Prolonged exposure of isolated islets to supraphysiologic concentrations of palmitate decreases insulin gene expression in the presence of elevated glucose levels. This study was designed to ...determine whether or not this phenomenon is associated with a glucose-dependent increase in esterification of fatty acids into neutral lipids. Gene expression of sn-glycerol-3-phosphate acyltransferase (GPAT), diacylglycerol acyltransferase (DGAT), and hormone-sensitive lipase (HSL), three key enzymes of lipid metabolism, was detected in isolated rat islets. Their levels of expression were not affected after a 72-h exposure to elevated glucose and palmitate. To determine the effects of glucose on palmitate-induced neutral lipid synthesis, isolated rat islets were cultured for 72 h with trace amounts of 14Cpalmitate with or without 0.5 mmol/l unlabeled palmitate, at 2.8 or 16.7 mmol/l glucose. Glucose increased incorporation of 14Cpalmitate into complex lipids. Addition of exogenous palmitate directed lipid metabolism toward neutral lipid synthesis. As a result, neutral lipid mass was increased upon prolonged incubation with elevated palmitate only in the presence of high glucose. The ability of palmitate to increase neutral lipid synthesis in the presence of high glucose was concentration-dependent in HIT cells and was inversely correlated to insulin mRNA levels. 2-Bromopalmitate, an inhibitor of fatty acid mitochondrial beta-oxidation, reproduced the inhibitory effect of palmitate on insulin mRNA levels. In contrast, palmitate methyl ester, which is not metabolized, and the medium-chain fatty acid octanoate, which is readily oxidized, did not affect insulin gene expression, suggesting that fatty-acid inhibition of insulin gene expression requires activation of the esterification pathway. These results demonstrate that inhibition of insulin gene expression upon prolonged exposure of islets to palmitate is associated with a glucose-dependent increase in esterification of fatty acids into neutral lipids.
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.
The recently discovered adipose cell-specific hormone called leptin
decreases food intake and increases energy expenditure in rodents
through a pathway involving hypothalamic leptin receptors, OB-R. ...In
addition, leptin decreases insulin circulating levels independent of
the reduction in food intake. Whether or not the hormone has a direct
effect on pancreatic β-cells is not clear, because previous in
vitro studies have led to controversial results depending on
the animal model used. The present study was designed to investigate
the effects of leptin in islets of Langerhans isolated from normal
rodents. Three isoforms of the leptin receptor, OB-Ra, b, and f, were
detected by RT-PCR analysis of total RNA from rat islets. In static
incubations, leptin (10 ng/ml) did not alter basal insulin secretion
nor insulin secretion stimulated by glucose alone, potassium chloride,
or ketoisocaproic acid. In contrast, insulin secretion stimulated by
glucose + 3-isobutyl 1-methylxanthine (IBMX) was inhibited by 34±
15% (n = 4, P < 0.05). This was further
substantiated in perifusion experiments, in which leptin decreased by
31 ± 3% (n = 5, P < 0.01) glucose +
IBMX-stimulated insulin release. Similarly, in mouse islets a
significant inhibitory effect of leptin (-31 ± 4%, n = 6,
P < 0.05) was observed only on glucose +
IBMX-stimulated insulin secretion, with no effect of the hormone on
basal nor glucose-stimulated secretion. Finally, leptin was totally
inefficient in islets isolated from obese fa/fa rats,
which bear a mutation in OB-R. These results suggest that, in normal
rodent islets, leptin specifically inhibits IBMX-potentiated
glucose-induced insulin secretion, through a direct effect involving at
least one of the three isoforms of OB-R expressed in islets.
Insulin receptor substrate-2 (IRS-2) plays an essential role in pancreatic islet β-cells by promoting growth and survival. IRS-2 turnover is rapid in primary β-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 β-cells.
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.
Glucose-induced IRS-2 expression occurred in pancreatic islet β-cells in vivo but not in liver. Modulating rat islet β-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.
The mechanism behind glucose-induced transcriptional control of IRS-2 gene expression specific to the islet β-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.
Appropriate regulation of insulin receptor substrate 2 (IRS-2) expression in pancreatic β-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 β-cells.
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.
In IRS-2 promoter-reporter assays conducted in isolated islets, removal of the IRE decreased basal IRS-2 promoter activity in β-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 β-cells. ChIP assays indicated that transcription factors FoxO1 and FoxO3a associated with the IRE on the IRS-2 promoter in β-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 β-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.
The molecular mechanism for feedback control of IRS signaling to decrease IRS-2 expression in liver and β-cells is quite distinct, with a predominant role played by FoxO3a in β-cells.
Free fatty acids (FFA) have been reported to reduce pancreatic β-cell mitogenesis and to increase apoptosis. Here we show that the FFA, oleic acid, increased apoptosis 16-fold in the pancreatic ...β-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 β-cell apoptosis was lessened compared with that at a basal 5 mmglucose. However, most strikingly, adenoviral mediated expression of a constitutively active PKB, but not a “kinase-dead” PKB variant, essentially prevented FFA-induced β-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-3α/β and the forkhead transcription factor, FoxO1, in protection of FFA-induced β-cell apoptosis. In addition, down-regulation of the pro-apoptotic tumor suppresser protein, p53, via PKB-mediated phosphorylation of MDM2 might also play a role in partially protecting β-cells from FFA-induced apoptosis. Adenoviral mediated expression of wild type p53 potentiated FFA-induced β-cell apoptosis, whereas expression of a dominant negative p53 partly inhibited β-cell apoptosis by ∼50%. Hence, these data demonstrate that PKB activation plays an important role in promoting pancreatic β-cell survival in part via inhibition of the pro-apoptotic proteins glycogen synthase kinase-3α/β, FoxO1, and p53. This, in turn, provides novel insight into the mechanisms involved in FFA-induced β-cell apoptosis.
Regulation of insulin receptor substrate (IRS)-2 expression is critical to β-cell survival, but the mechanisms that control this are complex and undefined. Here in pancreatic β-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 (mTORΔ) 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 β-cells expressing mTORΔ, 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 β-cell apoptosis. Thus, chronic activation of mTOR by glucose (and/or IGF-1) in β-cells leads to increased Ser/Thr phosphorylation of IRS-2 that targets it for proteasomal degradation, resulting in decreased IRS-2 expression and increased β-cell apoptosis. This may be a contributing mechanism as to how β-cell mass is decreased by chronic hyperglycemia in the pathogenesis of type-2 diabetes.
Proinsulin biosynthesis is regulated in response to nutrients, most notably glucose. In the short term (≤2h) this is due to increases in the translation of pre-existing mRNA. However, prolonging ...glucose stimulation (24 h) also increases preproinsulin mRNA levels. It has been proposed that secreted insulin from the pancreatic β-cell regulates its own synthesis through a positive autocrine feedback mechanism. Here the comparative contributions of translation and mRNA levels on the levels of proinsulin biosynthesis were examined in isolated pancreatic islets. Also, the autocrine role of insulin upon four β-cell functions (insulin secretion, proinsulin translation, preproinsulin mRNA levels, and total protein synthesis) was investigated in parallel. The results showed that proinsulin biosynthesis is regulated, in the short term (1 h), solely at the level of translation, through an ∼6-fold increase in response to glucose (2.8 mmversus 16.7 mm glucose). In the longer term, when preproinsulin mRNA levels have increased ∼2-fold, a corresponding increase was observed in the fold response of proinsulin translation to a stimulatory glucose concentration (≥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 β-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.
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."
Insulin receptor substrate 2 (IRS-2) plays a critical role in pancreatic β-cells. Increased IRS-2 expression promotes β-cell growth and survival, whereas decreased IRS-2 levels lead to apoptosis. It ...was found that IRS-2 turnover in rat islet β-cells was rapid, with mRNA and protein half-lives of ∼90 min and ∼2 h, respectively. However, this was countered by specific glucose-regulated IRS-2 expression mediated at the transcriptional level. Glucose (≥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 ≥12 mm glucose (p ≤ 0.01). Glucose (15 mm) regulation of islet β-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 β-cells followed that of IRS-2 mRNA. Glucose metabolism was necessary for increased IRS-2 expression in β-cells. Moreover, inhibition of a glucose-induced rise in islet β-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 β-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 β-cells, these data further the idea that controlled IRS-2 expression in β-cells could be a therapeutic means to promote β-cell survival and delay the onset of the disease.
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