Increased expression of GAD65 and GABA in pancreatic beta-cells impairs first-phase insulin secretion

The functional role of glutamate decarboxylase (GAD) and its product GABA in pancreatic islets has remained elusive. Mouse beta-cells express the larger isoform GAD67, whereas human islets express only the smaller isoform GAD65. We have generated two lines of transgenic mice expressing human GAD65 in pancreatic beta-cells (RIP7-hGAD65, Lines 1 and 2) to study the effect that GABA generated by this isoform has on islet cell function. The ascending order of hGAD65 expression and/or activity in beta-cells was Line 1 heterozygotes < Line 2 heterozygotes < Line 1 homozygotes. Line 1 heterozygotes have normal glucose tolerance, whereas Line 1 homozygotes and Line 2 heterozygotes exhibit impaired glucose tolerance and inhibition of insulin secretion in vivo in response to glucose. In addition, fasting levels of blood glucose are elevated and insulin is decreased in Line 1 homozygotes. Pancreas perfusion experiments suggest that GABA generated by GAD65 may function as a negative regulator of first-phase insulin secretion in response to glucose by affecting a step proximal to or at the K(ATP)(+) channel.     

Glucose transporter isotypes switch in T-antigen-transformed pancreatic beta cells growing in culture and in mice.

High-level expression of the low-Km glucose transporter isoform GLUT-1 is characteristic of many cultured tumor and oncogene-transformed cells. In this study, we tested whether induction of GLUT-1 occurs in tumors in vivo. Normal mouse beta islet cells express the high-Km (approximately 20 mM) glucose transporter isoform GLUT-2 but not the low-Km (1 to 3 mM) GLUT-1. In contrast, a beta cell line derived from an insulinoma arising in a transgenic mouse harboring an insulin-promoted simian virus 40 T-antigen oncogene (beta TC3) expressed very low levels of GLUT-2 but high levels of GLUT-1. GLUT-1 protein was not detectable on the plasma membrane of islets or tumors of the transgenic mice but was induced in high amounts when the tumor-derived beta TC3 cells were grown in tissue culture. GLUT-1 expression in secondary tumors formed after injection of beta TC3 cells into mice was reduced. Thus, high-level expression of GLUT-1 in these tumor cells is characteristic of culture conditions and is not induced by the oncogenic transformation; indeed, overnight culture of normal pancreatic islets causes induction of GLUT-1. We also investigated the relationship between expression of the different glucose transporter isoforms by islet and tumor cells and induction of insulin secretion by glucose. Prehyperplastic transgenic islet cells that expressed normal levels of GLUT-2 and no detectable GLUT-1 exhibited an increased sensitivity to glucose, as evidenced by maximal insulin secretion at lower glucose concentrations, compared with that exhibited by normal islets. Further, hyperplastic islets and primary and secondary tumors expressed low levels of GLUT-2 and no detectable GLUT-1 on the plasma membrane; these cells exhibited high basal insulin secretion and responded poorly to an increase in extracellular glucose. Thus, abnormal glucose-induced secretion of insulin in prehyperplastic islets in mice was independent of changes in GLUT-2 expression and did not require induction of GLUT-1 expression.     

Analysis of the glucose transporter content of islet cell lines: Implications for glucose-stimulated insulin release

Glucose transport across the plasma membrane of mammalian cells is mediated by a family of homologous proteins. Each glucose transporter isoform has a specific tissue distribution which relates to that tissue's demand for glucose. The β-cells of pancreatic islets are known to express a distinct glucose transporter isoform, termed GLUT 2, which has a high K_m for glucose. In this study, we examined the glucose transporter content of normal rat islets and three beta cell lines, β-TC, HIT and RIN cells. We show that at the protein level, GLUT 2 is the only detectable transporter isoform in normal islets, and that all three cell lines also express detectable GLUT 2. In contrast, all three cell lines expressed high levels of GLUT 1, but this isoform was not detected in normal islets. Neither the native islets nor any of the cell lines expressed GLUT 3. The insulin-responsive glucose transporter GLUT 4 was detected at very low levels in β-TC cells; to our knowledge, this is the only non-muscle or adipose cell line which expresses this isoform. We propose that the elevated level of GLUT 1 expression, together with a reduced expression of the high K_m transporter GLUT 2, may account for the characteristics aberrant patterns of glucose-stimulated insulin release in cell lines derived from β-cells.     

GAD65-mediated glutamate decarboxylation reduces glucose-stimulated insulin secretion in pancreatic beta cells

Mitochondrial metabolism plays a pivotal role in the pancreatic beta cell by generating signals that couple glucose sensing to insulin secretion. We have demonstrated previously that mitochondrially derived glutamate participates directly in the stimulation of insulin exocytosis. The aim of the present study was to impose altered cellular glutamate levels by overexpression of glutamate decarboxylase (GAD) to repress elevation of cytosolic glutamate. INS-1E cells infected with a recombinant adenovirus vector encoding GAD65 showed efficient overexpression of the GAD protein with a parallel increase in enzyme activity. In control cells glutamate levels were slightly increased by 7.5 mm glucose (1.4-fold) compared with the effect at 15 mm (2.3-fold) versus basal 2.5 mm glucose. Upon GAD overexpression, glutamate concentrations were no longer elevated by 15 mm glucose as compared with controls (-40%). Insulin secretion was stimulated in control cells by glucose at 7.5 mm (2.5-fold) and more efficiently at 15 mm (5.2-fold). INS-1E cells overexpressing GAD exhibited impaired insulin secretion on stimulation with 15 mm glucose (-37%). The secretory response to 30 mm KCl, used to raise cytosolic Ca(2+) levels, was unaffected. Similar results were obtained in perifused rat pancreatic islets following adenovirus transduction. This GAD65-mediated glutamate decarboxylation correlating with impaired glucose-induced insulin secretion is compatible with a role for glutamate as a glucose-derived factor participating in insulin exocytosis.     

Selective deletion of Pten in pancreatic beta cells leads to increased islet mass and resistance to STZ-induced diabetes

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a lipid phosphatase. PTEN inhibits the action of phosphatidylinositol-3-kinase and reduces the levels of phosphatidylinositol triphosphate, a crucial second messenger for cell proliferation and survival, as well as insulin signaling. In this study, we deleted Pten specifically in the insulin producing beta cells during murine pancreatic development. Pten deletion leads to increased cell proliferation and decreased cell death, without significant alteration of beta-cell differentiation. Consequently, the mutant pancreas generates more and larger islets, with a significant increase in total beta-cell mass. PTEN loss also protects animals from developing streptozotocin-induced diabetes. Our data demonstrate that PTEN loss in beta cells is not tumorigenic but beneficial. This suggests that modulating the PTEN-controlled signaling pathway is a potential approach for beta-cell protection and regeneration therapies.     

Inhibition of Notch activity promotes pancreatic cytokeratin 5-positive cell differentiation to beta cells and improves glucose homeostasis following acute pancreatitis

Some individuals develop prediabetes and/or diabetes following acute pancreatitis (AP). AP-induced beta-cell injury and the limited regenerative capacity of beta cells might account for pancreatic endocrine insufficiency. Previously, we found that only a few pancreatic cytokeratin 5 positive (Krt5⁺) cells differentiated into beta cells in the murine AP model, which was insufficient to maintain glucose homeostasis. Notch signaling determines pancreatic progenitor differentiation in pancreas development. This study aimed to examine whether Notch signaling inhibition could promote pancreatic Krt5⁺ cell differentiation into beta cells and improve glucose homeostasis following AP. Pancreatic tissues from patients with acute necrotizing pancreatitis (ANP) were used to evaluate beta-cell injury, Krt5⁺ cell activation and differentiation, and Notch activity. The murine AP model was induced by cerulein, and the effect of Notch inhibition on Krt5⁺ cell differentiation was evaluated both in vivo and in vitro. The results demonstrated beta-cell loss in ANP patients and AP mice. Krt5⁺ cells were activated in ANP pancreases along with persistently elevated Notch activity, which resulted in the formation of massive duct-like structures. AP mice that received Notch inhibitor showed that impaired glucose tolerance was reversed 7 and 15 days following AP, and increased numbers of newborn small islets due to increased differentiation of Krt5⁺ cells to beta cells to some extent. In addition, Krt5⁺ cells isolated from AP mice showed increased differentiation to beta cells by Notch inhibition. Collectively, these findings suggest that beta-cell loss contributes to pancreatic endocrine insufficiency following AP, and inhibition of Notch activity promotes pancreatic Krt5⁺ cell differentiation to beta cells and improves glucose homeostasis. The findings from this study may shed light on the potential treatment of prediabetes/diabetes following AP.Subject terms: Endocrine system and metabolic diseases, Pancreatitis     

Insulin secretion and islet endocrine cell content at onset and during the early stages of diabetes in the BB rat: effect of the level of glycemic control.

Although it is agreed that autoimmune destruction of pancreatic islets in diabetic BB rats is rapid, reports of endocrine cell content of islets from BB diabetic rats at the time of onset of diabetes vary considerably. Because of the rapid onset of the disease (hours) and the attendant changes in islet morphology and insulin secretion, it was the aim of this study to compare islet beta-cell numbers to other islet endocrine cells as close to the time of onset of hyperglycemia as possible (within 12 h). As it has been reported that hyperglycemia renders the beta cell insensitive to glucose, the early effects of different levels of insulin therapy (well-controlled vs. poorly controlled glycemia) on islet morphology and insulin secretion were examined. When measured within 12 h of onset, insulin content of BB diabetic islets, measured by morphometric analysis or pancreatic extraction, was 60% of insulin content of control islets. Despite significant amounts of insulin remaining in the pancreas, 1-day diabetic rats exhibited fasting hyperglycemia and were glucose intolerant. The insulin response from the isolated perfused pancreas to glucose and the glucose-dependent insulinotropic hormone, gastric inhibitory polypeptide (GIP), was reduced by 95%. Islet content of other endocrine peptides, glucagon, somatostatin, and pancreatic polypeptide, was normal at onset and at 2 weeks post onset. A group of diabetic animals, maintained in a hyperglycemic state for 7 days with low doses of insulin, were compared with a group kept normoglycemic by appropriate insulin therapy. No insulin could be detected in islets of poorly controlled diabetics, while well-controlled animals had 30% of the normal islet insulin content.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neurotensin is a regulator of insulin secretion in pancreatic beta-cells

Neurotensin (NT) is secreted from neurons and gastrointestinal endocrine cells. We previously reported that the three NT receptors (NTSRs) are expressed in pancreatic islets and beta cell lines on which we observed a protective effect of NT against cytotoxic agents. In this study, we explored the role of NT on insulin secretion in the endocrine pancreatic beta cells. We observed that NT stimulates insulin secretion at low glucose level and has a small inhibiting effect on stimulated insulin secretion from isolated islets or INS-1E cells. We studied the mechanisms by which NT elicited calcium concentration changes using fura-2 loaded islets or INS-1E cells. NT increases calcium influx through the opening of cationic channels. Similar calcium influxes were observed after treatment with NTSR selective ligands. NT-evoked calcium regulation involves PKC and the translocation of PKCα and PKC? to the plasma membrane. Part of NT effects appears to be also mediated by PKA but not via the Erk pathway. Taken together, these data provide evidence for an important endocrine role of NT in the regulation of the secretory function of beta cells.     

Cyclins D2 and D1 are essential for postnatal pancreatic beta-cell growth

Regulation of adult beta-cell mass in pancreatic islets is essential to preserve sufficient insulin secretion in order to appropriately regulate glucose homeostasis. In many tissues mitogens influence development by stimulating D-type cyclins (D1, D2, or D3) and activating cyclin-dependent kinases (CDK4 or CDK6), which results in progression through the G(1) phase of the cell cycle. Here we show that cyclins D2 and D1 are essential for normal postnatal islet growth. In adult murine islets basal cyclin D2 mRNA expression was easily detected, while cyclin D1 was expressed at lower levels and cyclin D3 was nearly undetectable. Prenatal islet development occurred normally in cyclin D2(-/-) or cyclin D1(+/-) D2(-/-) mice. However, beta-cell proliferation, adult mass, and glucose tolerance were decreased in adult cyclin D2(-/-) mice, causing glucose intolerance that progressed to diabetes by 12 months of age. Although cyclin D1(+/-) mice never developed diabetes, life-threatening diabetes developed in 3-month-old cyclin D1(-/+) D2(-/-) mice as beta-cell mass decreased after birth. Thus, cyclins D2 and D1 were essential for beta-cell expansion in adult mice. Strategies to tightly regulate D-type cyclin activity in beta cells could prevent or cure diabetes.     

Transgenic mice expressing Shb adaptor protein under the control of rat insulin promoter exhibit altered viability of pancreatic islet cells.

BACKGROUND: The Src-homology 2 domain-containing adaptor protein Shb was recently cloned as a serum-inducible gene in the insulin-producing beta-TC1 cell line. Subsequent studies have revealed an involvement of Shb for apoptosis in NIH3T3 fibroblasts and differentiation in the neuronal PC12 cells. To assess a role of Shb for beta-cell function, transgenic mice utilizing the rat insulin promoter to drive expression of Shb were generated. MATERIALS AND METHODS: A gene construct allowing the Shb cDNA to be expressed from the rat insulin 2 promoter was microinjected into fertilized mouse oocytes and implanted into pseudopregnant mice. Mice containing a low copy number of this transgene were bred and used for further experimentation. Shb expression was determined by Western blot analysis. The insulin-positive area of whole pancreas, insulin secretion of isolated islets and islet cell apoptosis, glucose tolerance tests, and in vivo sensitivity to multiple injections of the beta-cell toxin streptozotocin were determined in control CBA and Shb-transgenic mice. RESULTS: Western blot analysis revealed elevated islet content of the Shb protein. Shb-transgenic mice displayed enhanced glucose-disappearance rates in response to an intravenous glucose injection. The relative pancreatic beta-cell area neonatally and at 6 months of age were increased in the Shb-transgenic mice. Islets isolated from Shb-transgenic mice showed enhanced insulin secretion in response to glucose and increased insulin and DNA content. Apoptosis was increased in islets isolated from Shb-transgenic mice compared with control islets both under basal conditions and after incubation with IL-1 beta + IFN-gamma. Rat insulinoma RINm5F cells overexpressing Shb displayed decreased viability during culture in 0.1% serum and after exposure to a cytotoxic dose of nicotinamide. Shb-transgenic mice injected with multiple doses of streptozotocin showed increased blood glucose values compared with the corresponding controls, suggesting increased in vivo susceptibility to this toxin. CONCLUSION: The results suggest that Shb has dual effects on beta-cell growth: whereas Shb increases beta-cell formation during late embryonal stages, Shb also enhances beta-cell death under certain stressful conditions and may thus contribute to beta-cell destruction in type 1 diabetes.     

Insulin secretory responses and phospholipid composition of pancreatic islets from mice that do not express Group VIA phospholipase A2 and effects of metabolic stress on glucose homeostasis

Studies involving pharmacologic or molecular biologic manipulation of Group VIA phospholipase A(2) (iPLA(2)beta) activity in pancreatic islets and insulinoma cells suggest that iPLA(2)beta participates in insulin secretion. It has also been suggested that iPLA(2)beta is a housekeeping enzyme that regulates cell 2-lysophosphatidylcholine (LPC) levels and arachidonate incorporation into phosphatidylcholine (PC). We have generated iPLA(2)beta-null mice by homologous recombination and have reported that they exhibit reduced male fertility and defective motility of spermatozoa. Here we report that pancreatic islets from iPLA(2)beta-null mice have impaired insulin secretory responses to D-glucose and forskolin. Electrospray ionization mass spectrometric analyses indicate that the abundance of arachidonate-containing PC species of islets, brain, and other tissues from iPLA(2)beta-null mice is virtually identical to that of wild-type mice, and no iPLA(2)beta mRNA was observed in any tissue from iPLA(2)beta-null mice at any age. Despite the insulin secretory abnormalities of isolated islets, fasting and fed blood glucose concentrations of iPLA(2)beta-null and wild-type mice are essentially identical under normal circumstances, but iPLA(2)beta-null mice develop more severe hyperglycemia than wild-type mice after administration of multiple low doses of the beta-cell toxin streptozotocin, suggesting an impaired islet secretory reserve. A high fat diet also induces more severe glucose intolerance in iPLA(2)beta-null mice than in wild-type mice, but PLA(2)beta-null mice have greater responsiveness to exogenous insulin than do wild-type mice fed a high fat diet. These and previous findings thus indicate that iPLA(2)beta-null mice exhibit phenotypic abnormalities in pancreatic islets in addition to testes and macrophages.     

Targeted elimination of peroxisome proliferator-activated receptor gamma in beta cells leads to abnormalities in islet mass without compromising glucose homeostasis

The nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPAR gamma) is an important regulator of lipid and glucose homeostasis and cellular differentiation. Studies of many cell types in vitro and in vivo have demonstrated that activation of PPAR gamma can reduce cellular proliferation. We show here that activation of PPAR gamma is sufficient to reduce the proliferation of cultured insulinoma cell lines. We created a model with mice in which the expression of the PPARG gene in beta cells was eliminated (beta gamma KO mice), and these mice were found to have significant islet hyperplasia on a chow diet. Interestingly, the normal expansion of beta-cell mass that occurs in control mice in response to high-fat feeding is markedly blunted in these animals. Despite this alteration in beta-cell mass, no effect on glucose homeostasis in beta gamma KO mice was noted. Additionally, while thiazolidinediones enhanced insulin secretion from cultured wild-type islets, administration of rosiglitazone to insulin-resistant control and beta gamma KO mice revealed that PPAR gamma in beta cells is not required for the antidiabetic actions of these compounds. These data demonstrate a critical physiological role for PPAR gamma function in beta-cell proliferation and also indicate that the mechanisms controlling beta-cell hyperplasia in obesity are different from those that regulate baseline cell mass in the islet.     

Glucose-dependent expansion of pancreatic beta-cells by the protein p8 in vitro and in vivo

p8 protein expression is known to be upregulated in the exocrine pancreas during acute pancreatitis. Own previous work revealed glucose-dependent p8 expression also in endocrine pancreatic beta-cells. Here we demonstrate that glucose-induced INS-1 beta-cell expansion is preceded by p8 protein expression. Moreover, isopropylthiogalactoside (IPTG)-induced p8 overexpression in INS-1 beta-cells (p8-INS-1) enhances cell proliferation and expansion in the presence of glucose only. Although beta-cell-related gene expression (PDX-1, proinsulin I, GLUT2, glucokinase, amylin) and function (insulin content and secretion) are slightly reduced during p8 overexpression, removal of IPTG reverses beta-cell function within 24 h to normal levels. In addition, insulin secretion of p8-INS-1 beta-cells in response to 0-25 mM glucose is not altered by preceding p8-induced beta-cell expansion. Adenovirally transduced p8 overexpression in primary human pancreatic islets increases proliferation, expansion, and cumulative insulin secretion in vitro. Transplantation of mock-transduced control islets under the kidney capsule of immunosuppressed streptozotocin-diabetic mice reduces blood glucose and increases human C-peptide serum concentrations to stable levels after 3 days. In contrast, transplantation of equal numbers of p8-transduced islets results in a continuous decrease of blood glucose and increase of human C-peptide beyond 3 days, indicating p8-induced expansion of transplanted human beta-cells in vivo. This is underlined by a doubling of insulin content in kidneys containing p8-transduced islet grafts explanted on day 9. These results establish p8 as a novel molecular mediator of glucose-induced pancreatic beta-cell expansion in vitro and in vivo and support the notion of existing beta-cell replication in the adult organism.     

Normal islet vascularization is dispensable for expansion of beta-cell mass in response to high-fat diet induced insulin resistance

The inability to increase of islet mass adequately to compensate for the demand of insulin due to insulin resistance is an important pathophysiological feature of type 2 diabetes. Previous studies suggested a relationship between pancreatic beta-cell mass and islet vascularization, although no evidence has confirmed this association in response to insulin resistance. Vascular endothelial growth factor-A (VEGF-A) in islets is essential for maintaining normal islet blood vessels. Here, insulin resistance was induced in mice carrying a beta-cell-specific VEGF-A gene mutation (RIP-Cre:Vegf^fl/fl) by 20-week feeding of high-fat diet as a model of impaired islet vascularization. These mice showed only a modest decrease in glucose tolerance, compared with control mice. In addition, although the endothelial cell area in the islets of high-fat-fed RIP-Cre:Vegf^fl/fl mice remained diminished, the pancreatic beta-cell area was modestly more than in high-fat-fed control mice. Thus, normal islet vascularization does not seem to be essential for expansion of beta cell mass in response to insulin resistance.     

Modulation of endocrine pancreas development but not beta-cell carcinogenesis by Sprouty4

Sprouty (Spry) proteins modulate signal transduction pathways elicited by receptor tyrosine kinases (RTK). Depending on cell type and the particular RTK, Spry proteins exert dual functions: They can either repress RTK-mediated signaling pathways, mainly by interfering with the Ras/Raf/mitogen-activated protein kinase pathway or sustaining RTK signal transduction, for example by sequestering the E3 ubiquitin-ligase c-Cbl and thus preventing ubiquitylation, internalization, and degradation of RTKs. Here, by the inducible expression of murine Spry4 in pancreatic beta cells, we have assessed the functional role of Spry proteins in the development of pancreatic islets of Langerhans in normal mice and in the Rip1Tag2 transgenic mouse model of beta-cell carcinogenesis. beta cell-specific expression of mSpry4 provokes a significant reduction in islet size, an increased number of alpha cells per islet area, and impaired islet cell type segregation. Functional analysis of islet cell differentiation in cultured PANC-1 cells shows that mSpry4 represses adhesion and migration of differentiating pancreatic endocrine cells, most likely by affecting the subcellular localization of the protein tyrosine phosphatase PTP1B. In contrast, transgenic expression of mSpry4 during beta-cell carcinogenesis does not significantly affect tumor outgrowth and progression to tumor malignancy. Rather, tumor cells seem to escape mSpry4 transgene expression.     

Selective expansion of the beta-cell compartment in the pancreas of keratinocyte growth factor transgenic mice

Epithelial-mesenchymal interactions are essential for growth, differentiation, and regeneration of exocrine and endocrine cells in the pancreas. The keratinocyte growth factor (KGF) is derived from mesenchyme and has been shown to promote epithelial cell differentiation and proliferation in a paracrine fashion. Here, we have examined the effect of ectopic expression of KGF on pancreatic differentiation and proliferation in transgenic mice by using the proximal elastase promoter. KGF transgenic mice were generated following standard procedures and analyzed by histology, morphometry, immunohistochemistry, Western blot analysis, and glucose tolerance testing. In KGF transgenic mice, the number of islets, the average size of islets, and the relation of endocrine to exocrine tissue are increased compared with littermate controls. An expansion of the beta-cell population is responsible for the increase in the endocrine compartment. Ectopic expression of KGF results in proliferation of beta-cells and pancreatic duct cells most likely through activation of the protein kinase B (PKB)/Akt signaling pathway. Glucose tolerance and insulin secretion are impaired in transgenic animals. These results provide evidence that ectopic expression of KGF in acinar cells promotes the expansion of the beta-cell lineage in vivo through activation of the PKB/Akt pathway. Furthermore, the observed phenotype demonstrates that an increase in the beta-cell compartment does not necessarily result in an improved glucose tolerance in vivo.