FoxO feedback control of basal IRS-2 expression in pancreatic ß-cells is distinct from that in hepatocytes.

OBJECTIVE: Appropriate regulation of insulin receptor substrate 2 (IRS-2) expression in pancreatic ß-cells is essential to adequately compensate for insulin resistance. In liver, basal IRS-2 expression is controlled via a temporal negative feedback of sterol regulatory element-binding protein 1 (SREBP-1) to antagonize transcription factors forkhead box class O (FoxO)1/FoxO3a at an insulin response element (IRE) on the IRS-2 promoter. The purpose of the study was to examine if a similar mechanism controlled IRS-2 expression in ß-cells. RESEARCH DESIGN AND METHODS: IRS-2 mRNA and protein expression, as well as IRS-2 gene promoter activity, were examined in isolated rat islets. Specific transcription factor association with the IRE on the IRS-2 promoter was examined by chromatin immunoprecipitation (ChIP) assay, and their nuclear translocation was examined by immunofluorescence. A direct in vivo effect of insulin on control of IRS-2 expression in liver and pancreatic islets was also investigated. RESULTS: In IRS-2 promoter-reporter assays conducted in isolated islets, removal of the IRE decreased basal IRS-2 promoter activity in ß-cells up to 80%. Activation of IRS signaling in isolated rat islets by insulin/IGF-I (used as an experimental in vitro tool) or downstream constitutive activation of protein kinase B (PKB) significantly decreased IRS-2 expression. In contrast, inhibition of phosphatidylinositol 3-kinase (PI3K) or PKB significantly increased IRS-2 levels in ß-cells. ChIP assays indicated that transcription factors FoxO1 and FoxO3a associated with the IRE on the IRS-2 promoter in ß-cells in a PI3K/PKB-dependent manner, whereas others, such as SREBP-1, the transcription factor binding to immunoglobulin heavy chain enhancer 3', and the aryl hydrocarbon receptor nuclear translocator (ARNT), did not. However, only FoxO3a, not FoxO1, was capable of driving IRS-2 promoter activity via the IRE in ß-cells. In vivo studies showed insulin was able to suppress IRS-2 expression via activation of SREBP-1 in the liver, but this mechanism was not apparent in pancreatic islets from the same animal. CONCLUSIONS: The molecular mechanism for feedback control of IRS signaling to decrease IRS-2 expression in liver and ß-cells is quite distinct, with a predominant role played by FoxO3a in ß-cells.

Specific glucose-induced control of insulin receptor substrate-2 expression is mediated via Ca2+-dependent calcineurin/NFAT signaling in primary pancreatic islet ß-cells.

OBJECTIVE: Insulin receptor substrate-2 (IRS-2) plays an essential role in pancreatic islet ß-cells by promoting growth and survival. IRS-2 turnover is rapid in primary ß-cells, but its expression is highly regulated at the transcriptional level, especially by glucose. The aim was to investigate the molecular mechanism on how glucose regulates IRS-2 gene expression in ß-cells. RESEARCH DESIGN AND METHODS: Rat islets were exposed to inhibitors or subjected to adenoviral vector-mediated gene manipulations and then to glucose-induced IRS-2 expression analyzed by real-time PCR and immunoblotting. Transcription factor nuclear factor of activated T cells (NFAT) interaction with IRS-2 promoter was analyzed by chromatin immunoprecipitation assay and glucose-induced NFAT translocation by immunohistochemistry. RESULTS: Glucose-induced IRS-2 expression occurred in pancreatic islet ß-cells in vivo but not in liver. Modulating rat islet ß-cell Ca(2+) influx with nifedipine or depolarization demonstrated that glucose-induced IRS-2 gene expression was dependent on a rise in intracellular calcium concentration derived from extracellular sources. Calcineurin inhibitors (FK506, cyclosporin A, and a peptide calcineurin inhibitor [CAIN]) abolished glucose-induced IRS-2 mRNA and protein levels, whereas expression of a constitutively active calcineurin increased them. Specific inhibition of NFAT with the peptide inhibitor VIVIT prevented a glucose-induced IRS-2 transcription. NFATc1 translocation to the nucleus in response to glucose and association of NFATc1 to conserved NFAT binding sites in the IRS-2 promoter were demonstrated. CONCLUSIONS: The mechanism behind glucose-induced transcriptional control of IRS-2 gene expression specific to the islet ß-cell is mediated by the Ca(2+)/calcineurin/NFAT pathway. This insight into the IRS-2 regulation could provide novel therapeutic means in type 2 diabetes to maintain an adequate functional mass.

Specific regulation of IRS-2 expression by glucose in rat primary pancreatic islet beta-cells.

Insulin receptor substrate 2 (IRS-2) plays a critical role in pancreatic beta-cells. Increased IRS-2 expression promotes beta-cell growth and survival, whereas decreased IRS-2 levels lead to apoptosis. It was found that IRS-2 turnover in rat islet beta-cells was rapid, with mRNA and protein half-lives of approximately 90 min and approximately 2 h, respectively. However, this was countered by specific glucose-regulated IRS-2 expression mediated at the transcriptional level. Glucose (> or = 6 mM) increased IRS-2 mRNA and protein levels in a dose-dependent manner, reaching a maximum 4-fold increase in IRS-2 mRNA and a 5-6-fold increase in IRS-2 protein levels at > or = 12 mM glucose (p < or = 0.01). Glucose (15 mM) regulation of islet beta-cell IRS-2 gene expression was rapid, with a significant increase in IRS-2 mRNA levels within 2 h that reached a maximum 4-fold increase by 4 h. IRS-2 protein expression in beta-cells followed that of IRS-2 mRNA. Glucose metabolism was necessary for increased IRS-2 expression in beta-cells. Moreover, inhibition of a glucose-induced rise in islet beta-cell cytosolic [Ca2+]i prevented an increase in IRS-2 expression, indicating this was Ca2+-dependent. The glucose-induced rise in IRS-2 levels correlated with increased IRS-2 tyrosine phosphorylation and downstream activation of protein kinase B. These data indicate that fluctuations of glucose in the normal physiological range (5-15 mM) promote beta-cell survival via regulation of IRS-2 expression and a subsequent parallel protein kinase B activation. Given that the onset of type-2 diabetes is marked by loss of beta-cells, these data further the idea that controlled IRS-2 expression in beta-cells could be a therapeutic means to promote beta-cell survival and delay the onset of the disease.

Insulin receptor substrate-2 proteasomal degradation mediated by a mammalian target of rapamycin (mTOR)-induced negative feedback down-regulates protein kinase B-mediated signaling pathway in beta-cells.

Regulation of insulin receptor substrate (IRS)-2 expression is critical to beta-cell survival, but the mechanisms that control this are complex and undefined. Here in pancreatic beta-cells (INS-1), chronic exposure (>8 h) to 15 mm glucose and/or 5 nm IGF-1, increased Ser/Thr phosphorylation of IRS-2, which correlated with decreased IRS-2 levels. This glucose/IGF-1-induced decrease in IRS-2 levels was prevented by the proteasomal inhibitor, lactacystin. In addition, the glucose/IGF-1-induced increase in Ser/Thr phosphorylation of IRS-2 and the subsequent decrease in INS-1 cell IRS-2 protein levels was thwarted by the mammalian target of rapamycin(mTOR) inhibitor, rapamycin. Moreover, adenoviral-mediated expression of constitutively active mTOR (mTORDelta) further increased glucose/IGF-1-induced Ser/Thr phosphorylation of IRS-2 and decreased IRS-2 protein levels, whereas adenoviral-mediated expression of "kinase-dead" mTOR (mTOR-KD) conversely reduced Ser/Thr phosphorylation of IRS-2 and maintained IRS-2 protein levels. In adenoviral-infected beta-cells expressing mTORDelta, the decrease in IRS-2 protein levels was also prevented by rapamycin or lactacystin, further indicating a proteasomal mediated degradation of IRS-2 mediated via mTOR-induced Ser/Thr phosphorylation of IRS-2. Finally, we found that chronic activation of mTOR leading to decreased levels of IRS-2 in INS-1 cells led to a significant decrease in PKB activation and consequently increased beta-cell apoptosis. Thus, chronic activation of mTOR by glucose (and/or IGF-1) in beta-cells leads to increased Ser/Thr phosphorylation of IRS-2 that targets it for proteasomal degradation, resulting in decreased IRS-2 expression and increased beta-cell apoptosis. This may be a contributing mechanism as to how beta-cell mass is decreased by chronic hyperglycemia in the pathogenesis of type-2 diabetes.

Decreasing IRS-2 expression in pancreatic beta-cells (INS-1) promotes apoptosis, which can be compensated for by introduction of IRS-4 expression.

IRS-2 plays a pivotal role in the control of pancreatic beta-cell growth. Here, the effect of altering IRS-2 expression levels in the pancreatic beta-cell line, INS-1, was examined. Adenoviral-mediated increased in IRS-2 protein levels protected against fatty acid (FFA)-induced apoptosis, associated with increased activation of PKB and decreased levels of activated caspase-9. Conversely, decreasing endogenous IRS-2 in INS-1 cells, using adenoviral-mediated expression of IRS-2 antisense, caused a three-fold increase in baseline apoptosis that was further enhanced in the presence of FFA. This was associated with decreased activation of PKB and increased caspase-9 activation. Although IRS-4 is not normally expressed in beta-cells, it was found that adenoviral-mediated introduction of IRS-4 into INS-1 cells enhanced glucose/IGF-1 induced mitogenesis, and protected against FFA-induced apoptosis, similarly to IRS-2. Moreover, expression of IRS-4 in INS-1 cells depleted of IRS-2 levels by IRS-2 antisense, was able to compensate for the lack of IRS-2 and reduce apoptosis in these cells back to normal. Thus, in beta-cells IRS-4 and -2 have similar biological functions. Also, this study further emphasizes the importance of IRS-2 signaling in control of beta-cell survival.

Glucose-induced translational control of proinsulin biosynthesis is proportional to preproinsulin mRNA levels in islet beta-cells but not regulated via a positive feedback of secreted insulin.

Proinsulin biosynthesis is regulated in response to nutrients, most notably glucose. In the short term (/=10-fold). Importantly, neither exogenously added nor secreted insulin were found to play any role in regulating insulin secretion, proinsulin translation, preproinsulin mRNA levels, or total protein synthesis. The results presented here indicate that long term nutritional state sets the preproinsulin mRNA level in the beta-cell at which translation control regulates short term changes in rates of proinsulin biosynthesis in response to glucose, but this is not mediated by any autocrine effect of insulin.

Differential activation mechanisms of Erk-1/2 and p70(S6K) by glucose in pancreatic beta-cells.

Glucose can activate the mitogen-activated kinases, Erk-1/2, and the ribosomal-S6 kinase, p70(S6K), in beta-cells, contributing to an increase in mitogenesis. However, the signaling mechanism by which glucose induces Erk-1/2 and p70(S6K) phosphorylation activation is undefined. Increased glucose metabolism increases [Ca(2+)](i) and [cAMP], and it was investigated if these secondary signals were linked to glucose-induced Erk-1/2 and p70(S6K) activation in pancreatic beta-cells. Blocking Ca(2+) influx with verapamil, or inhibiting protein kinase A (PKA) with H89, prevented glucose-induced Erk-1/2 phosphorylation. Increasing cAMP levels by GLP-1 potentiated glucose-induced Erk-1/2 phosphorylation via PKA activation. Elevation of [Ca(2+)](i) by glyburide potentiated Erk-1/2 phosphorylation, which was also inhibited by H89, suggesting increased [Ca(2+)](i) preceded PKA for glucose-induced Erk-1/2 activation. Adenoviral-mediated expression of dominant negative Ras in INS-1 cells decreased IGF-1-induced Erk-1/2 phosphorylation but had no effect on that by glucose. Collectively, our study indicates that a glucose-induced rise in [Ca(2+)](i) leads to cAMP-induced activation of PKA that acts downstream of Ras and upstream of the MAP/Erk kinase, MEK, to mediate Erk-1/2 phosphorylation via phosphorylation activation of Raf-1. In contrast, glucose-induced p70(S6K) activation, in the same beta-cells, was mediated by a distinct signaling pathway independent of Ca(2+)/cAMP, most likely via mTOR-kinase acting as an "ATP-sensor."

Protein kinase B/Akt prevents fatty acid-induced apoptosis in pancreatic beta-cells (INS-1).

Free fatty acids (FFA) have been reported to reduce pancreatic beta-cell mitogenesis and to increase apoptosis. Here we show that the FFA, oleic acid, increased apoptosis 16-fold in the pancreatic beta-cell line, INS-1, over a 18-h period as assessed by Hoechst 33342/propidium iodide staining and caspase-3 and -9 activation, with negligible necrosis. A parallel analysis of the phosphorylation activation of protein kinase B (PKB) showed this was reduced in the presence of FFA that correlated with the incidence of apoptosis. At stimulatory 15 mm glucose and/or in the added presence of insulin-like growth factor 1, FFA-induced beta-cell apoptosis was lessened compared with that at a basal 5 mm glucose. However, most strikingly, adenoviral mediated expression of a constitutively active PKB, but not a "kinase-dead" PKB variant, essentially prevented FFA-induced beta-cell apoptosis under all glucose/insulin-like growth factor 1 conditions. Further analysis of pro-apoptotic downstream targets of PKB, implicated a role for PKB-mediated phosphorylation inhibition of glycogen synthase kinase-3alpha/beta and the forkhead transcription factor, FoxO1, in protection of FFA-induced beta-cell apoptosis. In addition, down-regulation of the pro-apoptotic tumor suppressor protein, p53, via PKB-mediated phosphorylation of MDM2 might also play a role in partially protecting beta-cells from FFA-induced apoptosis. Adenoviral mediated expression of wild type p53 potentiated FFA-induced beta-cell apoptosis, whereas expression of a dominant negative p53 partly inhibited beta-cell apoptosis by approximately 50%. Hence, these data demonstrate that PKB activation plays an important role in promoting pancreatic beta-cell survival in part via inhibition of the pro-apoptotic proteins glycogen synthase kinase-3alpha/beta, FoxO1, and p53. This, in turn, provides novel insight into the mechanisms involved in FFA-induced beta-cell apoptosis.

Ca2+-dependent dephosphorylation of kinesin heavy chain on beta-granules in pancreatic beta-cells. Implications for regulated beta-granule transport and insulin exocytosis.

The specific biochemical steps required for glucose-regulated insulin exocytosis from beta-cells are not well defined. Elevation of glucose leads to increases in cytosolic [Ca2+]i and biphasic release of insulin from both a readily releasable and a storage pool of beta-granules. The effect of elevated [Ca2+]i on phosphorylation of isolated beta-granule membrane proteins was evaluated, and the phosphorylation of four proteins was found to be altered by [Ca2+]i. One (a 18/20-kDa doublet) was a Ca2+-dependent increase in phosphorylation, and, surprisingly, three others (138, 42, and 36 kDa) were Ca2+-dependent dephosphorylations. The 138-kDa beta-granule phosphoprotein was found to be kinesin heavy chain (KHC). At low levels of [Ca2+]i KHC was phosphorylated by casein kinase 2, but KHC was rapidly dephosphorylated by protein phosphatase 2B beta (PP2Bbeta) as [Ca2+]i increased. Inhibitors of PP2B specifically reduced the second, microtubule-dependent, phase of insulin secretion, suggesting that dephosphorylation of KHC was required for transport of beta-granules from the storage pool to replenish the readily releasable pool of beta-granules. This is distinct from synaptic vesicle exocytosis, because neurotransmitter release from synaptosomes did not require a Ca2+-dependent KHC dephosphorylation. These results suggest a novel mechanism for regulating KHC function and beta-granule transport in beta-cells that is mediated by casein kinase 2 and PP2B. They also implicate a novel regulatory role for PP2B/calcineurin in the control of insulin secretion downstream of a rise in [Ca2+]i.

Differential effects of hyperlipidemia on insulin secretion in islets of langerhans from hyperglycemic versus normoglycemic rats.

Chronic elevations in plasma levels of fatty acids (FAs) adversely affect pancreatic beta-cell function in type 2 diabetes. In vitro, we have previously shown that deleterious effects of prolonged exposure of isolated islets to FAs were dependent on the presence of elevated glucose concentration. This led us to hypothesize that both hyperlipidemia and hyperglycemia must be present simultaneously for FAs to affect beta-cell function. To test this hypothesis in vivo, we administered a high-fat diet for 6 weeks to Goto-Kakizaki (GK) rats. High-fat feeding had no effect on insulin secretion, insulin content, or insulin mRNA levels in islets from normoglycemic Wistar rats. In contrast, high-fat feeding markedly impaired glucose-induced insulin secretion in islets from GK rats. High-fat feeding did not affect triglyceride (TG) content or the rate of glucose oxidation in islets. It was, however, accompanied by a twofold increase in uncoupling protein (UCP)-2 levels in GK rat islets. Insulin treatment completely normalized glucose-induced insulin secretion and prevented the increase in UCP-2 expression in islets from high-fat-fed GK rats. We conclude that hyperlipidemia induced by high-fat feeding affects insulin secretion in islets from hyperglycemic GK rats only, by a mechanism which may involve, at least in part, modulation of UCP-2 expression.