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V Poitout

Publications and source records attributed to V Poitout.

At least 19 recordsLinked to original sources
PubMed2002

Proinsulin processing in the diabetic Goto-Kakizaki rat.

The biosynthesis and processing of proinsulin was investigated in the diabetic Goto-Kakizaki (GK) rat. Immunofluorescence microscopy comparing GK and Wistar control rat pancreata revealed marked changes in the distribution of alpha-cells and pronounced beta-cell heterogeneity in the expression patterns of insulin, prohormone convertases PC1, PC2, carboxypeptidase E (CPE) and the PC-binding proteins 7B2 and ProSAAS. Western blot analyses of isolated islets revealed little difference in PC1 and CPE expression but PC2 immunoreactivity was markedly lower in the GK islets. The processing of the PC2-dependent substrate chromogranin A was reduced as evidenced by the appearance of intermediates. No differences were seen in the biosynthesis and post-translational modification of PC1, PC2 or CPE following incubation of islets in 16.7 mM glucose, but incubation in 3.3 mM glucose resulted in decreased PC2 biosynthesis in the GK islets. The rates of biosynthesis, processing and secretion of newly synthesized (pro)insulin were comparable. Circulating insulin immunoreactivity in both Wistar and GK rats was predominantly insulin 1 and 2 in the expected ratios with no (pro)insulin evident. Thus, the marked changes in islet morphology and PC2 expression did not impact the rate or extent of proinsulin processing either in vitro or in vivo in this experimental model.

Animals↗
PubMed2001

Antecedent hyperglycemia, not hyperlipidemia, is associated with increased islet triacylglycerol content and decreased insulin gene mRNA level in Zucker diabetic fatty rats.

Type 2 diabetes is caused by a combination of beta-cell dysfunction and insulin resistance. Over time, hyperglycemia worsens, a phenomenon that has been attributed to deleterious effects of chronic hyperglycemia (glucotoxicity) or chronic hyperlipidemia (lipotoxicity) on beta-cell function and is often accompanied by increased islet triacylglycerol (TAG) content and decreased insulin gene expression. To examine these two potentially pathogenic forces, we studied Zucker rats (leptin receptor wild type, +/+; heterozygous, +/-; and mutant, -/-). First, +/+ and +/- Zucker rats were compared metabolically. At 6 weeks of age, the +/- rats had a lower level of islet insulin mRNA compared with +/+. At 12 weeks of age, differences were found in body weight and islet TAG content; however, levels of insulin mRNA were equivalent. Second, we examined whether worsening of the diabetic state in the homozygous mutant (-/-) Zucker diabetic fatty (ZDF) rat is related more to chronic hyperglycemia or to hyperlipidemia. The ZDF rats were treated for 6 weeks with either bezafibrate, a lipid-lowering drug that does not affect plasma glucose levels, or phlorizin, a drug that reduces plasma glucose without lowering lipid levels. Bezafibrate treatment lessened the rise in plasma TAG observed in nontreated rats (239 +/- 16 vs. 388 +/- 36 mg/dl, treated versus nontreated; P < 0.0001) but did not prevent the rise in fasting plasma glucose. Despite lowering plasma TAG, bezafibrate was not effective in preventing an increased islet TAG content and did not prevent the associated decrease in insulin mRNA levels. Phlorizin treatment prevented hyperglycemia (61 +/- 2 vs. 145 +/- 7 mg/dl, treated versus nontreated; P < 0.0001) and lowered islet TAG content (32.7 +/- 0.7 vs. 47.8 +/- 2.7 ng/islet, treated versus nontreated; P < 0.0001) and preserved insulin mRNA levels without preventing hypertriglyceridemia. Plasma free fatty acid level did not correlate with changes in islet TAG or insulin mRNA levels. We conclude that antecedent elevated plasma glucose levels, not plasma lipid levels, are associated with elevated islet TAG content and decreased insulin mRNA levels in ZDF animals.

Alleles↗
PubMed2001

PPAR-alpha-null mice are protected from high-fat diet-induced insulin resistance.

Peroxisome proliferator-activated receptor (PPAR)-alpha controls the expression of genes involved in lipid metabolism. PPAR-alpha furthermore participates to maintain blood glucose during acute metabolic stress, as shown in PPAR-alpha-null mice, which develop severe hypoglycemia when fasted. Here, we assessed a potential role for PPAR-alpha in glucose homeostasis in response to long-term high-fat feeding. When subjected to this nutritional challenge, PPAR-alpha-null mice remained normoglycemic and normoinsulinemic, whereas wild-type mice became hyperinsulinemic (190%; P < 0.05) and slightly hyperglycemic (120%; NS). Insulin tolerance tests (ITTs) and glucose tolerance tests (GTTs) were performed to evaluate insulin resistance (IR). Under standard diet, the response to both tests was similar in wild-type and PPAR-alpha-null mice. Under high-fat diet, however, the efficiency of insulin in ITT was reduced and the amount of hyperglycemia in GTT was increased only in wild-type and not in PPAR-alpha-null mice. The IR index, calculated as the product of the areas under glucose and insulin curves in GTT, increased fourfold in high-fat-fed wild-type mice, whereas it remained unchanged in PPAR-alpha-null mice. In contrast, PPAR-alpha deficiency allowed the twofold rise in adiposity and blood leptin levels elicited by the diet. Thus, the absence of PPAR-alpha dissociates IR from high-fat diet-induced increase in adiposity. The effects of PPAR-alpha deficiency on glucose homeostasis seem not to occur via the pancreas, because glucose-stimulated insulin secretion of islets was not influenced by the PPAR-alpha genotype. These data suggest that PPAR-alpha plays a role for the development of IR in response to a Western-type high-fat diet.

1-Methyl-3-isobutylxanthine↗
PubMed2001

Lipotoxicity of the pancreatic beta-cell is associated with glucose-dependent esterification of fatty acids into neutral lipids.

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 [14C]palmitate with or without 0.5 mmol/l unlabeled palmitate, at 2.8 or 16.7 mmol/l glucose. Glucose increased incorporation of [14C]palmitate 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.

Acyltransferases↗
PubMed2000

Inhibition of insulin gene expression by long-term exposure of pancreatic beta cells to palmitate is dependent on the presence of a stimulatory glucose concentration.

Long-term exposure of pancreatic beta cells to elevated levels of fatty acids (FAs) impairs glucose-induced insulin secretion. However, the effects of FAs on insulin gene expression are controversial. We hypothesized that FAs adversely affect insulin gene expression only in the presence of elevated glucose concentrations. To test this hypothesis, isolated rat islets were cultured for up to 1 week in the presence of 2.8 or 16.7 mmol/L glucose with or without 0.5 mmol/L palmitate. Insulin release, insulin content, and insulin mRNA levels were determined at the end of each culture period. Palmitate increased insulin release at each time point independently of the glucose concentration. In contrast, insulin content was unchanged in the presence of palmitate at 2.8 mmol/L glucose, but was markedly decreased in the presence of 0.5 mmol/L palmitate and 16.7 mmol/L glucose after 2, 3, and 7 days of culture. In the presence of a basal concentration of glucose, insulin mRNA levels were transiently increased by palmitate at 24 hours but were unchanged thereafter. In contrast, palmitate significantly inhibited the stimulatory effects of 16.7 mmol/L glucose on insulin mRNA levels after 2, 3, and 7 days. To determine whether the inhibitory effect of palmitate on glucose-stimulated insulin mRNA levels was associated with decreased insulin promoter activity, HIT-T15 cells were cultured for 24 hours in 11.1 mmol/L glucose in the presence or absence of palmitate, and insulin gene promoter activity was measured in transient transfection experiments using the insulin promoter-reporter construct INSLUC. INSLUC activity was decreased more than 2-fold after 24 hours of exposure to 0.5 mmol/L palmitate. We conclude that long-term exposure of pancreatic beta cells to palmitate decreases insulin gene expression only in the presence of elevated glucose concentrations, in part through inhibition of insulin gene promoter activity.

Animals↗
PubMed2000

Glucose-induced insulin mRNA accumulation is impaired in islets from neonatal streptozotocin-treated rats.

According to the "glucose toxicity" hypothesis, hyperglycemia contributes to defective beta-cell function in type 2, non-insulin-dependent diabetes mellitus. This concept is supported by substantial data in rodent models of diabetes. However, the ability of glucose to stimulate the accumulation of insulin mRNA, a critical feature of normal beta-cell physiology, has not been investigated in in vivo models with chronic hyperglycemia. The aim of this study was to determine whether glucose-induced insulin mRNA accumulation is impaired in the neonatal streptozotocin-treated rat (n0-STZ rat), a model of non-obese, non-insulin-dependent diabetes mellitus. Islets of Langerhans isolated from n0-STZ and control rats were cultured for 24 h in the presence of 2.8 or 16.7 mmol/l glucose, and insulin mRNA levels were measured by Northern analysis. Insulin mRNA levels were increased more than twofold by glucose in control islets. In contrast, no significant effect of glucose was found on insulin mRNA levels in n0-STZ islets. We conclude that insulin gene regulation by glucose is impaired in n0-STZ rat islets.

Animals↗
PubMed2000

Glucose-induced insulin mRNA accumulation is impaired in islets from neonatal streptozotocin-treated rats.

According to the glucose toxicity hypothesis, hyperglycemia contributes to defective beta-cell function in type 2, non-insulin-dependent diabetes mellitus. This concept is supported by substantial data in rodent models of diabetes. However, the ability of glucose to stimulate the accumulation of insulin mRNA, a critical feature of normal beta-cell physiology, has not been investigated in in vivo models of chronic hyperglycemia. The aim of this study was to determine whether glucose-induced insulin mRNA accumulation is impaired in the neonatal streptozotocin-treated rat (n0-STZ rat), a model of non-obese, non-insulin-dependent diabetes mellitus. Islets of Langerhans isolated from n0-STZ and control rats were cultured for 24 h in the presence of 2.8 or 16.7 mmol/L glucose, and insulin mRNA levels were measured by Northern analysis. Insulin mRNA levels were increased more than twofold by glucose in control islets. In contrast, no significant effect of glucose was found on insulin mRNA levels in n0-STZ islets. We conclude that insulin gene regulation by glucose is impaired in n0-STZ rat islets.

Animals↗
PubMed1999

Prostaglandin E(2) mediates inhibition of insulin secretion by interleukin-1beta.

Interleukin-1beta (IL-1beta) and prostaglandin E(2) (PGE(2)), frequently co-participants in inflammatory states, are two well recognized inhibitors of glucose-induced insulin secretion. Previous reports have concluded that the inhibitory effects of these two autacoids on pancreatic beta cell function are not related because indomethacin, a potent prostaglandin synthesis inhibitor, does not prevent IL-1beta effects. However, indomethacin is not a specific cyclooxygenase inhibitor, and its other pharmacologic effects are likely to inhibit insulin secretion independently. Since we recently observed that IL-1beta induces cyclooxygenase-2 (COX-2) gene expression and PGE(2) synthesis in islet beta cells, we have reassessed the possibility that PGE(2) mediates IL-1beta effects on beta function. By using two cell lines (HIT-T15 and betaHC13) as well as Wistar rat isolated pancreatic islets, we examined the ability of two COX-2-specific antagonists, NS-398 and SC-236, to prevent IL-1beta inhibition of insulin secretion. Both drugs prevented IL-1beta from inducing PGE(2) synthesis and inhibiting insulin secretion; adding back exogenous PGE(2) re-established inhibition of insulin secretion in the presence of IL-1beta. We also found that EP3, the PGE(2) receptor subtype whose post-receptor effect is to decrease adenylyl cyclase activity and, thereby, insulin secretion, is the dominant mRNA subtype expressed. We conclude that endogenous PGE(2) mediates the inhibitory effects of exogenous IL-1beta on beta cell function.

Adenylyl Cyclases↗
PubMed1999

Mode of regulation of the extracellular signal-regulated kinases in the pancreatic beta-cell line MIN6 and their implication in the regulation of insulin gene transcription.

Physiological concentrations of glucose that lead to Ca2+ entry and insulin secretion activate extracellular signal-regulated protein kinases (ERK1 and ERK2) in the MIN6 pancreatic beta-cell line. Here we show that this activation is inhibited by the down-regulation of protein kinase C (PKC) and by genistein, an inhibitor of protein tyrosine kinases. In contrast with results obtained in other cell types, neither the epidermal growth factor activity nor the Src family protein tyrosine kinases seem to be involved in the Ca2+-dependent activation of ERKs. inhibition of tyrosine phosphatases by vanadate leads to the activation of ERKs. As observed in the response to glucose, this activation is dependent on Ca2+ entry through L-type voltage-dependent Ca2+ channels. Thus the activation of ERKs in response to glucose depends on PKC and possibly on a tyrosine kinase/tyrosine phosphatase couple. To define the role of ERK activation by glucose we studied the regulation of transcription of the insulin gene. We found that this transcription is regulated in the MIN6 cells in the same range of glucose concentration as in primary islets, and that specific inhibition of mitogen-activated protein kinase kinase, the direct activator of ERK, impaired the response of the insulin gene to glucose. This was observed by analysis of the transfected rat insulin I gene promoter activity and a Northern blot of endogenous insulin mRNA.

Animals↗
PubMed1999

Long-term exposure of isolated rat islets of Langerhans to supraphysiologic glucose concentrations decreases insulin mRNA levels.

Chronic hyperglycemia has been postulated to contribute to beta-cell dysfunction in type 2 diabetic patients. A deleterious effect of prolonged exposure to high glucose concentrations on insulin gene expression has been demonstrated in insulin-secreting cell lines. This study was designed to investigate in isolated rat islets the effects of long-term exposure to supraphysiologic glucose concentrations on insulin, GLUT2, and glucokinase gene expression. The acute effects of glucose on gene expression were investigated by culturing rat islets in 2.8 or 16.7 mmol/L glucose for 24 hours. Insulin, GLUT2, and glucokinase mRNA levels were assessed by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). As expected, glucose acutely increased relative insulin and GLUT2 mRNA levels by 2.8- +/- 0.5-fold (n = 5, P < .005) and 1.8- +/- 0.3-fold (n = 5, P < .05), respectively, but had no effect on glucokinase gene expression (1.1- +/- 0.1-fold increase, n = 4, NS). These results validate the use of semiquantitative RT-PCR to detect changes in gene expression in rat islets. Islets were then cultured in 5.6 or 16.7 mmol/L glucose for 2, 4, or 6 weeks. Relative insulin mRNA levels were higher in islets cultured in high glucose after 2 weeks (1.8+/-0.1 v 1.0+/-0.1, n = 4, P < .05), identical after 4 weeks (0.9+/-0.1 v 1.00+/-0.2, n = 4, NS), and significantly lower after 6 weeks (0.6+/-0.1 v 1.0+/-0.2, n = 6, P < .05). Relative GLUT2 mRNA levels were higher in islets cultured in high glucose after 2 weeks (1.7+/-0.2 v 1.0+/-0.2, n = 3, P < .05) and then identical in both groups after 4 weeks (1.0+/-0.1 v 1.0+/-0.1, n = 3, NS) and 6 weeks (1.0+/-0.2 v 1.0+/-0.1, n = 6, NS). Relative glucokinase mRNA levels were identical under both culture conditions at 2 (1.4+/-0.4 v 1.0+/-0.2, n = 3, NS), 4 (0.8+/-0.5 v 1.0+/-0.3, n = 3, NS), and 6 (0.9+/-0.2 v 1.0+/-0.1, n = 6, NS) weeks. These results indicate that a 6-week exposure of rat islets to supraphysiologic glucose concentrations decreases insulin mRNA levels without affecting GLUT2 and glucokinase gene expression. We conclude that the phenomenon of glucose toxicity decreasing insulin gene expression is not restricted to transformed cells, and might provide insight into the mechanisms by which chronic hyperglycemia adversely affects beta-cell function.

Animals↗
PubMed1998

[Use of islet cells in cell therapy].

Insulin dependent diabetes mellitus is a common disease affecting 1.5 million patients in Europe. It is currently treated by multiple injections of insulin associated with blood glucose self monitoring. The goal of the treatment is to obtain near normal glucose concentration, in order to prevent the later complications (retinopathy, nephropathy, neuropathy, macroangiopathy) of the disease--which may be severe--while avoiding severe hypoglycaemia. Although diabetes therapy has improved enormously in the last few decades, intensive research is currently aimed at replacing not only the missing hormone but also the cells which normally produce insulin in the pancreas. Transplantation of insulin secreting cells as a treatment of diabetes mellitus therefore has a special significance among other applications of cell therapy, since it deals with a disease which already has an efficient therapy. The aim of this article is the discussion of the objectives and the hopes in this field.

Cell- and Tissue-Based Therapy↗
PubMed1998

Inhibition of insulin secretion by leptin in normal rodent islets of Langerhans.

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 beta-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.

1-Methyl-3-isobutylxanthine↗
PubMed1998

Glucose rapidly and reversibly decreases INS-1 cell insulin gene transcription via decrements in STF-1 and C1 activator transcription factor activity.

We have reported that chronic exposure of HIT-T15 cells to supraphysiological concentrations of glucose over many months leads to decreased insulin gene transcription and decreased binding activities of two beta-cell-specific transcription factors, STF-1 and C1 activators, and have postulated that these events may provide a mechanism for glucose toxicity on beta-cell function. We now report that culturing the highly differentiated rat insulinoma cell line, INS-1, in glucose concentrations above 8.0 mM caused a marked decrease in insulin mRNA levels within 24 h. The decrease in insulin mRNA levels was reversed by further incubation of the cells in 4.0 mM glucose. Transient transfection of a chloramphenicol acetyltransferase reporter gene regulated by the 5'-regulatory sequences of the human insulin gene showed that elevated glucose concentrations caused a large decrease in insulin gene promoter activity. The decrease in insulin gene promoter activity was associated with reductions in the binding activities of both STF-1 and C1 activator, and these were partially reversed by lowering the glucose concentration. The decrease in STF-1 binding activity was associated with decreased STF-1 mRNA and occurred independently of changes in STF-1 promoter activity, suggesting a posttranscriptional regulatory mechanism. Furthermore, the decrease in insulin gene expression was found to occur independently of changes in cell proliferation. We conclude that physiologically relevent elevations in glucose can reversibly diminish insulin gene transcription by reducing the expression and/or binding activity of two critical beta-cell transcription factors.

Animals↗
PubMed1998

Does leptin regulate insulin secretion?

The hormone leptin secreted by adipocytes plays a major role in body weight homeostasis. Its main target is the hypothalamus, but it also affects several peripheral tissues directly. The direct effect of leptin on insulin secretion by pancreatic beta cells has been investigated in several studies, though with controversial results. Interpretation of these data must take into account the animal model and the leptin concentrations used. Experiments carried out on islets from ob/ob mice harbouring a mutation in the leptin gene are not representative of the leptin effect in normal animals because ob/ob islets are very sensitive to the hormone and show altered regulation of insulin secretion. In normal rodent islets, physiological concentrations of leptin seem to inhibit insulin secretion only when the islets are maximally stimulated with high concentrations of glucose associated with secretion potentiators. Several isoforms of the leptin receptor are expressed in pancreatic beta cells. Indirect experimental evidence suggests that leptin signalling in islets requires the long isoform of the receptor. The molecular mechanisms underlying the effect of leptin on insulin secretion are unknown. Our hypothesis is that physiological concentrations of leptin in normal rodents do not affect the direct pathway (coupling a rise in glucose concentration to insulin secretion) but modulate a potentiation of glucose-induced insulin secretion involving cyclic AMP or phospholipase C/protein kinase C activation.

Adipocytes↗
PubMed1997

Differentiation of glucose toxicity from beta cell exhaustion during the evolution of defective insulin gene expression in the pancreatic islet cell line, HIT-T15.

Chronic exposure of HIT-T15 cells to supraphysiologic glucose concentration diminishes insulin gene expression and decreased binding of two critical insulin gene transcription factors, STF-1 and RIPE-3b1 activator. To distinguish whether these changes are caused by glucose toxicity or beta cell exhaustion, HIT-T15 cells grown from passage 75 through 99 in media containing 11.1 mM glucose were switched to 0.8 mM glucose at passage 100. They regained binding of STF-1 and RIPE-3b1 activator and had a partial but minimal return of insulin mRNA expression. In a second study, inclusion of somatostatin in the media-containing 11.1 mM glucose inhibited insulin secretion; however, despite this protection against beta cell exhaustion, dramatic decreases in insulin gene expression, STF-1 and RIPE-3b1 binding, and insulin gene promoter activity still occurred. These data indicate that the glucotoxic effects caused by chronic exposure to supraphysiologic concentration of glucose are only minimally reversible and that they are not due simply to beta cell exhaustion. These observations carry with them the clinical implication that Type II diabetic patients who remain hyperglycemic for prolonged periods may have secondary glucose toxic effects on the beta cell that could lead to defective insulin gene expression and worsening of hyperglycemia.

Animals↗
PubMed1997

The defective glucagon response from transplanted intrahepatic pancreatic islets during hypoglycemia is transplantation site-determined.

The optimal site for pancreatic islet cell transplantation is presently unclear, although the liver has been the most commonly used. However, glucagon secretion from islets that have been autotransplanted in liver has been reported to be unresponsive to hypoglycemia yet responsive to arginine. To determine whether this selective glucagon secretory defect is related to the intrahepatic site of islet implantation or to the process of transplantation per se, we studied counterregulatory responses to hypoglycemia in dogs with pancreatic islet autotransplantation in the hepatic parenchyma (the intrahepatic [IH] group, n = 9) or the peritoneal cavity (the intraperitoneal [IP] group, n = 9), following total pancreatectomy, and compared them with the responses in normal controls (n = 10). Dogs were subjected to a hypoglycemic hyperinsulinemic (5 mU x kg-1 x min-1) clamp for 90 min under general anesthesia. Arterial glucose concentrations were clamped at 2.7 mmol/l for the final 45 min of the clamp. Immediately following the clamp, glucagon responses to IV arginine (5 g) were also assessed. During hypoglycemia, glucagon responses in the IH group (maximal incremental glucagon = 33 +/- 21 ng/l; glucagon area under curve [AUC] = 713 +/- 1,022 ng x l-1 x min-1) were significantly lower than either the IP (maximal incremental glucagon = 92 +/- 32 ng/l; glucagon AUC = 4,090 +/- 1,600 ng x l-1 x min-1) or control (maximal incremental glucagon = 154 +/- 71 ng/l; glucagon AUC = 6,943 +/- 2,842 ng x l-1 x min-1) group (IH vs. IP group, P < 0.05; control vs. IH group, P < 0.01). Glucagon responses in the IP group did not differ significantly from the control group. Epinephrine responses to hypoglycemia were similar in all groups, whereas neither of the transplanted groups (IH and IP) had pancreatic polypeptide responses. There was a prompt rise in plasma glucagon after intravenous arginine in all groups. These data indicate that glucagon unresponsiveness to hypoglycemia is specific to intrahepatically transplanted islets, rendering the liver a disadvantageous site for optimal alpha-cell function.

Animals↗
PubMed1996

Chronic exposure of betaTC-6 cells to supraphysiologic concentrations of glucose decreases binding of the RIPE3b1 insulin gene transcription activator.

We have shown previously that chronic exposure of HIT-T15 cells to supraphysiologic glucose concentrations causes decreased insulin gene transcription and decreased binding activities of two beta-cell specific transcription factors, STF-1 and the RIPE3b1 activator, and have suggested that these events may provide a mechanism for glucose toxicity on beta-cell function. However, this contention can be criticized because it is not clear whether these observations are unique to the HIT-T15 cell or generalizable to other beta-cell lines and the islet. Therefore, we cultured betaTC-6 cells for up to 41 wk in either 11.1 or 0.8 mM glucose. We observed a passage-dependent decrease in insulin content and insulin mRNA levels in betaTC-6 cells chronically cultured in 11.1 mM glucose. Cells chronically cultured in 0.8 mM glucose had higher insulin mRNA levels than cells chronically cultured in 11.1 mM glucose. The relative activity of a chloramphenicol acetyl transferase (CAT) reporter gene controlled by the 5' regulatory region of the human insulin gene was decreased in late passage betaTC-6 cells chronically cultured in 11.1 mM glucose, but was preserved in late passages of cells chronically cultured in 0.8 mM glucose. Electromobility shift assays demonstrated that binding of a specific nuclear protein that recognizes the RIPE3b1 binding site of the insulin gene was markedly diminished in late passage cells chronically exposed to 11.1 mM glucose, whereas binding activities of STF-1 and ICE activators were unchanged. RIPE3b1 binding activity was preserved in late passage cells chronically exposed to 0.8 mM glucose. Mutation of the RIPE3b1 binding site almost completely abolished insulin gene transcription as well as binding activity. We conclude that chronic exposure of betaTC-6 cells to high glucose concentrations paradoxically decreases insulin gene transcription, in part, by decreasing activity of the trans-activating factor which binds to the RIPE3b1 sequence. This study uniquely demonstrates that altered binding to the RIPE3b1 sequence mediates glucose toxicity in betaTC-6 cells, thus reinforcing the importance of this cis-acting element in the regulation of insulin gene transcription. We conclude that the phenomenon of glucose toxicity decreasing binding of transcription factors and thereby reducing insulin gene expression is not a feature solely of HIT-T15 cells and may be demonstrable generally in beta-cell lines.

Animals↗