Insulin mRNA Content Research Articles

Oxidative and endoplasmic reticulum stress develop adverse metabolic effects due to the high-fat high-fructose diet consumption from birth to young adulthood

AimsThe early postnatal dietary intake has been considered a crucial factor affecting the offspring later life metabolic status. Consistently, this study investigated the oxidative and endoplasmic reticulum (ER) stress interventions in the induction of adverse metabolic effects due to the high-fat high-fructose diet (HFHFD) consumption from birth to young adulthood in rat offspring. Materials and methodsAfter delivery, the dams with their pups were randomly allocated into the normal diet (ND) and HFHFD groups. At weaning, the male offspring were divided into ND-None, ND-DMSO, ND-4-phenyl butyric acid (4-PBA), HFHFD-None, HFHFD-DMSO, and HFHFD-4-PBA groups and fed on their respected diets for five weeks. Then, the drug was injected for ten days. Subsequently, glucose and lipid metabolism parameters, oxidative and ER stress markers, and Wolfram syndrome1 (Wfs1) expression were assessed. Key findingsIn the HFHFD group, anthropometrical parameters, plasma high-density lipoprotein (HDL), and glucose-stimulated insulin secretion and content were decreased. Whereas, the levels of plasma leptin, low-density lipoprotein (LDL) and glucose, hypothalamic leptin, pancreatic catalase activity and glutathione (GSH), pancreatic and hypothalamic malondialdehyde (MDA), binding immunoglobulin protein (BIP) and C/EBP homologous protein (CHOP), and pancreatic WFS1 protein were increased. 4-PBA administration in the HFHFD group, decreased the hypothalamic and pancreatic MDA, BIP and CHOP levels, while, increased the Insulin mRNA and glucose-stimulated insulin secretion and content. SignificanceHFHFD intake from birth to young adulthood through the development of pancreatic and hypothalamic oxidative and ER stress, increased the pancreatic WFS1 protein and impaired glucose and lipid homeostasis in male rat offspring.

Inactivating the permanent neonatal diabetes gene Mnx1 switches insulin-producing β-cells to a δ-like fate and reveals a facultative proliferative capacity in aged β-cells.

Homozygous Mnx1 mutation causes permanent neonatal diabetes in humans, but via unknown mechanisms. Our systematic and longitudinal analysis of Mnx1 function during murine pancreas organogenesis and into the adult uncovered novel stage-specific roles for Mnx1 in endocrine lineage allocation and β-cell fate maintenance. Inactivation in the endocrine-progenitor stage shows that Mnx1 promotes β-cell while suppressing δ-cell differentiation programs, and is crucial for postnatal β-cell fate maintenance. Inactivating Mnx1 in embryonic β-cells (Mnx1(Δbeta)) caused β-to-δ-like cell transdifferentiation, which was delayed until postnatal stages. In the latter context, β-cells escaping Mnx1 inactivation unexpectedly upregulated Mnx1 expression and underwent an age-independent persistent proliferation. Escaper β-cells restored, but then eventually surpassed, the normal pancreatic β-cell mass, leading to islet hyperplasia in aged mice. In vitro analysis of islets isolated from Mnx1(Δbeta) mice showed higher insulin secretory activity and greater insulin mRNA content than in wild-type islets. Mnx1(Δbeta) mice also showed a much faster return to euglycemia after β-cell ablation, suggesting that the new β-cells derived from the escaper population are functional. Our findings identify Mnx1 as an important factor in β-cell differentiation and proliferation, with the potential for targeting to increase the number of endogenous β-cells for diabetes therapy.

In vivo studies on non-viral transdifferentiation of liver cells towards pancreatic β cells

Transdifferentiation in vivo is an attractive option for autologous replacement of pancreatic β cells in patients with type 1 diabetes. It has been achieved by adenoviral delivery of genes for transcription factors in the liver and pancreas of hyperglycaemic mice. However, these viral approaches are not clinically applicable. We used the hydrodynamic approach to deliver genes Pdx1, Ngn3 (Neurog3) and MafA singly and in combination to livers of normoglycaemic rats. Five expression plasmids were evaluated. Livers were removed 1, 3, 7, 14 and 28 days after gene delivery and assayed by quantitative PCR, semi-quantitative PCR and immunohistology. Functional studies on hyperglycaemic rats were performed. The highest and most sustained expression was from a CpG-depleted plasmid (pCpG) and a plasmid with an in-frame scaffold/matrix attachment region ((pEPI(CMV)). When Pdx1, Ngn3 and MafA were delivered together to normoglycaemic rats with these plasmids, insulin mRNA was detected at all time points and was ~50-fold higher with pCpG. Insulin mRNA content of livers at days 3 and 7 was equivalent to that of a pancreas, with scattered insulin-positive cells detected by immunohistology, but levels declined thereafter. Prohormone convertase 1/3 was elevated at days 3 and 7. In hyperglycaemic rats, fasting blood glucose was lower at days 1, 3 and 7 but not thereafter, and body weight was maintained to day 28. We conclude that hydrodynamic gene delivery of multiple transcription factors to rat liver can initiate transdifferentiation to pancreatic β cells, but the process is reversible and probably requires more sustained transcription factor expression.

Treinamento resistido reduz inflamação em músculo esquelético e melhora a sensibilidade à insulina periférica em ratos obesos induzidos por dieta hiperlipídica

To determine if resistive exercise protocol can modulate Tnf-α, SOCS3 and glucose transporter GLUT4 genes expression in skeletal muscle, and peripheral insulin sensitivity in obese rats induced by hyperlipidic diet. Wistar obese rats induced by hyperlipidic diet were subjected a resistive exercise protocol as jump squat. Insulin sensitivity and mRNA content of Tnf-α, SOCS3 and GLUT4 were assayed and compared among the groups: obese sedentary (OS) and exercised (OE), control sedentary (CS) and exercised (CE). The mRNA content of Tnf-α and SOCS3 has increased in skeletal muscle from OS and has decreased in OE group. The protein and GLUT4 mRNA contents were correlated but they did not change among the groups. Peripheral insulin sensitivity has increased in the OE compared to OS group. The resistive exercise reverses the peripheral insulin resistance and the inflammatory state in skeletal muscle from diet-induced obese rats.

High Glucose Suppresses Human Islet Insulin Biosynthesis by Inducing miR-133a Leading to Decreased Polypyrimidine Tract Binding Protein-Expression

BackgroundProlonged periods of high glucose exposure results in human islet dysfunction in vitro. The underlying mechanisms behind this effect of high glucose are, however, unknown. The polypyrimidine tract binding protein (PTB) is required for stabilization of insulin mRNA and the PTB mRNA 3′-UTR contains binding sites for the microRNA molecules miR-133a, miR-124a and miR-146. The aim of this study was therefore to investigate whether high glucose increased the levels of these three miRNAs in association with lower PTB levels and lower insulin biosynthesis rates.Methodology/Principal FindingsHuman islets were cultured for 24 hours in the presence of low (5.6 mM) or high glucose (20 mM). Islets were also exposed to sodium palmitate or the proinflammatory cytokines IL-1β and IFN-γ, since saturated free fatty acids and cytokines also cause islet dysfunction. RNA was then isolated for real-time RT-PCR analysis of miR-133a, miR-124a, miR-146, insulin mRNA and PTB mRNA contents. Insulin biosynthesis rates were determined by radioactive labeling and immunoprecipitation. Synthetic miR-133a precursor and inhibitor were delivered to dispersed islet cells by lipofection, and PTB was analyzed by immunoblotting following culture at low or high glucose. Culture in high glucose resulted in increased islet contents of miR-133a and reduced contents of miR-146. Cytokines increased the contents of miR-146. The insulin and PTB mRNA contents were unaffected by high glucose. However, both PTB protein levels and insulin biosynthesis rates were decreased in response to high glucose. The miR-133a inhibitor prevented the high glucose-induced decrease in PTB and insulin biosynthesis, and the miR-133a precursor decreased PTB levels and insulin biosynthesis similarly to high glucose.ConclusionProlonged high-glucose exposure down-regulates PTB levels and insulin biosynthesis rates in human islets by increasing miR-133a levels. We propose that this mechanism contributes to hyperglycemia-induced beta-cell dysfunction.

Mechanisms of Pancreatic β-Cell Growth and Regeneration: Studies on Rat Insulinoma Cells

Information about the mechanism of beta-cell growth and regeneration may be obtained by studies of insulinoma cells. In the present study the growth and function of the rat insulinoma cell lines RINm5F and 5AH were evaluated by addition of serum, hormones, and growth factors. It was found that transferrin is the only obligatory factor whereas growth hormone, epidermal growth factor, fibroblast growth factor, and TRH had modulating effects. A heat-labile heparin binding serum factor which stimulated thymidine incorporation but not cell proliferation was demonstrated in human serum. Measurements of insulin mRNA content showed that the insulinoma cells only contained about 2% of that of normal rat beta-cells. These results are discussed in relation to the role of growth factors, oncogenes, and differentiation in the growth and regeneration of beta-cells.

Betacellulin and nicotinamide sustain PDX1 expression and induce pancreatic β-cell differentiation in human embryonic stem cells

The major obstacle in cell therapy of diabetes mellitus is the limited source of insulin-producing β cells. Very recently, it was shown that a five-stage protocol recapitulating in vivo pancreatic organogenesis induced pancreatic β cells in vitro; however, this protocol is specific to certain cell lines and shows much line-to-line variation in differentiation efficacy. Here, we modified the five-stage protocol for the human embryonic stem cell line SNUhES3 by the addition of betacellulin and nicotinamide. We reproduced in vivo pancreatic islet differentiation by directing the cells through stages that resembled in vivo pancreatic organogenesis. The addition of betacellulin and nicotinamide sustained PDX1 expression and induced β-cell differentiation. C-peptide—a genuine marker of de novo insulin production—was identified in the differentiated cells, although the insulin mRNA content was very low. Further studies are necessary to develop more efficient and universal protocols for β-cell differentiation.

Diminished β-cell replication contributes to reduced β-cell mass in fetal sheep with intrauterine growth restriction

Human fetuses with severe intrauterine growth restriction (IUGR) have less pancreatic endocrine tissue and exhibit beta-cell dysfunction, which may limit beta-cell function in later life and contribute to their increased incidence of noninsulin-dependent diabetes mellitus. Three factors, replication, apoptosis, and neoformation, contribute to fetal beta-cell mass. We studied an ovine model of IUGR to understand whether nutrient deficits lead to decreased rates of fetal pancreatic beta-cell replication, increased rates of apoptosis, or lower rates of differentiation. At 90% of term gestation, IUGR fetal and pancreatic weights were 58% and 59% less than pair-fed control, respectively. We identified a selective impairment of beta-cell mass compared with other pancreatic cell types in IUGR fetuses. Insulin and insulin mRNA contents were less than other pancreatic endocrine hormones in IUGR fetuses, as were pancreatic insulin positive area (42%) and beta-cell mass (76%). Pancreatic beta-cell apoptosis was not different between treatments. beta-cell capacity for cell cycling, determined by proliferating cell nuclear antigen (PCNA) immunostaining, was not different between treatment groups. However, the percentage of beta-cells actually undergoing mitosis was 72% lower in IUGR fetuses. These results indicate that in utero nutrient deficits decrease the population of pancreatic beta-cells by lengthening G1, S, and G2 stages of interphase and decreasing mitosis near term. Diminished beta-cell mass in IUGR infants at birth, if not adequately compensated for after birth, may contribute to insufficient insulin production in later life and, thus, a predisposition to noninsulin-dependent diabetes.

Modulation of adipoinsular axis in prediabetic zucker diabetic fatty rats by diazoxide.

Dysregulation of the adipoinsular axis in male obese Zucker diabetic fatty (ZDF; fa/fa) rats, a model of type 2 diabetes, results in chronic hyperinsulinemia and increased de novo lipogenesis in islets, leading to beta-cell failure and diabetes. Diazoxide (DZ; 150 mg/kg.d), an inhibitor of insulin secretion, was administered to prediabetic ZDF animals for 8 wk as a strategy for prevention of diabetes. DZ reduced food intake (P < 0.02) and rate of weight gain only in ZDF rats (P < 0.01). Plasma insulin response to glucose load was attenuated in DZ-Zucker lean rats (ZL; P < 0.01), whereas DZ-ZDF had higher insulin response to glucose than controls (P < 0.001). DZ improved hemoglobin A1c (P < 0.001) and glucose tolerance in ZDF (P < 0.001), but deteriorated hemoglobin A1c in ZL rats (P < 0.02) despite normal tolerance in the fasted state. DZ lowered plasma leptin (P < 0.001), free fatty acid, and triglyceride (P < 0.001) levels, but increased adiponectin levels (P < 0.02) only in ZDF rats. DZ enhanced beta3-adrenoreceptor mRNA (P < 0.005) and adenylate cyclase activity (P < 0.01) in adipose tissue from ZDF rats only, whereas it enhanced islet beta3- adrenergic receptor mRNA (P < 0.005) but paradoxically decreased islet adenylate cyclase activity (P < 0.005) in these animals. Islet fatty acid synthase mRNA (P < 0.03), acyl coenzyme A carboxylase mRNA (P < 0.01), uncoupling protein-2 mRNA (P < 0.01), and triglyceride content (P < 0.005) were only decreased in DZ-ZDF rats, whereas islet insulin mRNA and insulin content were increased in DZ-ZDF (P < 0.01) and DZ-ZL rats (P < 0.03). DZ-induced beta-cell rest improved the lipid profile, enhanced the metabolic efficiency of insulin, and prevented beta-cell dysfunction and diabetes in diabetes-prone animals. This therapeutic strategy may be beneficial in preventing beta-cell failure and progression to diabetes in humans.

Protein calorie restriction has opposite effects on glucose metabolism and insulin gene expression in fetal and adult rat endocrine pancreas.

We previously demonstrated that fetuses from undernourished pregnant rats exhibited increased beta-cell mass and hyperinsulinemia, whereas keeping food restriction until adult age caused reduced beta-cell mass, hypoinsulinemia, and decreased insulin secretion. Because these alterations can be related to insulin availability, we have now investigated early and long-term effects of protein calorie food restriction on insulin mRNA levels as well as the possible mechanisms that could modulate the endogenous insulin mRNA content. We used fetuses at 21.5 days of gestation proceeding from food-restricted rats during the last week of pregnancy and 70-day-old rats undernourished from day 14 of gestation until adult age and with respective controls. Insulin mRNA levels, glucose transporters, and total glycolysis and mitochondrial oxidative fluxes were evaluated. We additionally analyzed undernutrition effects on signals implicated in glucose-mediated insulin gene expression, especially pancreatic duodenal homeobox-1 (PDX-1), stress-activated protein kinase-2 (p38/SAPK2), and phosphatidylinositol 3-kinase. Undernourished fetuses showed increased insulin mRNA, oxidative glucose metabolism, and p38/SAPK2 levels, whereas undernutrition until adult age provoked a decrease in insulin gene expression, oxidative glucose metabolism, and PDX-1 levels. The results indicate that food restriction caused changes in insulin gene expression and content leading to alterations in glucose-stimulated insulin secretion. The molecular events, increased p38/SAPK2 levels in fetuses and decreased PDX-1 levels in adults, seem to be the responsible for the altered insulin mRNA expression. Moreover, because PDX-1 activation appears to be regulated by glucose-derived metabolite(s), the altered glucose oxidation caused by undernutrition could in some manner affect insulin mRNA expression.

A role for glutathione peroxidase in protecting pancreatic beta cells against oxidative stress in a model of glucose toxicity.

Antioxidant drugs have been reported to protect pancreatic islets from the adverse effects of chronic exposure to supraphysiological glucose concentrations. However, glucose has not been shown to increase intracellular oxidant load in islets, nor have the effects of increasing or inhibiting glutathione peroxidase (GPx) activity on islet resistance to sugar-induced oxidant stress been studied. We observed that high glucose concentrations increased intracellular peroxide levels in human islets and the pancreatic beta cell line, HIT-T15. Inhibition of gamma-glutamylcysteine synthetase (gammaGCS) by buthionine sulfoximine augmented the increase in islet peroxide and decrease in insulin mRNA levels, content, and secretion in islets and HIT-T15 cells induced by ribose. Adenoviral overexpression of GPx increased GPx activity and protected islets against adverse effects of ribose. These results demonstrate that glucose and ribose increase islet peroxide accumulation and that the adverse consequences of ribose-induced oxidative stress on insulin mRNA, content, and secretion can be augmented by a glutathione synthesis inhibitor and prevented by increasing islet GPx activity. These observations support the hypothesis that oxidative stress is one mechanism for glucose toxicity in pancreatic islets.

SOCS-1 Protein Prevents Janus Kinase/STAT-dependent Inhibition of β Cell Insulin Gene Transcription and Secretion in Response to Interferon-γ

In the pathogenesis of type I diabetes mellitus, activated leukocytes infiltrate pancreatic islets and induce beta cell dysfunction and destruction. Interferon (IFN)-gamma, tumor necrosis factor-alpha and interleukin (IL)-1 beta play important, although not completely defined, roles in these mechanisms. Here, using the highly differentiated beta Tc-Tet insulin-secreting cell line, we showed that IFN-gamma dose- and time-dependently suppressed insulin synthesis and glucose-stimulated secretion. As described previously IFN-gamma, in combination with IL-1 beta, also induces inducible NO synthase expression and apoptosis (Dupraz, P., Cottet, S., Hamburger, F., Dolci, W., Felley-Bosco, E., and Thorens, B. (2000) J. Biol. Chem. 275, 37672--37678). To assess the role of the Janus kinase/signal transducer and activator of transcription (STAT) pathway in IFN-gamma intracellular signaling, we stably overexpressed SOCS-1 (suppressor of cytokine signaling-1) in the beta cell line. We demonstrated that SOCS-1 suppressed cytokine-induced STAT-1 phosphorylation and increased cellular accumulation. This was accompanied by a suppression of the effect of IFN-gamma on: (i) reduction in insulin promoter-luciferase reporter gene transcription, (ii) decrease in insulin mRNA and peptide content, and (iii) suppression of glucose-stimulated insulin secretion. Furthermore, SOCS-1 also suppressed the cellular effects that require the combined presence of IL-1 beta and IFN-gamma: induction of nitric oxide production and apoptosis. Together our data demonstrate that IFN-gamma is responsible for the cytokine-induced defect in insulin gene expression and secretion and that this effect can be completely blocked by constitutive inhibition of the Janus kinase/STAT pathway.

Insulin gene expression pattern in rat pancreas during the estrous cycle

Sex steroid hormones influence insulin homeostasis and glucose metabolism, estradiol (E 2) and progesterone (P 4) induce changes in both fasting and postprandial insulinemia in rodents, however, insulin gene expression during estrous cycle is unknown. The aim of the present study was to determine an insulin gene expression pattern during the estrous cycle in the rat. Groups of 6 adult rats in each day of the estrous cycle were used. Serum P 4, E 2, testosterone (T) and insulin concentrations were determined by radioimmunoassay (RIA). A Northern blot analysis was performed to assess insulin gene expression in pancreatic tissue. We found a marked variation in insulin gene expression during the estrous cycle. The highest insulin expression was observed during the proestrus day. Interestingly, E 2 and P 4 but not T levels were correlated with changes in insulin mRNA content. The variations in serum insulin during the cycle were correlated with its mRNA content in pancreas. The overall results showed variations in serum insulin and insulin gene expression during estrous cycle of the rat that correlated with circulating E 2 and P 4 levels.

Islet amyloid polypeptide/amylin messenger RNA and protein expression in human insulinomas in relation to amyloid formation.

Islet amyloid polypeptide (IAPP), also named amylin, is the predominant protein component of amyloid deposits in human islet beta cell tumours of the pancreas (insulinomas). IAPP is co-produced with insulin by islet beta cells. We investigated IAPP expression in relation to insulin expression and to amyloid formation in eleven insulinomas. RNA and protein extracts were prepared from the same pieces of tumour tissue, and from specimens of two normal human pancreata. IAPP and insulin mRNA and peptide content were quantified using Northern blot analysis and radioimmunoassay (RIA) respectively. Molecular forms of IAPP immunoreactivity were analysed by reversed-phase high-performance liquid chromatography (HPLC). The presence of islet hormones and of amyloid was assessed by (immuno)histochemical staining of paraffin sections. Plasma levels of IAPP and insulin prior to tumour resection were determined by RIA. IAPP and insulin mRNA and peptide content varied widely between the tumour specimens, and there was considerable intratumour heterogeneity of peptide content. HPLC analysis indicated correct proteolytic processing of the IAPP precursor protein. Amyloid deposits were detected only in the three tumours with the highest IAPP content. In contrast to insulin, plasma levels of IAPP were not elevated in the insulinoma patients. The spectrum of hormone production by insulinomas cannot be inferred from only a few tissue sections due to intratumour heterogeneity. Expression of the IAPP and insulin genes is not coupled in insulinomas, which produce properly processed mature IAPP. In addition to IAPP overproduction, additional factors such as intracellular accumulation of IAPP are involved in amyloidogenesis in insulinomas.

Glucagon-like peptide-1 can reverse the age-related decline in glucose tolerance in rats.

Wistar rats develop glucose intolerance and have a diminished insulin response to glucose with age. The aim of this study was to investigate if these changes were reversible with glucagon-like peptide-1 (GLP-1), a peptide that we have previously shown could increase insulin mRNA and total insulin content in insulinoma cells. We infused 1.5 pmol/ kg-1.min-1 GLP-1 subcutaneously using ALZET microosmotic pumps into 22-mo-old Wistar rats for 48 h. Rat infused with either GLP-1 or saline were then subjected to an intraperitoneal glucose (1 g/kg body weight) tolerance test, 2 h after removing the pump. 15 min after the intraperitoneal glucose, GLP-1-treated animals had lower plasma glucose levels (9.04+/-0.92 mmol/liter, P < 0.01) than saline-treated animals (11.61+/-0.23 mmol/liter). At 30 min the plasma glucose was still lower in the GLP-1-treated animals (8.61+/-0.39 mmol/liter, P < 0.05) than saline-treated animals (10.36+/-0.43 mmol/liter). This decrease in glucose levels was reflected in the higher insulin levels attained in the GLP-1-treated animals (936+/-163 pmol/liter vs. 395+/-51 pmol/liter, GLP-1 vs. saline, respectively, P < 0.01), detected 15 min after glucose injection. GLP-1 treatment also increased pancreatic insulin, GLUT2, and glucokinase mRNA in the old rats. The effects of GLP-1 were abolished by simultaneous infusion of exendin [9-39], a specific antagonist of GLP-1. GLP-1 is therefore able to reverse some of the known defects that arise in the beta cell of the pancreas of Wistar rats, not only by increasing insulin secretion but also by inducing significant changes at the molecular level.

B cells of aging rats: Impaired stimulus-secretion coupling but normal susceptibility to adverse effects of a diabetic state

A diabetic state impairs B-cell function and survival. We tested whether the negative effects are exacerbated by the aging process. Islets were isolated from old (63.3 ± 2.3 weeks) and, young (11.3 ± 0.5 weeks) inbred Wistar rats. Age did not affect DNA and insulin content, yet both glucose-induced (27.8 mmol/L) and arginine-induced induced (10 mmol/L) insulin responses in old islets were significantly reduced. Islets were transplanted under the kidney capsule of recipients that were either nondiabetic or severely diabetic after streptozotocin (STZ) treatment (blood glucose > 20 mmol/L). Following 8 weeks' transplantation to nondiabetic recipients, perfused kidneys with grafts of old islets exhibited the same insulin responses to glucose as grafts of young islets. However, responses to arginine were reduced in grafts of old islets (28 ± 4 μU/min) relative to grafts of young islets. (70 ± 18 μU/min, P < .05). Insulin mRNA content was similar in grafts of old islets and grafts of young islets. Following 8 weeks' transplantation to diabetic recipients, 27.8 mmol/L glucose failed to induce insulin secretion in grafts of old islets and grafts of young islets alike, whereas arginine-induced insulin secretion was unaffected in grafts of old islets but reduced in grafts of young islets. Insulin mRNA content was reduced to a similar extent by the diabetic state (to 28% in grafts of old islets and to 27% in grafts of young islets grafts in nondiabetic recipients). We conclude that aging, although leading to impaired stimulus-secretion coupling, does not increase susceptibility to the negative effects of a diabetic state on B-cell function as presently tested.

Signals related to glucose metabolism regulate islet amyloid polypeptide (IAPP) gene expression in human pancreatic islets

Intracellular pathways involved in glucose stimulation of IAPP gene expression were studied in human pancreatic islets. Glucose (16.7 mM), but not mannose, caused a 2.3-fold increase in IAPP mRNA levels; this effect was inhibited by actinomycin D. In the presence of the non-metabolizable 6-deoxyglucose (16.7 mM) IAPP mRNA levels were markedly depleted. Both mannoheptulose and verapamil blocked glucose-induced stimulation of the IAPP gene. The magnitude of the insulin gene response to glucose was smaller (1.3-fold); none of the above-mentioned agents had significant effects on insulin mRNA content. Tunicamycin elicited a 2.4- and 2.7-fold increase in IAPP mRNA levels in the low and high glucose media, respectively; however, it did not change insulin mRNA. It had no effect on rat IAPP or insulin mRNAs, either. We conclude that IAPP gene expression is regulated by signals derived from glucose metabolism and that intracellular calcium may be involved in this response. IAPP and insulin genes are not co-regulated in cultured human pancreatic islets.

Glycation-dependent, reactive oxygen species-mediated suppression of the insulin gene promoter activity in HIT cells.

Prolonged poor glycemic control in non-insulin-dependent diabetes mellitus patients often leads to a decline in insulin secretion from pancreatic beta cells, accompanied by a decrease in the insulin content of the cells. As a step toward elucidating the pathophysiological background of the so-called glucose toxicity to pancreatic beta cells, we induced glycation in HIT-T15 cells using a sugar with strong deoxidizing activity, D-ribose, and examined the effects on insulin gene transcription. The results of reporter gene analyses revealed that the insulin gene promoter is more sensitive to glycation than the control beta-actin gene promoter; approximately 50 and 80% of the insulin gene promoter activity was lost when the cells were kept for 3 d in the presence of 40 and 60 mM D-ribose, respectively. In agreement with this, decrease in the insulin mRNA and insulin content was observed in the glycation-induced cells. Also, gel mobility shift analyses using specific antiserum revealed decrease in the DNA-binding activity of an insulin gene transcription factor, PDX-1/IPF1/STF-1. These effects of D-ribose seemed almost irreversible but could be prevented by addition of 1 mM aminoguanidine or 10 mM N-acetylcysteine, thus suggesting that glycation and reactive oxygen species, generated through the glycation reaction, serve as mediators of the phenomena. These observations suggest that protein glycation in pancreatic beta cells, which occurs in vivo under chronic hyperglycemia, suppresses insulin gene transcription and thus can explain part of the beta cell glucose toxicity.

Irreversible loss of normal beta-cell regulation by glucose in neonatally streptozotocin diabetic rats.

Animals with NIDDM display abnormal glucose regulation of insulin secretion and biosynthesis. We tested reversibility of abnormal regulation by normoglycaemia using an islet transplantation technique. Inbred non-diabetic and neonatally STZ diabetic rats (n-STZ) were used. Transplantations insufficient to normalize the blood glucose levels (200 islets under kidney capsule) were performed from diabetic to normal (D-N) and from diabetic to diabetic (D-D), as well as from normal to normal (N-N) and from normal to diabetic (N-D) rats. Four weeks after transplantation, graft bearing kidneys were isolated and perfused with Krebs-Henseleit bicarbonate buffer to measure insulin secretion in response to 27.8 mmol/l glucose and 10 mmol/l arginine. Four weeks of normoglycaemia failed to restore glucose-induced insulin secretion from n-STZ islets (glucose induced increment: -1.7 +/- 2.5 fmol/min in D-N, 1.2 +/- 7.1 fmol/min in D-D). In contrast to normal islets, normoglycaemia reduced insulin mRNA contents (60 +/- 24 in D-N, 496 +/- 119 in D-D; O.D.-arbitrary units). However, arginine-induced secretion was markedly enhanced by diabetic environment in both normal and n-STZ islet grafts. These results indicate that selected aspects of glucose recognition are irreversibly damaged by a long-term diabetic state or, alternatively, by a lasting effect of STZ administration.

Lack of long-term beta-cell glucotoxicity in vitro in pancreatic islets isolated from two mouse strains (C57BL/6J; C57BL/KsJ) with different sensitivities of the beta-cells to hyperglycaemia in vivo.

Previous studies have shown that 4 weeks after syngeneic transplantation of a suboptimal number of islets into either C57BL/6J (BL/6J) or C57BL/KsJ (BL/KsJ) diabetic mice there is an impaired insulin secretion by the perfused grafts. After normalization of the blood glucose level with a second islet graft, the BL/6J strain showed restored insulin secretion whilst that of the BL/KsJ strain remained impaired. The aim of the present work was to study the effects of glucose on the in-vitro function of islet beta-cells from these two mouse strains, with different sensitivities of their beta-cells to glucose in vivo. Isolated pancreatic islets from each strain were kept for 1 week in tissue culture at 5.6, 11, 28 or 56 mmol glucose/l and were subsequently analysed with regard to insulin release, (pro)-insulin and total protein biosynthesis, insulin, DNA and insulin mRNA contents and glucose metabolism. Islets from both strains cultured at 28 or 56 mmol glucose/l showed an increased accumulation of insulin in the culture medium and an enhanced glucose-stimulated insulin release compared with corresponding control islets cultured at 11 mmol glucose/l. After culture at either 5.6 or 56 mmol/l, rates of (pro)insulin biosynthesis were decreased in BL/KsJ islets in short-term incubations at 17 mmol glucose/l, whereas islets cultured at 56 mmol glucose/l showed a marked increase at 1.7 mmol glucose/l. In BL/6J islets, the (pro)insulin biosynthesis rates were similar to those of the BL/KsJ islets with one exception, namely that no decrease was observed at 56 mmol glucose/l. Islets of both strains showed a decreased insulin content after culture with 56 mmol glucose/l.(ABSTRACT TRUNCATED AT 250 WORDS)