yue; Hoang, Chuong D.; Diehn, Maximilian; Alizadeh, Ash A.

    2015-01-01

    We introduce CIBERSORT, a method for characterizing cell composition of complex tissues from their gene expression profiles. When applied to enumeration of hematopoietic subsets in RNA mixtures from fresh, frozen, and fixed tissues, including solid tumors, CIBERSORT outperformed other methods with respect to noise, unknown mixture content, and closely related cell types. CIBERSORT should enable large-scale analysis of RNA mixtures for cellular biomarkers and therapeutic targets (http://cibersort.stanford.edu). PMID:25822800

  11. Improved expression systems for regulated expression in Salmonella infecting eukaryotic cells.

    PubMed

    Medina, Carlos; Camacho, Eva María; Flores, Amando; Mesa-Pereira, Beatriz; Santero, Eduardo

    2011-01-01

    In this work we describe a series of improvements to the Salmonella-based salicylate-inducible cascade expression system comprised of a plasmid-borne expression module, where target gene expression is driven by the P(m) promoter governed by the XylS2 regulator, and a genome-integrated regulatory module controlled by the nahR/P(sal) system. We have constructed a set of high and low-copy number plasmids bearing modified versions of the expression module with a more versatile multiple cloning site and different combinations of the following elements: (i) the nasF transcriptional attenuator, which reduces basal expression levels, (ii) a strong ribosome binding site, and (iii) the Type III Secretion System (TTSS) signal peptide from the effector protein SspH2 to deliver proteins directly to the eukaryotic cytosol following bacterial infection of animal cells. We show that different expression module versions can be used to direct a broad range of protein production levels. Furthermore, we demonstrate that the efficient reduction of basal expression by the nasF attenuator allows the cloning of genes encoding highly cytotoxic proteins such as colicin E3 even in the absence of its immunity protein. Additionally, we show that the Salmonella TTSS is able to translocate most of the protein produced by this regulatory cascade to the cytoplasm of infected HeLa cells. Our results indicate that these vectors represent useful tools for the regulated overproduction of heterologous proteins in bacterial culture or in animal cells, for the cloning and expression of genes encoding toxic proteins and for pathogenesis studies. PMID:21829692

  12. Improved Expression Systems for Regulated Expression in Salmonella Infecting Eukaryotic Cells

    PubMed Central

    Medina, Carlos; Camacho, Eva María; Flores, Amando; Mesa-Pereira, Beatriz; Santero, Eduardo

    2011-01-01

    In this work we describe a series of improvements to the Salmonella-based salicylate-inducible cascade expression system comprised of a plasmid-borne expression module, where target gene expression is driven by the Pm promoter governed by the XylS2 regulator, and a genome-integrated regulatory module controlled by the nahR/Psal system. We have constructed a set of high and low-copy number plasmids bearing modified versions of the expression module with a more versatile multiple cloning site and different combinations of the following elements: (i) the nasF transcriptional attenuator, which reduces basal expression levels, (ii) a strong ribosome binding site, and (iii) the Type III Secretion System (TTSS) signal peptide from the effector protein SspH2 to deliver proteins directly to the eukaryotic cytosol following bacterial infection of animal cells. We show that different expression module versions can be used to direct a broad range of protein production levels. Furthermore, we demonstrate that the efficient reduction of basal expression by the nasF attenuator allows the cloning of genes encoding highly cytotoxic proteins such as colicin E3 even in the absence of its immunity protein. Additionally, we show that the Salmonella TTSS is able to translocate most of the protein produced by this regulatory cascade to the cytoplasm of infected HeLa cells. Our results indicate that these vectors represent useful tools for the regulated overproduction of heterologous proteins in bacterial culture or in animal cells, for the cloning and expression of genes encoding toxic proteins and for pathogenesis studies. PMID:21829692

  13. Interleukin-6 amplifies glucagon secretion: coordinated control via the brain and pancreas

    PubMed Central

    Barnes, Tammy M.; Otero, Yolanda F.; Elliott, Amicia D.; Locke, Alicia D.; Malabanan, Carlo M.; Coldren, Anastasia G.; Brissova, Marcela; Piston, David W.

    2014-01-01

    Inappropriate glucagon secretion contributes to hyperglycemia in inflammatory disease. Previous work implicates the proinflammatory cytokine interleukin-6 (IL-6) in glucagon secretion. IL-6-KO mice have a blunted glucagon response to lipopolysaccharide (LPS) that is restored by intravenous replacement of IL-6. Given that IL-6 has previously been demonstrated to have a transcriptional (i.e., slow) effect on glucagon secretion from islets, we hypothesized that the rapid increase in glucagon following LPS occurred by a faster mechanism, such as by action within the brain. Using chronically catheterized conscious mice, we have demonstrated that central IL-6 stimulates glucagon secretion uniquely in the presence of an accompanying stressor (hypoglycemia or LPS). Contrary to our hypothesis, however, we found that IL-6 amplifies glucagon secretion in two ways; IL-6 not only stimulates glucagon secretion via the brain but also by direct action on islets. Interestingly, IL-6 augments glucagon secretion from both sites only in the presence of an accompanying stressor (such as epinephrine). Given that both adrenergic tone and plasma IL-6 are elevated in multiple inflammatory diseases, the interactions of the IL-6 and catecholaminergic signaling pathways in regulating GCG secretion may contribute to our present understanding of these diseases. PMID:25205821

  14. Global gene expression response to telomerase in bovine adrenocortical cells

    SciTech Connect

    Perrault, Steven D.; Hornsby, Peter J.; Betts, Dean H. . E-mail: bettsd@uoguelph.ca

    2005-09-30

    The infinite proliferative capability of most immortalized cells is dependent upon the presence of the enzyme telomerase and its ability to maintain telomere length and structure. However, telomerase may be involved in a greater system than telomere length regulation, as recent evidence has shown it capable of increasing wound healing in vivo, and improving cellular proliferation rate and survival from apoptosis in vitro. Here, we describe the global gene expression response to ectopic telomerase expression in an in vitro bovine adrenocortical cell model. Telomerase-immortalized cells showed an increased ability for proliferation and survival in minimal essential medium above cells transgenic for GFP. cDNA microarray analyses revealed an altered cell state indicative of increased adrenocortical cell proliferation regulated by the IGF2 pathway and alterations in members of the TGF-B family. As well, we identified alterations in genes associated with development and wound healing that support a model that high telomerase expression induces a highly adaptable, progenitor-like state.

  15. Ikaros expression sensitizes leukemic cells to the chemotherapeutic drug doxorubicin

    PubMed Central

    He, Licai; Gao, Shenmeng; Zhu, Zhenfeng; Chen, Shang; Gu, Haihua

    2016-01-01

    Ikaros is an important transcription factor involved in the development and differentiation of hematopoietic cells. However, its role in the treatment of hematopoietic malignancies such as leukemia is less well understood. In the present study, it was observed by data mining of the Oncomine database that high expression levels of full-length Ikaros (IK1) is correlated with increased sensitivity of cancer cells to treatments with chemotherapeutic drugs, including doxorubicin (DOX). To examine the functional significance of this observation, the expression of IK1 in a leukemia cell line was altered, and the response of leukemic cells to DOX treatment was analyzed. It was observed that overexpression of IK1 could enhance DOX-induced apoptosis, while knockdown of IK1 attenuated DOX-induced apoptosis in leukemic cells. Further experiments demonstrated that IK1 sensitized leukemic cells to DOX-induced apoptosis, probably through upregulation of caspase-9. These data suggest that high expression levels of IK1 may be a potential biomarker to predict responses of leukemia patients to treatment with chemotherapy.

  16. Serial analysis of gene expression in a microglial cell line.

    PubMed

    Inoue, H; Sawada, M; Ryo, A; Tanahashi, H; Wakatsuki, T; Hada, A; Kondoh, N; Nakagaki, K; Takahashi, K; Suzumura, A; Yamamoto, M; Tabira, T

    1999-12-01

    We used the serial analysis of gene expression (SAGE) method to systematically analyze transcripts present in a microglial cell line. Over 10,000 SAGE tags were sequenced, and shown to represent 6,013 unique transcripts. Among the diverse transcripts that had not been previously detected in microglia were those for cytokines such as endothelial monocyte-activating polypeptide I (EMAP I), and for cell surface antigens, including adhesion molecules such as CD9, CD53, CD107a, CD147, CD162 and mast cell high affinity IgE receptor. In addition, we detected transcripts that were characteristic of hematopoietic cells or mesodermal structures, such as E3 protein, A1, EN-7, B94, and ufo. Furthermore, the profile contained a transcript, Hn1, that is important in hematopoietic cells and neurological development (Tang et al. Mamm Genome 8:695-696, 1997), suggesting the probable neural differentiation of microglia from the hematopoietic system in development. Messenger RNA expression of these genes was confirmed by RT-PCR in primary cultures of microglia. Significantly, this is the first systematic profiling of the genes expressed in a microglial cell line. The identification and further characterization of the genes described here should provide potential new targets for the study of microglial biology. PMID:10559785

  17. AIRE expressing marginal zone dendritic cells balances adaptive immunity and T-follicular helper cell recruitment.

    PubMed

    Lindmark, Evelina; Chen, Yunying; Georgoudaki, Anna-Maria; Dudziak, Diana; Lindh, Emma; Adams, William C; Loré, Karin; Winqvist, Ola; Chambers, Benedict J; Karlsson, Mikael C I

    2013-05-01

    Autoimmune polyendocrine syndrome Type I (APS I) results in multiple endocrine organ destruction and is caused by mutations in the Autoimmune regulator gene (AIRE). In the thymic stroma, cells expressing the AIRE gene dictate T cell education and central tolerance. Although this function is the most studied, AIRE is also expressed in the periphery in DCs and stromal cells. Still, how AIRE regulated transcription modifies cell behaviour in the periphery is largely unknown. Here we show that AIRE is specifically expressed by 33D1(+) DCs and dictates the fate of antibody secreting cell movement within the spleen. We also found that AIRE expressing 33D1(+) DCs expresses self-antigens as exemplified by the hallmark gene insulin. Also, as evidence for a regulatory function, absence of Aire in 33D1(+) DCs led to reduced levels of the chemokine CXCL12 and increased co-stimulatory properties. This resulted in altered activation and recruitment of T-follicular helper cells and germinal centre B cells. The altered balance leads to a change of the early response to a T cell-dependent antigen in Aire(-/-) mice. These findings add to the understanding of how specific DC subtypes regulate the early responses during T cell-dependent antibody responses within the spleen and further define the role of AIRE in the periphery as regulator of self-antigen expression and lymphocyte migration. PMID:23265639

  18. Gene cataloging and expression profiling in human gastric cancer cells by expressed sequence tags.

    PubMed

    Kim, Nam-Soon; Hahn, Yoonsoo; Oh, Jung-Hwa; Lee, Ju-Yeon; Oh, Kyung-Jin; Kim, Jeong-Min; Park, Hong-Seog; Kim, Sangsoo; Song, Kyu-Sang; Rho, Seung-Moo; Yoo, Hyang-Sook; Kim, Yong Sung

    2004-06-01

    To understand the molecular mechanism associated with gastric carcinogenesis, we identified genes expressed in gastric cancer cell lines and tissues. Of 97,609 high-quality ESTs sequenced from 36 cDNA libraries, 92,545 were coalesced into 10,418 human Unigene clusters (Build 151). The gene expression profile was produced by counting the cluster frequencies in each library. Although the profiles of highly expressed genes varied greatly from library to library, those genes related to cell structure formation, heat shock proteins, the glycolysis pathway, and the signaling pathway were highly represented in human gastric cancer cell lines and in primary tumors. Conversely, the genes encoding immunoglobulins, ribosomal proteins, and digestive proteins were down-regulated in gastric cancer cell lines and tissues compared to normal tissues. The transcription levels of some of these genes were confirmed by RT-PCR. We found that genes related to cell adhesion, apoptosis, and cytoskeleton formation were particularly up-regulated in the gastric cancer cell lines established from malignant ascites compared to those from primary tumors. This comprehensive molecular profiling of human gastric cancer should be useful for elucidating the genetic events associated with human gastric cancer. PMID:15177556

  19. Human clusterin gene expression is confined to surviving cells during in vitro programmed cell death.

    PubMed Central

    French, L E; Wohlwend, A; Sappino, A P; Tschopp, J; Schifferli, J A

    1994-01-01

    Clusterin is a serum glycoprotein endowed with cell aggregating, complement inhibitory, and lipid binding properties, and is also considered as a specific marker of dying cells, its expression being increased in various tissues undergoing programmed cell death (PCD). However, no study has so far directly shown that cells expressing clusterin in these tissues are actually apoptotic as defined by morphological and biochemical criteria. We have studied cellular clusterin gene expression in vitro using three different models of PCD: (a) ultraviolet B (UV-B) irradiation of human U937, HeLa, and A431 cell lines, (b) in vitro aging of human peripheral blood neutrophils (PMNs), and (c) dexamethasone-induced cell death of the human lymphoblastoid cell line CEM-C7. In all three models, the classical morphological and biochemical features of PCD observed did not correlate with an increase, but with either a marked decrease or an absence of clusterin gene expression as assessed by Northern blot analysis. In situ hybridization of U937 and A431 cells after UV-B irradiation revealed, in addition, that only morphologically normal cells that are surviving continue to express the clusterin gene. Our results demonstrate that in the human myeloid, lymphoid, and epithelial cell types studied, clusterin gene expression is not a prerequisite to their death by apoptosis. In addition, and most interestingly, in situ hybridization of U937 and A431 cells revealed that only surviving cells express the clusterin gene after the induction of PCD, thus providing novel evidence suggesting that clusterin may be associated with cell survival within tissues regressing as a consequence of PCD. Images PMID:8113419

  20. Activation of proglucagon gene transcription by protein kinase-A in a novel mouse enteroendocrine cell line.

    PubMed

    Drucker, D J; Jin, T; Asa, S L; Young, T A; Brubaker, P L

    1994-12-01

    The gene encoding proglucagon is expressed predominantly in the pancreas and intestine. The physiological importance of glucagon secreted from the islets of Langerhans has engendered considerable interest in the molecular control of proglucagon gene transcription in the endocrine pancreas. In contrast, little is known about the molecular control of proglucagon gene expression in the intestine. The recent demonstration that glucagon-like peptide-1 (GLP-1) secreted from the intestine is a potent regulator of insulin secretion and glucose homeostasis has stimulated renewed interest in the factors that control GLP-1 synthesis in the intestinal L-cell. To develop a model for the analysis of intestinal proglucagon gene expression, we have targeted expression of a proglucagon gene-simian virus-40 large T-antigen fusion gene to enteroendocrine cells in transgenic mice. These mice develop intestinal tumors that were used to derive a novel cell line, designated GLUTag, that expresses the proglucagon gene and secretes immunoreactive GLP-1 in vitro. GLUTag cells demonstrate morphological characteristics of enteroendocrine cells by electron microscopy and are plurihormonal, as shown by immunocytochemistry and RNA analyses. GLUTag cells express the proglucagon and cholecystokinin genes, consistent with the pattern of lineage-specific enteroendocrine differentiation described for mouse intestine. Proglucagon gene expression was induced by activators of the protein kinase-A pathway, and a combination of messenger RNA half-life and nuclear run-on experiments demonstrated that the protein kinase-A-induction is mediated by an increase in proglucagon gene transcription. In contrast, activators of protein kinase-C stimulated secretion, but not biosynthesis of the PGDPs in GLUTag cell cultures. Analysis of proglucagon processing in GLUTag cells demonstrated the liberation of glucagon, oxyntomodulin, glicentin, and multiple forms of GLP-1. These observations provide evidence for the

  1. PGC-1α Promotes Ureagenesis in Mouse Periportal Hepatocytes through SIRT3 and SIRT5 in Response to Glucagon

    PubMed Central

    Li, Lulu; Zhang, Ping; Bao, Zhengxi; Wang, Tongxin; Liu, Shuang; Huang, Feiruo

    2016-01-01

    Excess ammonia is produced during fasting when amino acids are used for glucogenesis. Together with ureagenesis, glucogenesis occurs in periportal hepatocytes mediated mainly through the peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α). In vivo experiments showed that fasting strongly stimulated mice glucagon secretion, hepatic PGC-1α, sirtuin 3 (SIRT3) and sirtuin 5 (SIRT5) expression and ureagenesis enzymatic activity such as carbamoyl phosphate synthetase 1 (CPS1) and ornithine transcarbamoylase (OTC). Interestingly, 15N-labeled urea and 13C-labeled glucose production in wild-type mice were significantly increased compared with PGC-1α null mice by [15N,13C]alanine perfused liver. Glucagon significantly stimulated ureagenesis, expression of SIRT3, SIRT5 and the activities of CPS1 and OCT but did not stimulate PGC-1α silencing hepatocytes in mice periportal hepatocytes. Contrarily, PGC-1α overexpression significantly increased the expression of SIRT3, SIRT5 and the activities of CPS1 and OTC, but induced no significant changes in CPS1 and OTC expression. Morever, SIRT3 directly deacetylates and upregulates the activity of OTC, while SIRT5 deacetylates and stimulates the activity of CPS1. During fasting, PGC-1α facilitates ureagenesis in mouse periportal hepatocytes by deacetylating CPS1 and OTC modulated by mitochondrial deacetylase, SIRT3 and SIRT5. This mechanism may be relevant to ammonia detoxification and metabolic homeostasis in liver during fasting. PMID:27052737

  2. PDGFR-{beta} expression in small cell lung cancer patients

    SciTech Connect

    Shinohara, Eric T.; Gonzalez, Adriana; Massion, Pierre P.; Olson, Sandra J.; Albert, Jeffrey M.; Shyr, Yu; Carbone, David P.; Johnson, David H.; Hallahan, Dennis E.; Lu Bo . E-mail: bo.lu@vanderbilt.edu

    2007-02-01

    Background: Platelet derived growth factor (PDGF) and PDGFR-{beta} are expressed and have been found to have prognostic value in several human cancers. Data in non-small-cell cancer cell lines have suggested that PDGFR is a therapeutic target for drug development. In the current study PDGFR-{beta} expression and prognostic value in small cell lung cancer (SCLC) was investigated. Methods and Materials: Paraffin-embedded tissue blocks from 53 patients with limited and extensive stage SCLC were obtained for immunohistochemical staining. Tumors from each patient were sampled 3 times and stained with PDGFR-{beta} specific antibody. Patients were divided into low and high staining groups based on intensity. Results: There was high intensity PDGFR-{beta} staining in 20 patients with SCLC. Another 29 expressed low intensity PDGFR-{beta} staining, with only 4 patients showing no PDGFR-{beta} staining. There was no statistically significant difference in 5 year overall survival between patients with low levels of PDGFR-{beta} staining vs. those with high level staining SCLC tumors (p = 0.538). Conclusions: The present study found that the majority of SCLC patients express, at least, a low level of PDGF-{beta}. However, the level of PDGFR-{beta} expression was not a statistically significant predictor of 5 year overall survival in SCLC.

  3. Live-Cell, Temporal Gene Expression Analysis of Osteogenic Differentiation in Adipose-Derived Stem Cells

    PubMed Central

    Desai, Hetal V.; Voruganti, Indu S.; Jayasuriya, Chathuraka; Chen, Qian

    2014-01-01

    Adipose-derived stem cells (ASCs) are a widely investigated type of mesenchymal stem cells with great potential for musculoskeletal regeneration. However, the use of ASCs is complicated by their cellular heterogeneity, which exists at both the population and single-cell levels. This study demonstrates a live-cell assay to investigate gene expression in ASCs undergoing osteogenesis using fluorescently tagged DNA hybridization probes called molecular beacons. A molecular beacon was designed to target the mRNA sequence for alkaline phosphatase (ALPL), a gene characteristically expressed during early osteogenesis. The percentage of cells expressing this gene in a population was monitored daily to quantify the uniformity of the differentiation process. Differentiating ASC populations were repeatedly measured in a nondestructive fashion over a 10-day period to obtain temporal gene expression data. Results showed consistent expression patterns for the investigated osteogenic genes in response to induction medium. Peak signal level, indicating when the most cells expressed ALPL at once, was observed on days 3–5. The differentiation response of sample populations was generally uniform when assessed on a well-by-well basis over time. The expression of alkaline phosphatase is consistent with previous studies of osteogenic differentiation, suggesting that molecular beacons are a viable means of monitoring the spatiotemporal gene expression of live, differentiating ASCs. PMID:24367991

  4. Disrupted dynamics of F-actin and insulin granule fusion in INS-1 832/13 beta-cells exposed to glucotoxicity: partial restoration by glucagon-like peptide 1.

    PubMed

    Quinault, Aurore; Gausseres, Blandine; Bailbe, Danielle; Chebbah, Nella; Portha, Bernard; Movassat, Jamileh; Tourrel-Cuzin, Cecile

    2016-08-01

    Actin dynamics in pancreatic β-cells is involved in insulin exocytosis but the molecular mechanisms of this dynamics and its role in biphasic insulin secretion in pancreatic β-cells is largely unknown. Moreover, the impact of a glucotoxic environment on the sub-cortical actin network dynamics is poorly studied. In this study, we investigate the behavior of insulin granules and the subcortical actin network dynamics in INS-1 832/13 β-cells submitted to a normal or glucotoxic environment. Our results show that glucose stimulation leads to a reorganization of the subcortical actin network with a rupture of its interactions with t-SNARE proteins (Syntaxin 1A and SNAP-25), promoting insulin secretion in INS-1 832/13 β-cells. Prolonged exposure of INS-1 832/13 β-cells to high-glucose levels (glucotoxicity) leads to the densification of the cortical actin network, which prevents its reorganization under acute glucose, and diminishes the glucose-stimulated insulin secretion, as shown by the decreased number of fusion events. The most interesting in our results is the partial restoration by GLP-1 of the insulin secretion ability from high-glucose treated INS-1 832/13 cells. This improved insulin exocytosis is associated with partial restored actin dynamics and fusion events during the two phases of the secretion, with a preferential involvement of Epac2 signaling in the first phase and a rather involvement of PKA signaling in the second phase of insulin exocytosis. All these data provide some new insights into the mechanism by which current therapeutics may be improving insulin secretion. PMID:27101990

  5. Association of luteinizing hormone receptor gene expression with cell cycle progression in granulosa cells

    PubMed Central

    Cannon, Jennifer D.; Seekallu, Srinivas V.; VandeVoort, Catherine A.; Chaffin, Charles L.

    2009-01-01

    During hormonally induced ovarian follicle growth, granulosa cell proliferation increases and returns to baseline prior to the administration of an ovulatory stimulus. Several key genes appear to follow a similar pattern, including the luteinizing hormone receptor (LHCGR), suggesting an association between cell cycle progression and gene expression. The expression of LHCGR mRNA in granulosa cells isolated from immature rats and treated in culture with FSH increased in a time-dependent manner, whereas administration of the cell cycle inhibitor mimosine completely suppressed expression. Although forskolin was able to induce luteinization in cells treated with mimosine, human chorionic gonadotropin had no effect, indicating the functional loss of LHCGR. The effects of mimosine on cell cycle progression and LHCGR mRNA expression were reversible within 24 h of mimosine removal. Cell cycle inhibition did not alter the stability of LHCGR mRNA, indicating that the primary effect was at the transcriptional level. To determine whether the relationship between LHCGR expression and cell cycle were relevant in vivo, immature rats were given a bolus of PMSG, followed by a second injection of either saline or PMSG 24 h later to augment levels of proliferation. The expression of LHCGR mRNA was elevated in the ovaries of animals receiving a supplement of PMSG. Mimosine also blocked cell cycle progression and LHCGR mRNA expression in macaque granulosa cells isolated following controlled ovarian stimulation cycles and in two different mouse Leydig tumor lines. These data collectively indicate that LHCGR mRNA is expressed as a function of the passage of cells across the G1-S phase boundary. PMID:19293332

  6. Calretinin mediates apoptosis in small cell lung cancer cells expressing tetraspanin CD9☆

    PubMed Central

    He, Ping; Kuhara, Hanako; Tachibana, Isao; Jin, Yingji; Takeda, Yoshito; Tetsumoto, Satoshi; Minami, Toshiyuki; Kohmo, Satoshi; Hirata, Haruhiko; Takahashi, Ryo; Inoue, Koji; Nagatomo, Izumi; Kida, Hiroshi; Kijima, Takashi; Naka, Tetsuji; Morii, Eiichi; Kawase, Ichiro; Kumanogoh, Atsushi

    2013-01-01

    A majority of small cell lung cancer (SCLC) cells lack a metastasis suppressor, tetraspanin CD9, and CD9 expression promotes their apoptosis. By a proteomics-based approach, we compared an SCLC cell line with its CD9 transfectant and found that a calcium-binding neuronal protein, calretinin, is upregulated in CD9-positive SCLC cells. Ectopic or anticancer drug-induced CD9 expression upregulated calretinin, whereas CD9 knockdown down-regulated calretinin in SCLC cells. When calretinin was knocked down, CD9-positive SCLC cells revealed increased Akt phosphorylation and decreased apoptosis. These results suggest that CD9 positively regulates the expression of calretinin that mediates proapoptotic effect in SCLC cells. PMID:23772398

  7. 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. PMID:27186352

  8. Firefly luciferase gene: structure and expression in mammalian cells.

    PubMed Central

    de Wet, J R; Wood, K V; DeLuca, M; Helinski, D R; Subramani, S

    1987-01-01

    The nucleotide sequence of the luciferase gene from the firefly Photinus pyralis was determined from the analysis of cDNA and genomic clones. The gene contains six introns, all less than 60 bases in length. The 5' end of the luciferase mRNA was determined by both S1 nuclease analysis and primer extension. Although the luciferase cDNA clone lacked the six N-terminal codons of the open reading frame, we were able to reconstruct the equivalent of a full-length cDNA using the genomic clone as a source of the missing 5' sequence. The full-length, intronless luciferase gene was inserted into mammalian expression vectors and introduced into monkey (CV-1) cells in which enzymatically active firefly luciferase was transiently expressed. In addition, cell lines stably expressing firefly luciferase were isolated. Deleting a portion of the 5'-untranslated region of the luciferase gene removed an upstream initiation (AUG) codon and resulted in a twofold increase in the level of luciferase expression. The ability of the full-length luciferase gene to activate cryptic or enhancerless promoters was also greatly reduced or eliminated by this 5' deletion. Assaying the expression of luciferase provides a rapid and inexpensive method for monitoring promoter activity. Depending on the instrumentation employed to detect luciferase activity, we estimate this assay to be from 30- to 1,000-fold more sensitive than assaying chloramphenicol acetyltransferase expression. Images PMID:3821727

  9. Profiling Eph receptor expression in cells and tissues

    PubMed Central

    Noberini, Roberta; Rubio de la Torre, Elena; Pasquale, Elena B.

    2012-01-01

    The Eph receptor tyrosine kinase family includes many members, which are often expressed together in various combinations and can promiscuously interact with multiple ephrin ligands, generating intricate networks of intracellular signals that control physiological and pathological processes. Knowing the entire repertoire of Eph receptors and ephrins expressed in a biological sample is important when studying their biological roles. Moreover, given the correlation between Eph receptor/ephrin expression and cancer pathogenesis, their expression patterns could serve important diagnostic and prognostic purposes. However, profiling Eph receptor and ephrin expression has been challenging. Here we describe a novel and straightforward approach to catalog the Eph receptors present in cultured cells and tissues. By measuring the binding of ephrin Fc fusion proteins to Eph receptors in ELISA and pull-down assays, we determined that a mixture of four ephrins is suitable for isolating both EphA and EphB receptors in a single pull-down. We then used mass spectrometry to identify the Eph receptors present in the pull-downs and estimate their relative levels. This approach was validated in cultured human cancer cell lines, human tumor xenograft tissue grown in mice, and mouse brain tissue. The new mass spectrometry approach we have developed represents a useful tool for the identification of the spectrum of Eph receptors present in a biological sample and could also be extended to profiling ephrin expression. PMID:22568954

  10. TPD52 expression increases neutral lipid storage within cultured cells.

    PubMed

    Kamili, Alvin; Roslan, Nuruliza; Frost, Sarah; Cantrill, Laurence C; Wang, Dongwei; Della-Franca, Austin; Bright, Robert K; Groblewski, Guy E; Straub, Beate K; Hoy, Andrew J; Chen, Yuyan; Byrne, Jennifer A

    2015-09-01

    Tumor protein D52 (TPD52) is amplified and/or overexpressed in cancers of diverse cellular origins. Altered cellular metabolism (including lipogenesis) is a hallmark of cancer development, and protein-protein associations between TPD52 and known regulators of lipid storage, and differential TPD52 expression in obese versus non-obese adipose tissue, suggest that TPD52 might regulate cellular lipid metabolism. We found increased lipid droplet numbers in BALB/c 3T3 cell lines stably expressing TPD52, compared with control and TPD52L1-expressing cell lines. TPD52-expressing 3T3 cells showed increased fatty acid storage in triglyceride (from both de novo synthesis and uptake) and formed greater numbers of lipid droplets upon oleic acid supplementation than control cells. TPD52 colocalised with Golgi, but not endoplasmic reticulum (ER), markers and also showed partial colocalisation with lipid droplets coated with ADRP (also known as PLIN2), with a proportion of TPD52 being detected in the lipid droplet fraction. Direct interactions between ADRP and TPD52, but not TPD52L1, were demonstrated using the yeast two-hybrid system, with ADRP-TPD52 interactions confirmed using GST pulldown assays. Our findings uncover a new isoform-specific role for TPD52 in promoting intracellular lipid storage, which might be relevant to TPD52 overexpression in cancer. PMID:26183179

  11. Cell surface expression and biosynthesis of epithelial Na+ channels.

    PubMed Central

    Prince, L S; Welsh, M J

    1998-01-01

    The epithelial Na+ channel (ENaC) complex is composed of three homologous subunits: alpha, beta and gamma. Mutations in ENaC subunits can increase the number of channels on the cell surface, causing a hereditary form of hypertension called Liddle's syndrome, or can decrease channel activity, causing pseudohypoaldosteronism type I, a salt-wasting disease of infancy. To investigate surface expression, we studied ENaC subunits expressed in COS-7 and HEK293 cells. Using surface biotinylation and protease sensitivity, we found that when individual ENaC subunits are expressed alone, they traffic to the cell surface. The subunits are glycosylated with high-mannose oligosaccharides, but seem to have the carbohydrate removed before they reach the cell surface. Moreover, subunits form a complex that cannot be disrupted by several non-ionic detergents. The pattern of glycosylation and detergent solubility/insolubility persists when the N-teminal and C-terminal cytoplasmic regions of ENaC are removed. With co-expression of all three ENaC subunits, the insoluble complex is the predominant species. These results show that ENaC and its family members are unique in their trafficking, biochemical characteristics and post-translational modifications. PMID:9841884

  12. Differential expression and interaction of host factors augment HIV-1 gene expression in neonatal mononuclear cells

    SciTech Connect

    Sundaravaradan, Vasudha; Mehta, Roshni; Harris, David T.; Zack, Jerome A.; Ahmad, Nafees

    2010-04-25

    We have previously shown a higher level of HIV-1 replication and gene expression in neonatal (cord) blood mononuclear cells (CBMC) compared with adult blood cells (PBMC), which could be due to differential expression of host factors. We performed the gene expression profile of CBMC and PBMC and found that 8013 genes were expressed at higher levels in CBMC than PBMC and 8028 genes in PBMC than CBMC, including 1181 and 1414 genes upregulated after HIV-1 infection in CBMC and PBMC, respectively. Several transcription factors (NF-kappaB, E2F, HAT-1, TFIIE, Cdk9, Cyclin T1), signal transducers (STAT3, STAT5A) and cytokines (IL-1beta, IL-6, IL-10) were upregulated in CBMC than PBMC, which are known to influence HIV-1 replication. In addition, a repressor of HIV-1 transcription, YY1, was down regulated in CBMC than PBMC and several matrix metalloproteinase (MMP-7, -12, -14) were significantly upregulated in HIV-1 infected CBMC than PBMC. Furthermore, we show that CBMC nuclear extracts interacted with a higher extent to HIV-1 LTR cis-acting sequences, including NF-kappaB, NFAT, AP1 and NF-IL6 compared with PBMC nuclear extracts and retroviral based short hairpin RNA (shRNA) for STAT3 and IL-6 down regulated their own and HIV-1 gene expression, signifying that these factors influenced differential HIV-1 gene expression in CBMC than PBMC.

  13. PROFILES OF GENE EXPRESSION ASSOCIATED WITH TETRACYCLINE OVER EXPRESSION OF HSP70 IN MCF-7 BREAST CANCER CELLS

    EPA Science Inventory

    Profiles of gene expression associated with tetracycline over expression of HSP70 in MCF-7 breast cancer cells.

    Heat shock proteins (HSPs) protect cells from damage through their function as molecular chaperones. Some cancers reveal high levels of HSP70 expression in asso...

  14. Enhancement of endothelial cell migration by constitutively active LPA{sub 1}-expressing tumor cells

    SciTech Connect

    Kitayoshi, Misaho; Kato, Kohei; Tanabe, Eriko; Yoshikawa, Kyohei; Fukui, Rie; Fukushima, Nobuyuki; Tsujiuchi, Toshifumi

    2012-06-01

    Highlights: Black-Right-Pointing-Pointer Mutated LPA{sub 1} stimulates cell migration of endothelial cells. Black-Right-Pointing-Pointer VEGF expressions are increased by mutated LPA{sub 1}. Black-Right-Pointing-Pointer LPA signaling via mutated LPA{sub 1} is involved in angiogenesis. Black-Right-Pointing-Pointer Mutated LPA{sub 1} promotes cancer cell progression. -- Abstract: Lysophosphatidic acid (LPA) receptors belong to G protein-coupled transmembrane receptors (LPA receptors; LPA{sub 1} to LPA{sub 6}). They indicate a variety of cellular response by the interaction with LPA, including cell proliferation, migration and differentiation. Recently, we have reported that constitutive active mutated LPA{sub 1} induced the strong biological effects of rat neuroblastoma B103 cells. In the present study, we examined the effects of mutated LPA{sub 1} on the interaction between B103 cells and endothelial F-2 cells. Each LPA receptor expressing B103 cells were maintained in serum-free DMEM and cell motility assay was performed with a Cell Culture Insert. When F-2 cells were cultured with conditioned medium from Lpar1 and Lpar3-expressing cells, the cell motility of F-2 cells was significantly higher than control cells. Interestingly, the motile activity of F-2 cells was strongly induced by mutated LPA{sub 1} than other cells, correlating with the expression levels of vascular endothelial growth factor (Vegf)-A and Vegf-C. Pretreatment of LPA signaling inhibitors inhibited F-2 cell motility stimulated by mutated LPA{sub 1}. These results suggest that activation of LPA signaling via mutated LPA{sub 1} may play an important role in the promotion of angiogenesis in rat neuroblastoma cells.

  15. Antitumor effects of celecoxib in COX-2 expressing and non-expressing canine melanoma cell lines

    PubMed Central

    Seo, Kyoung-won; Coh, Ye-rin; Rebhun, Robert B.; Ahn, Jin-ok; Han, Sei-Myung; Lee, Hee-woo; Youn, Hwa-Young

    2016-01-01

    Cyclooxygenase-2 (COX-2) is a potential target for chemoprevention and cancer therapy. Celecoxib, a selective COX-2 inhibitor, inhibits cell growth of various types of human cancer including malignant melanoma. In dogs, oral malignant melanoma represents the most common oral tumor and is often a fatal disease. Therefore, there is a desperate need to develop additional therapeutic strategies. The purpose of this study was to investigate the anticancer effects of celecoxib on canine malignant melanoma cell lines that express varying levels of COX-2. Celecoxib induced a significant anti-proliferative effect in both LMeC and CMeC-1 cells. In the CMeC cells, treatment of 50 µM celecoxib caused an increase in cells in the G0/G1 and a decreased proportion of cells in G-2 phase. In the LMeC cells, 50 µM of celecoxib led to an increase in the percentage of cells in the sub-G1 phase and a significant activation of caspase-3 when compared to CMeC-1 cells. In conclusion, these results demonstrate that celecoxib exhibits antitumor effects on canine melanoma LMeC and CMeC-1 cells by induction of G1-S cell cycle arrest and apoptosis. Our data suggest that celecoxib might be effective as a chemotherapeutic agent against canine malignant melanoma. PMID:24656746

  16. Immunotoxin BL22 induces apoptosis in mantle cell lymphoma (MCL) cells dependent on Bcl-2 expression.

    PubMed

    Bogner, Christian; Dechow, Tobias; Ringshausen, Ingo; Wagner, Michaela; Oelsner, Madlen; Lutzny, Gloria; Licht, Thomas; Peschel, Christian; Pastan, Ira; Kreitman, Robert J; Decker, Thomas

    2010-01-01

    Mantle cell lymphoma (MCL) is an incurable mature B cell proliferation, combining the unfavourable clinical features of aggressive and indolent lymphomas. The blastic variant of MCL has an even worse prognosis and new treatment options are clearly needed. We analysed the effects of BL22, an immunotoxin composed of the Fv portion of an anti- CD22 antibody fused to a 38-kDa Pseudomonas exotoxin-A fragment on four MCL cell lines as well as on primary cells of four MCL patients. Apoptosis induction by BL22 was much more pronounced in MCL cell lines with low Bcl-2 expression (NCEB-1, JeKo-1 and JVM-2) compared to Granta-519 cells with high Bcl-2 expression. While the expression of the antiapoptotic protein Mcl-1 declined (NCEB-1, Granta-519), Bcl-2 levels remained unchanged in Granta-519 cells. However transfection of BCL2 cDNA into NCEB-1, JeKo-1 and JVM-2 cells significantly reduced BL22-mediated toxicity. Accordingly we examined the effects of Bcl-2 inactivation in Granta-519 cells using siRNA. Indeed, apoptosis induction was strongly enhanced in Granta-519 cells with silenced Bcl-2. Our results were confirmed in freshly isolated MCL-cells from patients with leukaemic MCL. We conclude that Bcl-2 expression is important for mediating resistance against the immunotoxin BL22 in MCL cells. PMID:19821820

  17. Proteomics Based Identification of Cell Migration Related Proteins in HBV Expressing HepG2 Cells

    PubMed Central

    Feng, Huixing; Li, Xi; Chan, Vincent; Chen, Wei Ning

    2014-01-01

    Proteomics study was performed to investigate the specific protein expression profiles of HepG2 cells transfected with mutant HBV compared with wildtype HBV genome, aiming to identify the specific functions of SH3 binding domain (proline rich region) located in HBx. In addition to the cell movement and kinetics changes due to the expression of HBV genome we have observed previously, here we further targeted to explore the specific changes of cellular proteins and potential intracellular protein interactions, which might provide more information of the potential cellular mechanism of the differentiated cell movements. Specific changes of a number of proteins were shown in global protein profiling in HepG2 cells expressing wildtype HBV, including cell migration related proteins, and interestingly the changes were found recovered by SH3 binding domain mutated HBV. The distinctive expressions of proteins were validated by Western blot analysis. PMID:24763314

  18. Single-cell gene expression profiling reveals functional heterogeneity of undifferentiated human epidermal cells

    PubMed Central

    Tan, David W. M.; Jensen, Kim B.; Trotter, Matthew W. B.; Connelly, John T.; Broad, Simon; Watt, Fiona M.

    2013-01-01

    Human epidermal stem cells express high levels of β1 integrins, delta-like 1 (DLL1) and the EGFR antagonist LRIG1. However, there is cell-to-cell variation in the relative abundance of DLL1 and LRIG1 mRNA transcripts. Single-cell global gene expression profiling showed that undifferentiated cells fell into two clusters delineated by expression of DLL1 and its binding partner syntenin. The DLL1+ cluster had elevated expression of genes associated with endocytosis, integrin-mediated adhesion and receptor tyrosine kinase signalling. Differentially expressed genes were not independently regulated, as overexpression of DLL1 alone or together with LRIG1 led to the upregulation of other genes in the DLL1+ cluster. Overexpression of DLL1 and LRIG1 resulted in enhanced extracellular matrix adhesion and increased caveolin-dependent EGFR endocytosis. Further characterisation of CD46, one of the genes upregulated in the DLL1+ cluster, revealed it to be a novel cell surface marker of human epidermal stem cells. Cells with high endogenous levels of CD46 expressed high levels of β1 integrin and DLL1 and were highly adhesive and clonogenic. Knockdown of CD46 decreased proliferative potential and β1 integrin-mediated adhesion. Thus, the previously unknown heterogeneity revealed by our studies results in differences in the interaction of undifferentiated basal keratinocytes with their environment. PMID:23482486

  19. Higher expression of SIRT1 induced resistance of esophageal squamous cell carcinoma cells to cisplatin

    PubMed Central

    Shi, Qintong; Wang, Wengong

    2015-01-01

    Background High expression of Sirtuin type 1 (SIRT1) exists in some cancer cells. However, it is still unclear whether SIRT1 affects the sensitivity of esophageal cancer cells to cisplatin. This study was designed to explore the relationship between SIRT1 expression and resistance of esophageal squamous cell carcinoma (ESCC) cells to cisplatin and reveal the underlying mechanism. Methods The tissue samples of 68 ESCC patients were collected from Nanjing Drum Tower Hospital, China. All the patients had undergone cisplatin based combination chemotherapy. The expression of SIRT1and Noxa in tissue samples were analyzed by quantitative real-time reverse PCR (qRT-PCR) and Western blot. Human ESCC cell line (ECa9706 cells) was cultured and a cisplatin-resistant subline (ECa9706-CisR cells) was established by continuous exposure to cisplatin at different concentrations. The expression of SIRT1 and Noxa in both cell lines was analyzed by qRT-PCR and Western blot. siRNA technology was utilized to down-regulate the SIRT1 expression in ECa9706-CisR cells. The influence of SIRT1 silence on sensitivity of ECa9706-CisR cells to cisplatin was confirmed using CCK-8 assay and flow cytometry. Furthermore, the level change of Noxa after SIRT1 silence in ECa9706-CisR cells was determined by qRT-PCR and Western blot. Result SIRT1 and Noxa expression in chemo-resistant patients was significantly increased and decreased respectively, compared with chemo-sensitive patients. SIRT1 expression in ECa9706-CisR cells was significantly increased with a lower Noxa level, compared with normal ECa9706 cells. Cisplatin 5 µM could cause proliferation inhibition, G2/M phase arrest and apoptosis in ECa9706-CisR cells and these effects could be enhanced dramatically by SIRT1 silencing. Moreover, Noxa expression was increased after treated with SIRT1 siRNA. Conclusions Over-expression of SIRT1 may cause resistance of ESCC cells to cisplatin through the mechanism involved with Noxa expression. PMID

  20. Isolation of genes predominantly expressed in guard cells and epidermal cells of Nicotiana glauca.

    PubMed

    Smart, L B; Cameron, K D; Bennett, A B

    2000-04-01

    Guard cells are specialized and metabolically active cells which arise during the differentiation of the epidermis. Using Nicotiana glauca epidermal peels as a source of purified guard cells, we have constructed a cDNA library from guard cell RNA. In order to isolate genes that are predominantly expressed in guard cells, we performed a differential screen of this library, comparing the hybridization of a radiolabeled cDNA probe synthesized from guard cell RNA to that from a mesophyll cell cDNA probe. Sixteen clones were isolated based on their greater level of hybridization with the guard cell probe. Of these, eight had high homology to lipid transfer protein (LTP), two were similar to glycine-rich protein (GRP), and one displayed high homology to proline-rich proteins from Arabidopsis thaliana (AtPRP2, AtPRP4) and from potato guard cells (GPP). Northern analysis confirmed that one or more NgLTP genes, NgGRP1, and NgGPP1 are all differentially expressed, with highest levels in guard cells, and low or undetectable levels in mesophyll cells and in roots. In addition, all are induced to some degree in drought-stressed guard cells. NgLTP and NgGRP1 expression was localized by in situ hybridization to the guard cells and pavement cells in the epidermis. NgGRP1 expression was also detected in cells of the vasculature. Genomic Southern analysis indicated that LTP is encoded by a family of highly similar genes in N. glauca. This work has identified members of a subset of epidermis- and guard cell-predominant genes, whose protein products are likely to contribute to the unique properties acquired by guard cells and pavement cells during differentiation. PMID:10890533

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

    PubMed Central

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

    1994-01-01

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

  2. Vitamin H-regulated transgene expression in mammalian cells.

    PubMed

    Weber, Wilfried; Bacchus, William; Daoud-El Baba, Marie; Fussenegger, Martin

    2007-01-01

    Although adjustable transgene expression systems are considered essential for future therapeutic and biopharmaceutical manufacturing applications, the currently available transcription control modalities all require side-effect-prone inducers such as immunosupressants, hormones and antibiotics for fine-tuning. We have designed a novel mammalian transcription-control system, which is reversibly fine-tuned by non-toxic vitamin H (also referred to as biotin). Ligation of vitamin H, by engineered Escherichia coli biotin ligase (BirA), to a synthetic biotinylation signal fused to the tetracycline-dependent transactivator (tTA), enables heterodimerization of tTA to a streptavidin-linked transrepressor domain (KRAB), thereby abolishing tTA-mediated transactivation of specific target promoters. As heterodimerization of tTA to KRAB is ultimately conditional upon the presence of vitamin H, the system is vitamin H responsive. Transgenic Chinese hamster ovary cells, engineered for vitamin H-responsive gene expression, showed high-level, adjustable and reversible production of a human model glycoprotein in bench-scale culture systems, bioreactor-based biopharmaceutical manufacturing scenarios, and after implantation into mice. The vitamin H-responsive expression systems showed unique band pass filter-like regulation features characterized by high-level expression at low (0-2 nM biotin), maximum repression at intermediate (100-1000 nM biotin), and high-level expression at increased (>100 000 nM biotin) biotin concentrations. Sequential ON-to-OFF-to-ON, ON-to-OFF and OFF-to-ON expression profiles with graded expression transitions can all be achieved by simply increasing the level of a single inducer molecule without exchanging the culture medium. These novel expression characteristics mediated by an FDA-licensed inducer may foster advances in therapeutic cell engineering and manufacturing of difficult-to-produce protein therapeutics. PMID:17827215

  3. Human vascular smooth muscle cells express a urate transporter.

    PubMed

    Price, Karen L; Sautin, Yuri Y; Long, David A; Zhang, Li; Miyazaki, Hiroki; Mu, Wei; Endou, Hitoshi; Johnson, Richard J

    2006-07-01

    An elevated serum uric acid is associated with the development of hypertension and renal disease. Renal regulation of urate excretion is largely controlled by URAT1 (SLC22A12), a member of the organic anion transporter superfamily. This study reports the specific expression of URAT1 on human aortic vascular smooth muscle cells, as assessed by reverse transcription-PCR and Western blot analysis. Expression of URAT1 was localized to the cell membrane. Evidence that the URAT1 transporter was functional was provided by the finding that uptake of 14C-urate was significantly inhibited in the presence of probenecid, an organic anion transporter inhibitor. It is proposed that URAT1 may provide a mechanism by which uric acid enters the human vascular smooth muscle cell, a finding that may be relevant to the role of uric acid in cardiovascular disease. PMID:16775029

  4. Stem Cell-Associated Marker Expression in Canine Hair Follicles.

    PubMed

    Gerhards, Nora M; Sayar, Beyza S; Origgi, Francesco C; Galichet, Arnaud; Müller, Eliane J; Welle, Monika M; Wiener, Dominique J

    2016-03-01

    Functional hair follicle (HF) stem cells (SCs) are crucial to maintain the constant recurring growth of hair. In mice and humans, SC subpopulations with different biomarker expression profiles have been identified in discrete anatomic compartments of the HF. The rare studies investigating canine HF SCs have shown similarities in biomarker expression profiles to that of mouse and human SCs. The aim of our study was to broaden the current repertoire of SC-associated markers and their expression patterns in the dog. We combined analyses on the expression levels of CD34, K15, Sox9, CD200, Nestin, LGR5 and LGR6 in canine skin using RT-qPCR, the corresponding proteins in dog skin lysates, and their expression patterns in canine HFs using immunohistochemistry. Using validated antibodies, we were able to define the location of CD34, Sox9, Keratin15, LGR5 and Nestin in canine HFs and confirm that all tested biomarkers are expressed in canine skin. Our results show similarities between the expression profile of canine, human and mouse HF SC markers. This repertoire of biomarkers will allow us to conduct functional studies and investigate alterations in the canine SC compartment of different diseases, like alopecia or skin cancer with the possibility to extend relevant findings to human patients. PMID:26739040

  5. Expression of cell cycle regulator cdk2ap1 suppresses tumor cell phenotype by non-cell autonomous mechanisms

    PubMed Central

    Zolochevska, Olga; Figueiredo, Marxa L.

    2009-01-01

    We evaluated the effect of expressing the cell cycle regulator cdk2ap1 in epithelial or stromal cell compartments to reduce SCC growth in vitro and in vivo. Cell autonomous and/or non-cell autonomous expression of cdk2ap1 reduced tumor growth and invasion and altered cell cycle, adhesion, invasion, angiogenesis, and apoptotic gene expression, as assessed by several in vitro phenotype assays, quantitative real time PCR, and in vivo molecular imaging using a novel three-way xenograft animal model. Our findings suggest that the interactions between cancer cells and fibroblasts that promote abnormal growth can be minimized by expressing cdk2ap1, supporting a novel concept by which tumor/growth suppressor genes can impact tumorigenesis phenotypes from non-cell autonomous interactions within the tumor microenvironment. PMID:19515604

  6. Expression of the Bitter Taste Receptor, T2R38, in Enteroendocrine Cells of the Colonic Mucosa of Overweight/Obese vs. Lean Subjects

    PubMed Central

    Latorre, Rocco; Huynh, Jennifer; Mazzoni, Maurizio; Gupta, Arpana; Bonora, Elena; Clavenzani, Paolo; Chang, Lin; Mayer, Emeran A.; De Giorgio, Roberto; Sternini, Catia

    2016-01-01

    Bitter taste receptors (T2Rs) are expressed in the mammalian gastrointestinal mucosa. In the mouse colon, T2R138 is localized to enteroendocrine cells and is upregulated by long-term high fat diet that induces obesity. The aims of this study were to test whether T2R38 expression is altered in overweight/obese (OW/OB) compared to normal weight (NW) subjects and characterize the cell types expressing T2R38, the human counterpart of mouse T2R138, in human colon. Colonic mucosal biopsies were obtained during colonoscopy from 35 healthy subjects (20 OW/OB and 15 NW) and processed for quantitative RT-PCR and immunohistochemistry using antibodies to T2R38, chromogranin A (CgA), glucagon like peptide-1 (GLP-1), cholecystokinin (CCK), or peptide YY (PYY). T2R38 mRNA levels in the colonic mucosa of OW/OB were increased (> 2 fold) compared to NW subjects but did not reach statistical significance (P = 0.06). However, the number of T2R38 immunoreactive (IR) cells was significantly increased in OW/OB vs. NW subjects (P = 0.01) and was significantly correlated with BMI values (r = 0.7557; P = 0.001). In both OW/OB and NW individuals, all T2R38-IR cells contained CgA-IR supporting they are enteroendocrine. In both groups, T2R38-IR colocalized with CCK-, GLP1- or PYY-IR. The overall CgA-IR cell population was comparable in OW/OB and NW individuals. This study shows that T2R38 is expressed in distinct populations of enteroendocrine cells in the human colonic mucosa and supports T2R38 upregulation in OW/OB subjects. T2R38 might mediate host functional responses to increased energy balance and intraluminal changes occurring in obesity, which could involve peptide release from enteroendocrine cells. PMID:26866366

  7. Characterization of TLX Expression in Neural Stem Cells and Progenitor Cells in Adult Brains

    PubMed Central

    Li, Shengxiu; Sun, Guoqiang; Murai, Kiyohito; Ye, Peng; Shi, Yanhong

    2012-01-01

    TLX has been shown to play an important role in regulating the self-renewal and proliferation of neural stem cells in adult brains. However, the cellular distribution of endogenous TLX protein in adult brains remains to be elucidated. In this study, we used immunostaining with a TLX-specific antibody to show that TLX is expressed in both neural stem cells and transit-amplifying neural progenitor cells in the subventricular zone (SVZ) of adult mouse brains. Then, using a double thymidine analog labeling approach, we showed that almost all of the self-renewing neural stem cells expressed TLX. Interestingly, most of the TLX-positive cells in the SVZ represented the thymidine analog-negative, relatively quiescent neural stem cell population. Using cell type markers and short-term BrdU labeling, we demonstrated that TLX was also expressed in the Mash1+ rapidly dividing type C cells. Furthermore, loss of TLX expression dramatically reduced BrdU label-retaining neural stem cells and the actively dividing neural progenitor cells in the SVZ, but substantially increased GFAP staining and extended GFAP processes. These results suggest that TLX is essential to maintain the self-renewing neural stem cells in the SVZ and that the GFAP+ cells in the SVZ lose neural stem cell property upon loss of TLX expression.Understanding the cellular distribution of TLX and its function in specific cell types may provide insights into the development of therapeutic tools for neurodegenerative diseases by targeting TLX in neural stem/progenitors cells. PMID:22952666

  8. Expression of TIA-1 and TIA-2 in T cell malignancies and T cell lymphocytosis.

    PubMed Central

    Matutes, E; Coelho, E; Aguado, M J; Morilla, R; Crawford, A; Owusu-Ankomah, K; Catovsky, D

    1996-01-01

    OBJECTIVE: To investigate the reactivity with TIA-1 and TIA-2, two monoclonal antibodies that recognise, respectively, granular structures in T lymphocytes and the T cell receptor chain in cells from a variety of T cell disorders. METHODS: Cytoplasmic staining with TIA-1 and TIA-2 was carried out by the immunoalkaline phosphatase anti-alkaline phosphatase technique in 67 cases with a T cell disorder: 31 large granular lymphocyte (LGL) leukaemia, nine T-prolymphocytic leukaemia (T-PLL), five Sezary syndrome, four peripheral T cell lymphoma (PTCL), 13 T cell lymphocytosis, and five T-acute lymphoblastic leukaemia (T-ALL). All had over 75% abnormal T cells which were CD2+, CD3+, CD5+, CD7+, and negative with B cell markers. RESULTS: TIA-1 was positive in 77% cases of LGL leukaemia and half of the PTCL and T-ALL, whereas it was negative in all Sezary syndrome and most T-PLL (8/9) and reactive T-lymphocytosis (10/13). In LGL leukaemia, TIA-1 was positive irrespective of the membrane phenotype, whether CD8+, CD4- or CD4+, CD8-, and was more often positive in cases where cells were CD16+, CD56+, or CD57+. TIA-2 was positive in 60% of cases encompassing all diagnostic types of T cell disorder. There was no correlation between TIA-2 expression and that of other T cell markers, activation antigens, and natural killer markers. CONCLUSIONS: The pattern of TIA-1 expression in T cell malignancies may help in the differential diagnosis among LGL leukaemia (high expression), T cell lymphocytosis and other T cell diseases (low expression). As TIA-2 is expressed in over 95% mature T lymphocytes and thymic cells, its assessment may be useful to demonstrate aberrant phenotypes which can be exploited for detecting minimal residual disease. Images PMID:8655683

  9. [The expression and significance of hnRNPD in esophageal squamous cell carcinoma cells].

    PubMed

    Geng, Yangyang; Zhang, Lulu; Xu, Miaomiao; Sheng, Wenjiong; Dong, Aijing; Cao, Jinming; Cao, Jianping

    2015-12-01

    Objective To investigate the expression of heterogeneous nuclear ribonucleoprotein D (hnRNPD) in esophageal squamous cell carcinoma (ESCC) tissues and the relationship between hnRNPD expression and the clinicopathological features of ESCC, and to study the effect of down-regulated hnRNPD on the proliferation of ESCC cells and explore its potential mechanism. Methods The expression of hnRNPD protein in ESCC tissues and the normal paracancerous tissues were detected by immunohistochemistry. The siRNA-hnRNPD was transfected into ESCC cells and the silence effect was verified by Western blotting. MTT assay and clone formation assay were used to evaluate the proliferation of ESCC cells after down-regulation of hnRNPD genes. Cell apoptosis was examined by annexin V-phycoerythrin/7-aminoactinomycin D (annexin V-PE/7-AAD) staining and flow cytometry. Results The expression of hnRNPD protein in ESCC tissues was significantly higher than that of the normal paracancerous tissues, and the expression was closely related with neoplasm staging. Down-regulation of hnRNPD inhibited the proliferation and clonality of ESCC cells. Compared with the control group, siRNA targeting hnRNPD significantly promoted cell apoptosis. Conclusion Down-regulation of hnRNPD inhibits the proliferation of ESCC cells by promoting cell apoptosis. PMID:26648300

  10. 'Fluorescent Cell Chip' for immunotoxicity testing: Development of the c-fos expression reporter cell lines

    SciTech Connect

    Trzaska, Dominika; Zembek, Patrycja; Olszewski, Maciej; Adamczewska, Violetta; Ulleras, Erik; Dastych, JarosIaw . E-mail: jdastych@cbm.pan.pl

    2005-09-01

    The Fluorescent Cell Chip for in vitro immunotoxicity testing employs cell lines derived from lymphocytes, mast cells, and monocytes-macrophages transfected with various EGFP cytokine reporter gene constructs. While cytokine expression is a valid endpoint for in vitro immunotoxicity screening, additional marker for the immediate-early response gene expression level could be of interest for further development and refinement of the Fluorescent Cell Chip. We have used BW.5147.3 murine thymoma transfected with c-fos reporter constructs to obtain reporter cell lines expressing ECFP under the control of murine c-fos promoter. These cells upon serum withdrawal and readdition and incubation with heavy metal compounds showed paralleled induction of c-Fos expression as evidenced by Real-Time PCR and ECFP fluorescence as evidenced by computer-supported fluorescence microscopy. In conclusion, we developed fluorescent reporter cell lines that could be employed in a simple and time-efficient screening assay for possible action of chemicals on c-Fos expression in lymphocytes. The evaluation of usefulness of these cells for the Fluorescent Cell Chip-based detection of immunotoxicity will require additional testing with a larger number of chemicals.

  11. Epithelial-Mesenchymal Transition Protein Expression in Basal Cell Adenomas and Basal Cell Adenocarcinomas.

    PubMed

    Tesdahl, Brennan A; Wilson, Thomas C; Hoffman, Henry T; Robinson, Robert A

    2016-06-01

    Basal cell adenomas and basal cell adenocarcinomas show marked histomorphologic similarity and are separated microscopically primarily by the invasive characteristics of the adenocarcinomas. We wished to explore potential differences in the expression of epithelial-mesenchymal transition associated proteins in these two tumor types. A tissue microarray was constructed utilizing 29 basal cell adenomas and 16 basal cell adenocarcinomas. Immunohistochemical expression of E-cadherin, beta-catenin, Twist 1 and vimentin were investigated. Both tumors expressed all proteins in a relatively similar manner. Nuclear beta-catenin was essentially limited to the abluminal cell populations in both tumor types. E-cadherin was limited largely to luminal locations but was more prevalent in the adenocarcinomas as compared to the adenomas. Primarily abluminal expression for vimentin was seen, sometimes present in an apical dot-like pattern. Distinct populations of cellular expression of these four markers of epithelial mesenchymal transition were present but were similar in locations in both tumors with no patterns discerned to separate basal cell adenoma from basal cell adenocarcinoma. Given these findings, the mechanisms by which basal cell adenocarcinoma is able to invade while its counterpart, basal cell adenoma can not, may be more complex than in other tumor types. PMID:26442856

  12. Neural effects of gut- and brain-derived glucagon-like peptide-1 and its receptor agonist.

    PubMed

    Katsurada, Kenichi; Yada, Toshihiko

    2016-04-01

    Glucagon-like peptide-1 (GLP-1) is derived from both the enteroendocrine L cells and preproglucagon-expressing neurons in the nucleus tractus solitarius (NTS) of the brain stem. As GLP-1 is cleaved by dipeptidyl peptidase-4 yielding a half-life of less than 2 min, it is plausible that the gut-derived GLP-1, released postprandially, exerts its effects on the brain mainly by interacting with vagal afferent neurons located at the intestinal or hepatic portal area. GLP-1 neurons in the NTS widely project in the central nervous system and act as a neurotransmitter. One of the physiological roles of brain-derived GLP-1 is restriction of feeding. GLP-1 receptor agonists have recently been used to treat type 2 diabetic patients, and have been shown to exhibit pleiotropic effects beyond incretin action, which involve brain functions. GLP-1 receptor agonist administered in the periphery is stable because of its resistance to dipeptidyl peptidase-4, and is highly likely to act on the brain by passing through the blood-brain barrier (BBB), as well as interacting with vagal afferent nerves. Central actions of GLP-1 have various roles including regulation of feeding, weight, glucose and lipid metabolism, cardiovascular functions, cognitive functions, and stress and emotional responses. In the present review, we focus on the source of GLP-1 and the pathway by which peripheral GLP-1 informs the brain, and then discuss recent findings on the central effects of GLP-1 and GLP-1 receptor agonists. PMID:27186358

  13. Enzymatic mono-pegylation of glucagon-like peptide 1 towards long lasting treatment of type 2 diabetes

    PubMed Central

    Selis, Fabio; Schrepfer, Rodolfo; Sanna, Riccardo; Scaramuzza, Silvia; Tonon, Giancarlo; Dedoni, Simona; Onali, Pierluigi; Orsini, Gaetano; Genovese, Stefano

    2012-01-01

    Human glucagon-like peptide-1 (GLP-1) is a physiological gastrointestinal peptide with glucose-dependent insulinotropic effects which is therefore considered an interesting antidiabetic agent. However, after in vivo administration, exogenous GLP-1 does not exert its physiological action due to the combination of rapid proteolytic degradation by ubiquitous dipeptidyldipeptidase IV (DPP IV) enzyme and renal clearance resulting in an extremely short circulating half-life. In this work we describe the conjugation of GLP-1-(7-36)-amide derivatives with polyethylene glycol (PEG) by enzymatic site-specific transglutamination reaction as an approach to reduce both the proteolysis and the renal clearance rates. The compound GLP-1-(7-36)-amide-Q23-PEG 20 kDa monopegylated on the single glutamine residue naturally present in position 23 maintained the ability to activate the GLP-1 receptor expressed in the rat β-cell line RIN-m5F with nanomolar potency along with an increased in vitro resistance to DDP IV and a circulating half-life of about 12 h after subcutaneous administration in rats. These properties enabled GLP-(7-36)-amide-Q23-PEG 20 kDa to exert a glucose-stabilizing effect for a period as long as 8 h, as demonstrated by a single subcutaneous injection to diabetic mice concomitantly challenged with an oral glucose load. The results reported in this work indicate that GLP-(7-36)-amide-Q23-PEG 20 kDa could be a lead compound for the development of long-lasting anti-diabetic agents useful in the treatment of type 2 diabetes affected patients. PMID:25755995

  14. Polony analysis of gene expression in ES cells and blastocysts

    PubMed Central

    Rieger, C.; Poppino, R.; Sheridan, R.; Moley, K.; Mitra, R.; Gottlieb, D.

    2007-01-01

    Expression profiling of stem cells is challenging due to their small numbers and heterogeneity. The PCR colony (polony) approach has theoretical advantages as an assay for stem cells but has not been applied to small numbers of cells. An assay has been developed that is sensitive enough to detect mRNAs from small numbers of ES cells and from fractions of a single mouse blastocyst. Genes assayed include Oct3, Rex1, Nanog, Cdx2 and GLUT-1. The assay is highly sensitive so that multiple mRNAs from a single blastocyst were easily detected in the same assay. In its present version, the assay is an attractive alternative to conventional RT–PCR for profiling small populations of stem cells. The assay is also amenable to improvements that will increase its sensitivity and ability to analyze many cDNAs simultaneously. PMID:18073198

  15. Expression of Hyaluronidase by Tumor Cells Induces Angiogenesis in vivo

    NASA Astrophysics Data System (ADS)

    Liu, Dacai; Pearlman, Eric; Diaconu, Eugenia; Guo, Kun; Mori, Hiroshi; Haqqi, Tariq; Markowitz, Sanford; Willson, James; Sy, Man-Sun

    1996-07-01

    Hyaluronic acid is a proteoglycan present in the extracellular matrix and is important for the maintenance of tissue architecture. Depolymerization of hyaluronic acid may facilitate tumor invasion. In addition, oligosaccharides of hyaluronic acid have been reported to induce angiogenesis. We report here that a hyaluronidase similar to the one on human sperm is expressed by metastatic human melanoma, colon carcinoma, and glioblastoma cell lines and by tumor biopsies from patients with colorectal carcinomas, but not by tissues from normal colon. Moreover, angiogenesis is induced by hyaluronidase+ tumor cells but not hyaluronidase- tumor cells and can be blocked by an inhibitor of hyaluronidase. Tumor cells thus use hyaluronidase as one of the ``molecular saboteurs'' to depolymerize hyaluronic acid to facilitate invasion. As a consequence, breakdown products of hyaluronic acid can further promote tumor establishment by inducing angiogenesis. Hyaluronidase on tumor cells may provide a target for anti-neoplastic drugs.

  16. Expression of Aquaporin-6 in Rat Retinal Ganglion Cells.

    PubMed

    Jang, Sun Young; Lee, Eung Suk; Ohn, Young-Hoon; Park, Tae Kwann

    2016-08-01

    Several aquaporins (AQPs) have been identified to be present in the eyes, and it has been suggested that they are involved in the movement of water and small solutes. AQP6, which has low water permeability and transports mainly anions, was recently discovered in the eyes. In the present study, we investigate the localization of AQP6 in the rat retina and show that AQP6 is selectively localized to the ganglion cell layer and the outer plexiform layer. Along with the gradual decrease in retinal ganglion cells after a crushing injury of optic nerve, immunofluorescence signals of AQP6 gradually decreased. Confocal microscope images confirmed AQP6 expression in retinal ganglion cells and Müller cells in vitro. Therefore, AQP6 might participate in water and anion transport in these cells. PMID:26526333

  17. Altered expression of uncoupling protein 2 in GLP-1-producing cells after chronic high glucose exposure: implications for the pathogenesis of diabetes mellitus.

    PubMed

    Urbano, Francesca; Filippello, Agnese; Di Pino, Antonino; Barbagallo, Davide; Di Mauro, Stefania; Pappalardo, Alessandro; Rabuazzo, Agata Maria; Purrello, Michele; Purrello, Francesco; Piro, Salvatore

    2016-04-01

    Glucagon-like peptide-1 (GLP-1) is a gut L-cell hormone that enhances glucose-stimulated insulin secretion. Several approaches that prevent GLP-1 degradation or activate the GLP-1 receptor are being used to treat type 2 diabetes mellitus (T2DM) patients. In T2DM, GLP-1 secretion has been suggested to be impaired, and this defect appears to be a consequence rather than a cause of impaired glucose homeostasis. However, although defective GLP-1 secretion has been correlated with insulin resistance, little is known about the direct effects of chronic high glucose concentrations, which are typical in diabetes patients, on GLP-1-secreting cell function. In the present study, we demonstrate that glucotoxicity directly affects GLP-1 secretion in GLUTag cells chronically exposed to high glucose. Our results indicate that this abnormality is associated with a decrease in ATP production due to the elevated expression of mitochondrial uncoupling protein 2 (UCP2). Furthermore, UCP2 inhibition using small interfering RNA (siRNA) and the application of glibenclamide, an ATP-sensitive potassium (KATP(+)) channel blocker, reverse the GLP-1 secretion defect induced by chronic high-glucose treatment. These results show that glucotoxicity diminishes the secretory responsiveness of GLP-1-secreting cells to acute glucose stimulation. We conclude that the loss of the incretin effect, as observed in T2DM patients, could at least partially depend on hyperglycemia, which is typical in diabetes patients. Such an understanding may not only provide new insight into diabetes complications but also ultimately contribute to the identification of novel molecular targets within intestinal L-cells for controlling and improving endogenous GLP-1 secretion. PMID:26739488

  18. Stem cell and neurogenic gene-expression profiles link prostate basal cells to aggressive prostate cancer

    PubMed Central

    Zhang, Dingxiao; Park, Daechan; Zhong, Yi; Lu, Yue; Rycaj, Kiera; Gong, Shuai; Chen, Xin; Liu, Xin; Chao, Hsueh-Ping; Whitney, Pamela; Calhoun-Davis, Tammy; Takata, Yoko; Shen, Jianjun; Iyer, Vishwanath R.; Tang, Dean G.

    2016-01-01

    The prostate gland mainly contains basal and luminal cells constructed as a pseudostratified epithelium. Annotation of prostate epithelial transcriptomes provides a foundation for discoveries that can impact disease understanding and treatment. Here we describe a genome-wide transcriptome analysis of human benign prostatic basal and luminal epithelial populations using deep RNA sequencing. Through molecular and biological characterizations, we show that the differential gene-expression profiles account for their distinct functional properties. Strikingly, basal cells preferentially express gene categories associated with stem cells, neurogenesis and ribosomal RNA (rRNA) biogenesis. Consistent with this profile, basal cells functionally exhibit intrinsic stem-like and neurogenic properties with enhanced rRNA transcription activity. Of clinical relevance, the basal cell gene-expression profile is enriched in advanced, anaplastic, castration-resistant and metastatic prostate cancers. Therefore, we link the cell-type-specific gene signatures to aggressive subtypes of prostate cancer and identify gene signatures associated with adverse clinical features. PMID:26924072

  19. The Drosophila ortholog of TMEM18 regulates insulin and glucagon-like signaling.

    PubMed

    Wiemerslage, Lyle; Gohel, Priya A; Maestri, Giulia; Hilmarsson, Torfi G; Mickael, Michel; Fredriksson, Robert; Williams, Michael J; Schiöth, Helgi B

    2016-06-01

    Transmembrane protein 18 (TMEM18) is an ill-described, obesity-related gene, but few studies have explored its molecular function. We found single-nucleotide polymorphism data, suggesting that TMEM18 may be involved in the regulation/physiology of metabolic syndrome based on associations with insulin, homeostatic model assessment-β (HOMAβ), triglycerides, and blood sugar. We then found an ortholog in the Drosophila genome, knocked down Drosophila Tmem18 specifically in insulin-producing cells, and tested for its effects on metabolic function. Our results suggest that TMEM18 affects substrate levels through insulin and glucagon signaling, and its downregulation induces a metabolic state resembling type 2 diabetes. This work is the first to experimentally describe the metabolic consequences of TMEM18 knockdown, and further supports its association with obesity. PMID:27029472

  20. Characterization of the hypotensive effects of glucagon-like peptide-2 in anesthetized rats.

    PubMed

    Iwai, Takashi; Kaneko, Maki; Sasaki-Hamada, Sachie; Oka, Jun-Ichiro

    2013-08-29

    Glucagon-like peptide-2 (GLP-2) is a proglucagon-derived peptide released from enteroendocrine cells and neurons. We recently reported that GLP-2 induced hypotension. In the present study, we characterized the mechanisms of GLP-2-induced hypotension. GLP-2 was administered peripherally or centrally to male Wistar rats anesthetized with urethane and α-chloralose. The rats were vagotomized or systemically pretreated with atropine, prazosin, or propranolol before the GLP-2 administration. The central and peripheral administration of GLP-2 reduced mean arterial blood pressure (MAP). The maximum change of MAP (maximum ΔMAP) was reduced by vagotomy or prazosin, but not propranolol. The effects of the central but not peripheral administration of GLP-2 were reduced by atropine. These results suggest that GLP-2 modulates vagal afferent inputs and inhibits the sympathetic nervous system in the brain to induce hypotension. PMID:23867714

  1. Glucagon like peptide-1 receptor agonists may ameliorate the metabolic adverse effect associated with antiretroviral therapy.

    PubMed

    Culha, Mehmet Gokhan; Inkaya, Ahmet Cagkan; Yildirim, Emre; Unal, Serhat; Serefoglu, Ege Can

    2016-09-01

    The number of people living with HIV and AIDS (PLWHA) reached to almost 40 million, half of which are under antiretroviral treatment (ART). Although the introduction of this therapy significantly improved the life span and quality of PLWHA, metabolic complications of these people remains to be an important issue. These metabolic complications include hyperlipidemia, abnormal fat redistribution and diabetes mellitus, which are defined as lipodystrophy syndrome. Glucagon-like peptide-1 (GLP-1) is a neuropeptide secreted from intestinal L cells and recently developed GLP-1 receptor agonists (GLP-1RAs) stimulate insulin secretion, improve weight control and reduce cardiovascular outcomes. This class of drugs may be a valuable medication in the treatment of HIV-associated metabolic adverse effects and extend the life expectancy of patients infected with HIV. PMID:27515222

  2. Resveratrol Represses Pokemon Expression in Human Glioma Cells.

    PubMed

    Yang, Yutao; Cui, Jiajun; Xue, Feng; Overstreet, Anne-Marie; Zhan, Yiping; Shan, Dapeng; Li, Hui; Li, Hui; Wang, Yongjun; Zhang, Mengmeng; Yu, Chunjiang; Xu, Zhi-Qing David

    2016-03-01

    POK erythroid myeloid ontogenic factor (Pokemon), an important proto-oncoprotein, is a transcriptional repressor that regulates the expression of many genes and plays an important role in tumorigenesis. Resveratrol (RSV), a natural polyphenolic compound, has many beneficial biological effects on health. In this study, we investigated the role of Pokemon in RSV-induced biological effects and the effect of RSV on the expression of Pokemon in glioma cells. We found that overexpression of Pokemon decreased RSV-induced cell apoptosis, senescence, and anti-proliferative effects. Moreover, we showed that RSV could efficiently decrease the activity of the Pokemon promoter and the expression of Pokemon. Meanwhile, RSV also inhibited Sp1 DNA binding activity to the Pokemon promoter; whereas, it did not influence the expression and nuclear translocation of Sp1. In addition, we found that RSV could increase the recruitment of HDAC1, but decreased p300 to the Pokemon promoter. Taken together, all these results extended our understanding on the anti-cancer mechanism of RSV in glioma cells. PMID:25875864

  3. Tangeretin induces cell cycle arrest and apoptosis through upregulation of PTEN expression in glioma cells.

    PubMed

    Ma, Li-Li; Wang, Da-Wei; Yu, Xu-Dong; Zhou, Yan-Ling

    2016-07-01

    Tangeretin (TANG), present in peel of citrus fruits, has been shown to various medicinal properties such as chemopreventive and neuroprotective. However, the chemopreventive effect of TANG on glioblastoma cells has not been examined. The present study was designed to explore the anticancer potential of TANG in glioblastoma cells and to investigate the related mechanism. Human glioblastoma U-87MG and LN-18 cells were treated with 45μM concentration of TANG and cell growth was measured by MTT assay. The cell cycle distribution and cell death were measured by flow cytometry. The expression of cell cycle and apoptosis related genes were analyzed by quantitative RT-PCR and western blot. The cells treated with TANG were significantly increased cell growth suppression and cell death effects than vehicle treated cells. Further, TANG treatment increases G2/M arrest and apoptosis by modulating PTEN and cell-cycle regulated genes such as cyclin-D and cdc-2 mRNA and protein expressions. Moreover, the ability of TANG to decrease cell growth and to induce cell death was compromised when PTEN was knockdown by siRNA. Taken together, the chemopreventive effect of TANG is associated with regulation of cell-cycle and apoptosis in glioblastoma, thereby attenuating glioblastoma cell growth. Hence, the present findings suggest that TANG may be a therapeutic agent for glioblastoma treatment. PMID:27261630

  4. Stem cells and germ cells: microRNA and gene expression signatures.

    PubMed

    Dyce, Paul William; Toms, Derek; Li, Julang

    2010-04-01

    The study of primordial germ cell development in vivo is hampered by their low numbers and inaccessibility. Recent research has shown the ability of embryonic and adult stem cells to differentiate into primordial germ cells and more mature gametes and this generation of germ cells in vitro may be an attractive model for their study. One of the biggest challenges facing in vitro differentiation of stem cells into primordial germ cells is the lack of markers to clearly distinguish the two. As both cell types originate early in embryonic development they share many pluripotent markers such as OCT4, VASA, FRAGILIS, and NANOG. Genome wide microarray profiling has been used to identify transcriptome patterns unique to primordial germ cells. A more thorough analysis of the temporal and quantitative expression of a panel of genes may be more robust in distinguishing these two cell populations. MicroRNAs, short RNA molecules that have been shown to regulate translation through interactions with mRNA transcripts, have also recently come under investigation for the role they may play in pluripotency. Attempts to elucidate key microRNAs responsible for both stem cell and primordial germ cell characteristics have recently been undertaken. Unique microRNAs, either individually or as global profiles, may also help to distinguish differentiated primordial germ cells from stem cells in vitro. This review will examine gene expression and microRNA signatures in stem cells and germ cells as ways to distinguish these closely related cell types. PMID:20183803

  5. Cell-free protein expression based on extracts from CHO cells.

    PubMed

    Brödel, Andreas K; Sonnabend, Andrei; Kubick, Stefan

    2014-01-01

    Protein expression systems are widely used in biotechnology and medicine for the efficient and economic production of therapeutic proteins. Today, cultivated Chinese hamster ovary (CHO) cells are the market dominating mammalian cell-line for the production of complex therapeutic proteins. Despite this outstanding potential of CHO cells, no high-yield cell-free system based on translationally active lysates from these cells has been reported so far. To date, CHO cell extracts have only been used as a foundational research tool for understanding mRNA translation (Lodish et al., 1974; McDowell et al., 1972). In the present study, we address this fact by establishing a novel cell-free protein expression system based on extracts from cultured CHO cells. Lysate preparation, adaptation of in vitro reaction conditions and the construction of particular expression vectors are considered for high-yield protein production. A specific in vitro expression vector, which includes an internal ribosome entry site (IRES) from the intergenic region (IGR) of the Cricket paralysis virus (CrPV), has been constructed in order to obtain optimal performance. The IGR IRES is supposed to bind directly to the eukaryotic 40S ribosomal subunit thereby bypassing the process of translation initiation, which is often a major bottleneck in cell-free systems. The combination of expression vector and optimized CHO cell extracts enables the production of approximately 50 µg/mL active firefly luciferase within 4 h. The batch-type cell-free coupled transcription-translation system has the potential to perform post-translational modifications, as shown by the glycosylation of erythropoietin. Accordingly, the system contains translocationally active endogenous microsomes, enabling the co-translational incorporation of membrane proteins into biological membranes. Hence, the presented in vitro translation system is a powerful tool for the fast and convenient optimization of expression constructs, the

  6. Single-cell transcriptome analysis reveals coordinated ectopic gene expression patterns in medullary thymic epithelial cells

    PubMed Central

    Brennecke, Philip; Reyes, Alejandro; Pinto, Sheena; Rattay, Kristin; Nguyen, Michelle; Küchler, Rita; Huber, Wolfgang; Kyewski, Bruno; Steinmetz, Lars M.

    2015-01-01

    Expression of tissue-restricted self-antigens (TRAs) in medullary thymic epithelial cells (mTECs) is essential for self-tolerance induction and prevents autoimmunity, with each TRA being expressed in only a few mTECs. How this process is regulated in single mTECs and coordinated at the population level, such that the varied single-cell patterns add up to faithfully represent TRAs, is poorly understood. Here we used single-cell RNA-sequencing and provide evidence for numerous recurring TRA co-expression patterns, each present in only a subset of mTECs. Co-expressed genes clustered in the genome and showed enhanced chromatin accessibility. Our findings characterize TRA expression in mTECs as a coordinated process, which might involve local re-modeling of chromatin and thus ensures a comprehensive representation of the immunological self. PMID:26237553

  7. CD40 antigen is expressed by endothelial cells and tumor cells in Kaposi's sarcoma.

    PubMed Central

    Pammer, J.; Plettenberg, A.; Weninger, W.; Diller, B.; Mildner, M.; Uthman, A.; Issing, W.; Stürzl, M.; Tschachler, E.

    1996-01-01

    The CD40 antigen is a member of the tumor necrosis factor receptor/nerve growth factor receptor superfamily and is involved in cell proliferation, differentiation, and survival. Using different monoclonal antibodies, we found CD40 expression by immunohistochemistry on CD31- and CD34-positive Kaposi's sarcoma spindle cells in all tumors of 18 HIV-1 seropositive and 4 HIV-1 seronegative patients. Western blot analysis of tumor lysates detected a 48- to 50-kd glycoprotein corresponding to the CD40 antigen expressed by B lymphocytes. CD40 expression was also detectable in one of four cultures of spindle cells derived from Kaposi sarcoma tissue. Treatment of the CD40-positive spindle cells but not of the CD40-negative ones with interferon-gamma up-regulated CD40 surface expression. Besides on Kaposi sarcoma tumor cells, CD40 was distinctly present on vascular endothelial cells in areas within and adjacent to the tumors and in benign inflammatory lesions such as granulation tissue of HIV-1-negative patients. In contrast, CD34-negative endothelia of thin walled vessels, most likely lymphatics, were predominantly CD40 negative. Only faint or no CD40 expression was found on endothelial cells in normal skin. We conclude from our data that expression of the CD40 antigen by endothelial cells is up-regulated during tissue inflammation. As signaling through CD40 is able to increase cell survival, expression of CD40 by Kaposi sarcoma tumor cells might play an important role in the pathogenesis of this neoplasm. Images Figure 1 Figure 2 Figure 3 Figure 5 PMID:8623911

  8. Expression of cubilin in mouse testes and Leydig cells.

    PubMed

    Oh, Y S; Seo, J T; Ahn, H S; Gye, M C

    2016-04-01

    Cubilin (cubn) is a receptor for vitamins and various protein ligands. Cubn lacks a transmembrane domain but anchors to apical membranes by forming complexes with Amnionless or Megalin. In an effort to better understand the uptake of nutrients in testis, we analysed cubn expression in the developing mice testes. In testes, cubn mRNA increased from birth to adulthood. In the inter-stitium and isolated seminiferous tubules, neonatal increase in cubn mRNA until 14 days post-partum (pp) was followed by a marked increase at puberty (28 days pp). Cubn was found in the gonocytes, spermatogonia, spermatocytes and spermatids in the developing testes. In adult testes, strong Cubn immunoreactivity was found in the elongating spermatids, suggesting the role of Cubn in endocytosis during early spermiogenesis. In Sertoli cells and peritubular cells, Cubn immunoreactivity was weak throughout the testis development. In the inter-stitium, Cubn immunoreactivity was found in foetal Leydig cells, was weak to negligible in the stem cells and progenitor Leydig cells and was strong in immature and adult Leydig cells, demonstrating a positive association between Cubn and steroidogenic activity of Leydig cells. Collectively, these results suggest that Cubn may participate in the endocytotic uptake of nutrients in germ cells and somatic cells, supporting the spermatogenesis and steroidogenesis in mouse testes. PMID:26148765

  9. Lipopolysaccharide induces autotaxin expression in human monocytic THP-1 cells

    SciTech Connect

    Li Song; Zhang Junjie

    2009-01-09

    Autotaxin (ATX) is a secreted enzyme with lysophospholipase D (lysoPLD) activity, which converts lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA), a bioactive phospholipid involved in numerous biological activities, including cell proliferation, differentiation, and migration. In the present study, we found that bacterial lipopolysaccharide (LPS), a well-known initiator of the inflammatory response, induced ATX expression in monocytic THP-1 cells. The activation of PKR, JNK, and p38 MAPK was required for the ATX induction. The LPS-induced ATX in THP-1 cells was characterized as the {beta} isoform. In the presence of LPC, ATX could promote the migrations of THP-1 and Jurkat cells, which was inhibited by pertussis toxin (PTX), an inhibitor of Gi-mediated LPA receptor signaling. In summary, LPS induces ATX expression in THP-1 cells via a PKR, JNK and p38 MAPK-mediated mechanism, and the ATX induction is likely to enhance immune cell migration in proinflammatory response by regulating LPA levels in the microenvironment.

  10. Multiple melanocortin receptors are expressed in bone cells

    NASA Technical Reports Server (NTRS)

    Zhong, Qing; Sridhar, Supriya; Ruan, Ling; Ding, Ke-Hong; Xie, Ding; Insogna, Karl; Kang, Baolin; Xu, Jianrui; Bollag, Roni J.; Isales, Carlos M.

    2005-01-01

    Melanocortin receptors belong to the seven transmembrane domain, G-protein coupled family of receptors. There are five members of this receptor family labeled MC1R-MC5R. These receptors are activated by fragments derived from a larger molecule, proopiomelanocortin (POMC) and include ACTH, alpha beta and gamma-MSH and beta-endorphin. Because of in vitro and in vivo data suggesting direct effects of these POMC molecules on bone and bone turnover, we examined bone and bone derived cells for the presence of the various members of the melanocortin receptor family. We report that the five known melanocortin receptors are expressed to varying degrees in osteoblast-like and osteoclastic cells. POMC fragments increased proliferation and expression of a variety of genes in osteoblastic cells. Furthermore, POMC mRNA was detected in osteoclastic cells. These data demonstrate that POMC-derived peptide hormones acting through high affinity melanocortin receptors have specific effects on bone cells. Thus, in addition to the indirect effects of POMC-derived hormones on bone turnover through their modulation of steroid hormone secretion, POMC fragments may have direct and specific effects on bone cell subpopulations.

  11. Expression of CD34 on human B cell precursors.

    PubMed Central

    Schmitt, C; Eaves, C J; Lansdorp, P M

    1991-01-01

    CD34 is a 110-kD glycoprotein previously shown by a variety of monoclonal antibodies (MoAbs) to be expressed selectively on immature hematopoietic cells. However, more detailed characterization of CD34+ cells has been hampered by lack of anti-CD34 MoAbs that can be labelled directly with fluorochromes to facilitate subpopulation analysis by multi-parameter flow cytometry. We have recently isolated a murine anti-CD34 MoAb, designated as 8G12, that can be directly labelled with fluorochromes such as FITC. In this study, we have exploited this property of 8G12 to compare the reactivity of 8G12 and My10 with normal and leukaemic human marrow cells and to characterize normal early human B cell precursors by two- and three-colour immunofluorescence analysis. Comparison of three-colour staining profiles of normal bone marrow cells incubated with both 8G12 and MY10, and either anti-CD10 or anti-CD19 MoAb revealed the reactivity patterns of 8G12 and MY10 to be indistinguishable. This conclusion was confirmed by a similar comparative analysis of 8G12 and MY10 staining of blood and bone marrow cells from 4 patients with B lineage acute lymphoblastic leukaemia (ALL). Of interest, both 8G12 and MY10 detected a CD34+CD10+CD19- population in normal adult bone marrow. To determine whether a CD34+CD10+CD19- precursor population previously reported by others to exist in fetal liver could also be identified, CD10+CD16- marrow cells were first isolated by FACS and the sorted cells then re-analysed for expression of CD19 and CD34. These studies showed that all of the sorted CD10+ cells that expressed CD34 appeared to coexpress CD19. No CD34+CD10+CD19- cells were detected (at a sensitivity of less than or equal to 0.1%). Further studies will be required to determine whether a very minor population of CD34+CD10+CD19- cells may still be generated in the normal development of B cells in adult human marrow. PMID:1712682

  12. Gene expression profiling of cancer stem cell in human lung adenocarcinoma A549 cells

    PubMed Central

    Seo, Dong-Cheol; Sung, Ji-Min; Cho, Hee-Jung; Yi, Hee; Seo, Kun-Ho; Choi, In-Soo; Kim, Dong-Ku; Kim, Jin-Suk; El-Aty AM, Abd; Shin, Ho-Chul

    2007-01-01

    Background The studies on cancer-stem-cells (CSCs) have attracted so much attention in recent years as possible therapeutic implications. This study was carried out to investigate the gene expression profile of CSCs in human lung adenocarcinoma A549 cells. Results We isolated CSCs from A549 cell line of which side population (SP) phenotype revealed several stem cell properties. After staining the cell line with Hoechst 33342 dye, the SP and non-side population (non-SP) cells were sorted using flow cytometric analysis. The mRNA expression profiles were measured using an Affymetrix GeneChip® oligonucleotide array. Among the sixty one differentially expressed genes, the twelve genes inclusive three poor prognostic genes; Aldo-keto reductase family 1, member C1/C2 (AKR1C1/C2), Transmembrane 4 L six family member 1 nuclear receptor (TM4SF1), and Nuclear receptor subfamily 0, group B, member 1 (NR0B1) were significantly up-regulated in SP compared to non-SP cells. Conclusion This is the first report indicating the differences of gene expression pattern between SP and non-SP cells in A549 cells. We suggest that the up-regulations of the genes AKR1C1/C2, TM4SF1 and NR0B1 in SP of human adenocarcinoma A549 cells could be a target of poor prognosis in anti-cancer therapy. PMID:18034892

  13. Effects of space flight exposure on cell growth, tumorigenicity and gene expression in cancer cells

    NASA Astrophysics Data System (ADS)

    Yang, Cheng; Li, Yuehui; Zhang, Zhijie; Luo, Chen; Tong, Yongqing; Zhou, Guohua; Xie, Pingli; Hu, Jinyue; Li, Guancheng

    2008-12-01

    It is well recognized that harsh outer space environment, consisting of microgravity and radiation, poses significant health risks for human cells. To investigate potential effects of the space environment exposure on cancer cells we examined the biological changes in Caski cells carried by the "Shen Zhou IV" spaceship. After exposure for 7 days in spaceflight, 1440 survival subclonal cell lines were established and 4 cell lines were screened. 44F10 and 17E3 were selected because of their increased cell proliferation and tumorigenesis, while 48A9 and 31F2 had slower cytological events. Experiments with cell proliferation assay, flow cytometry, soft agar assay, tumorigenesis assay and DNA microarray analysis have shown that selected cell lines presented multiple biological changes in cell morphology, cell growth, tumorigenicity and gene expression. These results suggest that space environment exposure can make significant biological impact on cancer cells and provide an entry point to find the immunological target of tumorigenesis.

  14. Gene expression and cell turnover in human renal dysplasia.

    PubMed

    Woolf, A S; Winyard, P J

    2000-01-01

    Kidney malformations are common causes of chronic renal failure in children. Dysplastic kidneys represent a unique model of perturbed epithelial-mesenchymal interaction which leads to the formation of malformed branching tubules surrounded by undifferentiated and metaplastic mesenchymal cells. We have found that human dysplastic epithelia express PAX2 (a transcription factor), BCL2 (a survival factor) and galectin-3 (a cell adhesion/signaling molecule). These genes are implicated in oncogenesis and their persistent expression may drive proliferation of dysplastic cysts, hence explaining the massive growth of some multicystic dysplastic kidneys. We have also detected prominent apoptosis in undifferentiated tissues around dysplastic epithelia, and this may provide a potential mechanism for the well-documented regression of dysplastic kidneys. Hence, although these kidneys may not have any excretory function, it is incorrect to consider them as 'end stage organs' because they are highly active in terms of cell turnover and gene expression; furthermore, these processes can be correlated with patterns of tissue growth and involution. Further elucidation of 'molecular lesions' in renal malformations may lead to novel therapies to enhance the differentiation of progenitor cells. PMID:10668206

  15. Interferon-γ Reduces Melanosomal Antigen Expression and Recognition of Melanoma Cells by Cytotoxic T Cells

    PubMed Central

    Le Poole, I. Caroline; Riker, Adam I.; Quevedo, M. Eugenia; Stennett, Lawrence S.; Wang, Ena; Marincola, Francesco M.; Kast, W. Martin; Robinson, June K.; Nickoloff, Brian J.

    2002-01-01

    In malignant melanoma, tumor-infiltrating lymphocytes are frequently reactive with melanosomal antigens. Achieving complete remissions by peptide therapy is frequently hampered by metastases evading immune recognition. The tumor microenvironment seems to favor reduced expression of target antigens by melanoma cells. Among candidate factors, interferon-γ (IFN-γ) (102 to 103 U/ml) suppressed expression of antigens MART-1, TRP-1, and gp100 by M14 melanoma cells as shown by immunohistology and fluorescence-activated cell sorting analysis, reducing MART-1 expression by >65%. Northern blot analysis revealed that reduced expression was regulated at the transcriptional level, demonstrating a 79% reduction in MART-1 transcript abundance after 32 hours of IFN-γ treatment. To evaluate consequences of IFN-γ exposure for immune recognition, MART-1-responsive T cells were reacted with pretreated HLA-matched melanoma cells. Cytotoxicity was reduced up to 78% by IFN-γ pretreatment, and was restored by addition of MART-1 peptide AAGIGILTV for 2 hours. Examination of melanoma lesions by quantitative reverse transcriptase-polymerase chain reaction revealed up to 188-fold more abundant IFN-γ transcripts when compared to control skin. Laser capture microdissection and immunohistology localized most IFN-γ-producing T cells to the tumor stroma. Reduced MART-1 expression was frequently observed in adjacent tumor cells. Consequently, IFN-γ may enhance inflammatory responses yet hamper effective recognition of melanoma cells. PMID:11839572

  16. Immortalized bovine mammary epithelial cells express stem cell markers and differentiate in vitro.

    PubMed

    Hu, Han; Zheng, Nan; Gao, Haina; Dai, Wenting; Zhang, Yangdong; Li, Songli; Wang, Jiaqi

    2016-08-01

    The bovine mammary epithelial cell is a secretory cell, and its cell number and secretory activity determine milk production. In this study, we immortalized a bovine mammary epithelial cell line by SV40 large T antigen gene using a retrovirus based on Chinese Holstein primary mammary epithelial cells (CMEC) cultured in vitro. An immortalized bovine mammary epithelial cell line surpassed the 50-passage mark and was designated the CMEC-H. The immortalized mammary epithelial cells grew in close contact with each other and exhibited the typical cobblestone morphology characteristic with obvious boundaries. The telomerase expression of CMEC-H has consistently demonstrated the presence of telomerase activity as an immortalized cell line, but the cell line never induced tumor formation in nude mice. CMEC-H expressed epithelial (cytokeratins CK7, CK8, CK18, and CK19), mesenchymal (vimentin), and stem/progenitor (CD44 and p63) cell markers. The induced expression of milk proteins, αS1 -casein, β-casein, κ-casein, and butyrophilin, indicated that CMEC-H maintained the synthesis function of the mammary epithelial cells. The established immortalized bovine mammary epithelial cell line CMEC-H is capable of self-renewal and differentiation and can serve as a valuable reagent for studying the physiological mechanism of the mammary gland. PMID:27189858

  17. Triggering Receptor Expressed on Myeloid Cells in Cutaneous Melanoma.

    PubMed

    Nguyen, Austin Huy; Koenck, Carleigh; Quirk, Shannon K; Lim, Victoria M; Mitkov, Mario V; Trowbridge, Ryan M; Hunter, William J; Agrawal, Devendra K

    2015-10-01

    The tumor microenvironment plays an important role in the progression of melanoma, the prototypical immunologic cutaneous malignancy. The triggering receptor expressed on myeloid cells (TREM) family of innate immune receptors modulates inflammatory and innate immune signaling. It has been investigated in various neoplastic diseases, but not in melanoma. This study examines the expression of TREM-1 (a proinflammatory amplifier) and TREM-2 (an anti-inflammatory modulator and phagocytic promoter) in human cutaneous melanoma and surrounding tissue. Indirect immunofluorescence staining was performed on skin biopsies from 10 melanoma patients and staining intensity was semiquantitatively scored. Expression of TREM-1 and TREM-2 was higher in keratinocytes than melanoma tissue (TREM-1: p < 0.01; TREM-2: p < 0.01). Whereas TREM-2 was the dominant isoform expressed in normal keratinocytes, TREM-1 expression predominated in melanoma tissue (TREM-1 to TREM-2 ratio: keratinocytes = 0.78; melanoma = 2.08; p < 0.01). The increased TREM ratio in melanoma tissue could give rise to a proinflammatory and protumor state of the microenvironment. This evidence may be suggestive of a TREM-1/TREM-2 paradigm in which relative levels dictate inflammatory and immune states, rather than absolute expression of one or the other. Further investigation regarding this paradigm is warranted and could carry prognostic or therapeutic value in treatment for melanoma. PMID:26184544

  18. Expression of functional leptin receptors in rodent Leydig cells.

    PubMed

    Caprio, M; Isidori, A M; Carta, A R; Moretti, C; Dufau, M L; Fabbri, A

    1999-11-01

    Several studies indicate that the size of body fat stores and the circulating levels of the adipocyte-derived hormone leptin are able to influence the activity of the hypothalamic-pituitary-gonadal axis. The leptin-hypothalamic-pituitary-gonadal interactions have been mainly studied at the level of the central nervous system. In this study, we investigated the possibility that leptin may have direct effects on the rodent Leydig cell function. To probe this hypothesis, we first analyzed the expression of leptin receptors (OB-R) in rodent Leydig cells in culture. RT-PCR studies showed that rat Leydig cells express both the long (OB-Rb) and short isoform (OB-Ra) of leptin receptor, whereas MLTC-1 cells (a murine Leydig tumor cell line) express only the long isoform. Short-term (30-90 min) incubation of rat Leydig cells with increasing concentrations ofleptin (2-500 ng/ml) led to a significant and dose-dependent inhibition of human (h)CG-stimulated testosterone (T) production (approximately 60% reduction, IC50 = 20 ng/ml) but no change in basal androgen release. Also, leptin (150 ng/ml) amplified hCG-induced intracellular cAMP formation (1- to 2-fold) without modifying basal cAMP levels. Subsequent experiments showed that leptin inhibited 8Br-cAMP-stimulated T production, indicating that leptin's effect is exerted beyond cAMP. The inhibitory effect of leptin on hCG-induced T secretion was accompanied by a significant reduction of androstenedione and a concomitant rise of the precursor metabolites pregnenolone, progesterone, and 17-OH-progesterone, conceivable with a leptin-induced lesion of 17,20 lyase activity. Separate experiments performed with the MLTC-1 cells (not expressing cytochrome P450-17alpha) showed that leptin, though amplifying hCG-stimulated cAMP production, did not modify hCG-stimulated pregnenolone and progesterone release. These results further indicate that leptin action on steroidogenesis occurs downstream of progesterone synthesis. Northern Blot

  19. Constitutive and functional expression of YB-1 in microglial cells.

    PubMed

    Keilhoff, G; Titze, M; Esser, T; Langnaese, K; Ebmeyer, U

    2015-08-20

    Y-box-binding protein (YB-1) is a member of the cold-shock protein family and participates in a wide variety of DNA/RNA-dependent cellular processes including DNA repair, transcription, mRNA splicing, packaging, and translation. At the cellular level, YB-1 is involved in cell proliferation and differentiation, stress responses, and malignant cell transformation. A general role for YB-1 during inflammation has also been well described; however, there are minimal data concerning YB-1 expression in microglia, which are the immune cells of the brain. Therefore, we studied the expression of YB-1 in a clinically relevant global ischemia model for neurological injury following cardiac arrest. This model is characterized by massive neurodegeneration of the hippocampal CA1 region and the subsequent long-lasting activation of microglia. In addition, we studied YB-1 expression in BV-2 cells, which are an accepted microglia culture model. BV-2 cells were stressed by oxygen/glucose deprivation (OGD), OGD-relevant mediators, lipopolysaccharide (LPS), and phagocytosis-inducing cell debris and nanoparticles. Using quantitative polymerase chain reaction (PCR), we show constitutive expression of YB-1 transcripts in unstressed BV-2 cells. The functional upregulation of the YB-1 protein was demonstrated in microglia in vivo and in BV-2 cells in vitro. All stressors except for LPS were potent enhancers of the level of YB-1 protein, which appears to be regulated primarily by proteasomal degradation and, to a lesser extent, by the activation (phosphorylation) of the translation initiation factor eIF4E. The proteasome of BV-2 cells is impaired by OGD, which results in decreased protein degradation and therefore increased levels of YB-1 protein. LPS induces proteasome activity, which enables the level of YB-1 protein to remain at control levels despite enhanced protein ubiquitination. The proteasome inhibitor MG-132 was able to increase YB-1 protein levels in control and LPS

  20. ELK3 Expression Correlates With Cell Migration, Invasion, and Membrane Type 1-Matrix Metalloproteinase Expression in MDA-MB-231 Breast Cancer Cells.

    PubMed

    Heo, Sun-Hee; Lee, Je-Yong; Yang, Kyung-Min; Park, Kyung-Soon

    2015-01-01

    ELK3 is a member of the Ets family of transcription factors. Its expression is associated with angiogenesis, vasculogenesis, and chondrogenesis. ELK3 inhibits endothelial migration and tube formation through the regulation of MT1-MMP transcription. This study assessed the function of ELK3 in breast cancer (BC) cells by comparing its expression between basal and luminal cells in silico and in vitro. In silico analysis showed that ELK3 expression was higher in the more aggressive basal BC cells than in luminal BC cells. Similarly, in vitro analysis showed that ELK3 mRNA and protein expression was higher in basal BC cells than in normal cells and luminal BC cells. To investigate whether ELK3 regulates basal cell migration or invasion, knockdown was achieved by siRNA in the basal BC cell line MDA-MB-231. Inhibition of ELK3 expression decreased cell migration and invasion and downregulated MT1-MMP, the expression of which is positively correlated with tumor cell invasion. In silico analysis revealed that ELK3 expression was associated with that of MT1-MMP in several BC cell lines (0.98 Pearson correlation coefficient). Though MT1-MMP expression was upregulated upon ELK3 nuclear translocation, ELK3 did not directly bind to the 1.3-kb promoter region of the MT1-MMP gene. These results suggest that ELK3 plays a positive role in the metastasis of BC cells by indirectly regulating MT1-MMP expression. PMID:26637400

  1. Mesenchymal stem cells expressing neural antigens instruct a neurogenic cell fate on neural stem cells.

    PubMed

    Croft, Adam P; Przyborski, Stefan A

    2009-04-01

    The neurogenic response to injury in the postnatal brain is limited and insufficient for restoration of function. Recent evidence suggests that transplantation of mesenchymal stem cells (MSCs) into the injured brain is associated with improved functional recovery, mediated in part through amplification in the endogenous neurogenic response to injury. In the current study we investigate the interactions between bone marrow-derived MSCs and embryonic neural stem cells (NSCs) plus their differentiated progeny using an in vitro co-culture system. Two populations of MSCs were used, MSCs induced to express neural antigens (nestin+, Tuj-1+, GFAP+) and neural antigen negative MSCs. Following co-culture of induced MSCs with differentiating NSC/progenitor cells a significant increase in Tuj-1+ neurons was detected compared to co-cultures of non-induced MSCs in which an increase in astrocyte (GFAP+) differentiation was observed. The effect was mediated by soluble interactions between the two cell populations and was independent of any effect on cell death and proliferation. Induced and non-induced MSCs also promoted the survival of Tuj-1+ cell progeny in long-term cultures and both promoted axonal growth, an effect also seen in differentiating neuroblastoma cells. Therefore, MSCs provide instructive signals that are able to direct the differentiation of NSCs and promote axonal development in neuronal progeny. The data indicates that the nature of MSC derived signals is dependent not only on their microenvironment but on the developmental status of the MSCs. Pre-manipulation of MSCs prior to transplantation in vivo may be an effective means of enhancing the endogenous neurogenic response to injury. PMID:19159625

  2. Effect of glucagon on the diagnostic accuracy of double-contrast barium enema examinations.

    PubMed

    Thoeni, R F; Vandeman, F; Wall, S D

    1984-01-01

    The effect of glucagon-induced hypotonicity on the diagnostic accuracy of double-contrast barium enema examinations was determined in 133 consecutive patients in a double-blind crossover study. All patients underwent colonoscopy and served as their own controls by undergoing a double-contrast study after intravenous injection of 1 mg of g