Response Of Islets Research Articles

Beneficial effects of tigerinin-1R on glucose homeostasis and beta cell function in mice with diet-induced obesity-diabetes.

This paper investigates the anti-diabetic effects of tigerinin-1R (RVCSAIPLPICH.NH2), a previously described amphibian host defence peptide, in mice with diet-induced obesity-diabetes. Proteolytic degradation of synthetic tigerinin-1R was investigated by reversed-phase HPLC and MALDI-TOF mass spectrometry. Changes in glycaemic responses and metabolic parameters were measured in mice with high fat diet-induced obesity-diabetes treated with twice-daily with of tigerinin-1R (75 nmol/kg bw) for 15 days. Indirect calorimetry and body composition were measured by CLAMS and DEXA whole body scanning. Insulin secretory responses of islets isolated from treated and untreated mice were examined. Tigerinin-1R was resistant to in vitro degradation by plasma enzymes. Twice-daily injection of tigerinin-1R for 15 days had no significant effect on food intake or body weight. Non-fasting glucose levels were significantly lowered, and insulin levels were elevated compared to saline treated controls. Glycaemic responses to both oral and intraperitoneal glucose administration were significantly improved by tigerinin-1R treatment. Plasma insulin was also significantly elevated. The peptide had no significant effect on insulin sensitivity but the beta cell responses of islets isolated from treated mice to a range of nutrients and peptidergic secretagogues were significantly improved. Oxygen consumption, CO2 production, respiratory exchange ratio, energy expenditure and body composition were not significantly altered by treatment with tigerinin-1R. Tigerinin-1R significantly improves glucose homeostasis and may have potential as a novel antidiabetic agent.

Magainin-AM2 improves glucose homeostasis and beta cell function in high-fat fed mice

BackgroundMagainin-AM2, a previously described amphibian host-defense peptide, stimulates insulin- and glucagon-like peptide-1-release in vitro. This study investigated anti-diabetic effects of the peptide in mice with diet-induced obesity and glucose intolerance. MethodsMale National Institute of Health Swiss mice were maintained on a high-fat diet for 12-weeks prior to the daily treatment with magainin-AM2. Various indices of glucose tolerance were monitored together with insulin secretory responsiveness of islets at conclusion of study. ResultsFollowing twice daily treatment with magainin-AM2 for 15days, no significant difference in body weight and food intake was observed compared with saline-treated high fat control animals. However, non-fasting blood glucose was significantly (P<0.05) decreased while plasma insulin concentrations were significantly (P<0.05) increased. Oral and intraperitoneal glucose tolerance and insulin secretion following glucose administration via both routes were significantly (P<0.05) enhanced. The peptide significantly (P<0.001) improved insulin sensitivity as well as the beta cell responses of islets isolated from treated mice to a range of insulin secretagogues. Oxygen consumption, CO2 production, respiratory exchange ratio and energy expenditure were not significantly altered by sub-chronic administration of magainin-AM2 but a significant (P<0.05) reduction in fat deposition was observed. ConclusionThese results indicate that magainin-AM2 improves glucose tolerance, insulin sensitivity and islet beta cells secretory responsiveness in mice with obesity-diabetes. General significanceThe activity of magainin-AM2 suggests the possibility of exploiting this peptide for treatment of type 2 diabetes.

12-lipoxygenase promotes obesity-induced oxidative stress in pancreatic islets.

High-fat diets lead to obesity, inflammation, and dysglycemia. 12-Lipoxygenase (12-LO) is activated by high-fat diets and catalyzes the oxygenation of cellular arachidonic acid to form proinflammatory intermediates. We hypothesized that 12-LO in the pancreatic islet is sufficient to cause dysglycemia in the setting of high-fat feeding. To test this, we generated pancreas-specific 12-LO knockout mice and studied their metabolic and molecular adaptations to high-fat diets. Whereas knockout mice and control littermates displayed identical weight gain, body fat distribution, and macrophage infiltration into fat, knockout mice exhibited greater adaptive islet hyperplasia, improved insulin secretion, and complete protection from dysglycemia. At the molecular level, 12-LO deletion resulted in increases in islet antioxidant enzymes Sod1 and Gpx1 in response to high-fat feeding. The absence or inhibition of 12-LO led to increases in nuclear Nrf2, a transcription factor responsible for activation of genes encoding antioxidant enzymes. Our data reveal a novel pathway in which islet 12-LO suppresses antioxidant enzymes and prevents the adaptive islet responses in the setting of high-fat diets.

Assessment of re‐aggregated human pancreatic islets for secondary drug screening

Insulin secretion from isolated pancreatic islets is a pivotal assay in developing novel insulin secretagogues, given its good correlation with in vivo efficacy. Because the supply of human islets is limited, this assay is typically run with rodent islets, which do not address species differences and are low-throughput, because of the size matching or volume normalization required. Here we have evaluated the suitability of human re-aggregated islets for this assay. We generated re-aggregated human islets of a consistent size, using micromolds and compared their responses with those of native human and rat islets, to known secretagogues and inhibitors of insulin release. Insulin secretion from rat islets, human islets and human re-aggregated cell clusters was concentration-dependently increased by glucose. The calcium channel agonist, Bay K 8644, stimulated insulin secretion in native rat islets and human re-aggregated islets, but not native human islets. Glibenclamide and tolbutamide were more effective and potent in re-aggregated human clusters compared with the other two preparations. Rat islets outperformed both human preparations of islets in response to caffeine, carbachol and glucagon-like peptide-1. Re-aggregated human islet clusters were more sensitive to somatostatin, diazoxide and sodium azide, but rodent islets were more sensitive to nifedipine. Human re-aggregated clusters of islet cells, of a constant size were more responsive to all compounds tested than native human islets. Importantly, the assay variability was less in the re-aggregated cluster preparations, which suggests that such re-aggregated cells could be useful for drug development.

CNX-011-67, a novel GPR40 agonist, enhances glucose responsiveness, insulin secretion and islet insulin content in n-STZ rats and in islets from type 2 diabetic patients

BackgroundGPR40 is a G-protein coupled receptor regulating free fatty acid induced and also glucose induced insulin secretion. We generated neonatally-streptozotocin-treated female rats (n-STZ) and treated them with CNX-011-67, a GPR40 agonist to examine the role of GPR40 in modulation of glucose metabolism, insulin secretion and content.MethodsFemale n-STZ animals were orally administered with CNX-011-67 (15 mg/kg body weight, twice daily) or with vehicle for 8 weeks (n = 8 per group). Glucose tolerance in treated animals and insulin secretion, islet insulin content and gene expression in isolated islets were determined. Islets from type 2 diabetic mellitus (T2DM) patients were treated with different concentrations of glucose in presence or absence of CNX-011-67 and insulin secretion was measured.ResultsTreatment of n-STZ rats with GPR40 agonist CNX-011-67 enhanced insulin secretion in response to oral glucose load on day 0 and this response persisted during the treatment period. The treatment also produced a ‘memory effect’ during which insulin secretion in response to oral glucose load remained enhanced, for a week, even in absence of the agonist. Activation of GPR40 enhanced responsiveness of islets to glucose and increased glucose induced insulin secretion and islet insulin content. An increase in islet mRNA expression of GCK, PDX1, insulin and PC was also observed. Acute treatment of islets from n-STZ rats with GPR40 agonist enhanced cellular ATP content. Activation of GPR40 enhanced mitochondrial calcium level in NIT-1 insulinoma cells. CNX-011-67 increased insulin secretion in islets from T2DM patients which were non-responsive to increased glucose concentrationConclusionsOur data provide evidence that activation of GPR40 with CNX-011-67 stimulates glucose metabolism, enhances glucose responsiveness, increases insulin secretion and content and that pharmacological activation of GPR40 will prove beneficial for treatment of T2DM.

The proapoptotic BH3-only proteins Bim and Puma are downstream of endoplasmic reticulum and mitochondrial oxidative stress in pancreatic islets in response to glucotoxicity

Apoptosis of pancreatic beta cells is a feature of type 2 diabetes and its prevention may have therapeutic benefit. High glucose concentrations induce apoptosis of islet cells, and this requires the proapoptotic Bcl-2 homology domain 3 (BH3)-only proteins Bim and Puma. We studied the stress pathways induced by glucotoxicity in beta cells that result in apoptosis. High concentrations of glucose or ribose increased expression of the transcription factor CHOP (C/EBP homologous protein) but not endoplasmic reticulum (ER) chaperones, indicating activation of proapoptotic ER stress signaling. Inhibition of ER stress prevented ribose-induced upregulation of Chop and Puma mRNA, and partially protected islets from glucotoxicity. Loss of Bim or Puma partially protected islets from the canonical ER stressor thapsigargin. The antioxidant N-acetyl-cysteine also partially protected islets from glucotoxicity. Islets deficient in both Bim and Puma, but not Bim or Puma alone, were significantly protected from killing induced by the mitochondrial reactive oxygen species donor rotenone. Our data demonstrate that high concentrations of glucose induce ER and oxidative stress, which causes cell death mediated by Bim and Puma. We observed significantly higher Bim and Puma mRNA in islets of human donors with type 2 diabetes. This indicates that inhibition of Bim and Puma, or their inducers, may prevent beta-cell destruction in type 2 diabetes.

Physical Exercise Introduced After Weaning Enhances Pancreatic Islet Responsiveness to Glucose and Potentiating Agents in Adult MSG-Obese Rats

Physical exercise represents an alternative way to prevent and/or ameliorate chronic metabolic diseases. Disruption of sympathetic nervous system (SNS) activity contributes to adiposity in obese subjects. Here, we verified the preventive effect of swimming training upon adiposity, adrenal catecholamine storage, and pancreatic islet function in obese monosodium glutamate (MSG)-treated rats. Male neonatal Wistar rats received MSG (4 mg/g body weight) during the first 5 days of life and, at weaning, half of the rats were submitted to swimming training, 30 min/day, 3 days a week, until 90 days of age (exercised rats: MSGex). Half of the rats were used as controls (sedentary group, MSGsd). Exercise training (ET) decreased insulinemia and fat deposition in MSGex, and increased adrenal catecholamine content, compared with MSGsd rats. Insulinemia during the ivGTT was lower in MSGex rats, despite a lack of difference in glycemia. Swimming training enhanced insulin release in islets challenged by 2.8-8.3 mmol/l glucose, whereas, at supraphysiological glucose concentrations (11.1-16.7 mmol/l), MSGex islets secreted less insulin than MSGsd. No differences in insulin secretion were observed following l-arginine (Arg) or K(+) stimuli. In contrast, islets from MSGex rats secreted more insulin when exposed to carbachol (100 μmol/l), forskolin (10 μmol/l), or IBMX (1 mmol/l) at 8.3 mmol/l glucose. Additionally, MSGex islets presented a better epinephrine inhibition upon insulin release. These results demonstrate that ET prevented the onset of obesity in MSG rats, probably by enhancing adrenal catecholamine levels. ET ameliorates islet responsiveness to several compounds, as well as insulin peripheral action.

Glucose-stimulated Single Pancreatic Islets Sustain Increased Cytosolic ATP Levels during Initial Ca2+ Influx and Subsequent Ca2+ Oscillations

In pancreatic islets, insulin secretion occurs via synchronous elevation of Ca(2+) levels throughout the islets during high glucose conditions. This Ca(2+) elevation has two phases: a quick increase, observed after the glucose stimulus, followed by prolonged oscillations. In these processes, the elevation of intracellular ATP levels generated from glucose is assumed to inhibit ATP-sensitive K(+) channels, leading to the depolarization of membranes, which in turn induces Ca(2+) elevation in the islets. However, little is known about the dynamics of intracellular ATP levels and their correlation with Ca(2+) levels in the islets in response to changing glucose levels. In this study, a genetically encoded fluorescent biosensor for ATP and a fluorescent Ca(2+) dye were employed to simultaneously monitor the dynamics of intracellular ATP and Ca(2+) levels, respectively, inside single isolated islets. We observed rapid increases in cytosolic and mitochondrial ATP levels after stimulation with glucose, as well as with methyl pyruvate or leucine/glutamine. High ATP levels were sustained as long as high glucose levels persisted. Inhibition of ATP production suppressed the initial Ca(2+) increase, suggesting that enhanced energy metabolism triggers the initial phase of Ca(2+) influx. On the other hand, cytosolic ATP levels did not fluctuate significantly with the Ca(2+) level in the subsequent oscillation phases. Importantly, Ca(2+) oscillations stopped immediately before ATP levels decreased significantly. These results might explain how food or glucose intake evokes insulin secretion and how the resulting decrease in plasma glucose levels leads to cessation of secretion.

Rotavirus acceleration of murine type 1 diabetes is associated with increased MHC class I-restricted antigen presentation by B cells and elevated proinflammatory cytokine expression by T cells

Rotavirus infection has been proposed to enhance progression towards type 1 diabetes in at-risk children. Rhesus monkey rotavirus (RRV) accelerates diabetes onset in non-obese diabetic (NOD) and T cell receptor transgenic NOD8.3 mice. Infected NOD mice show virus spread to pancreatic lymph nodes (PLN) and mesenteric lymph nodes (MLN), induction of a serum T helper 1-biased specific antibody response and proinflammatory cytokine mRNA expression in PLN and islets. Here, we analysed the effects of RRV infection on intestinal responses and the activation of antigen presenting cells (APC), T cells and B cells in PLN, MLN, spleen and islets. Diabetes acceleration by RRV was associated with minimal immune activation in Peyer's patches. Increased proinflammatory cytokine expression by APC, including dendritic cells, was observed exclusively in the PLN, while cytokine expression by T cells was detected in islets, PLN, MLN and spleen. RRV infection of NOD8.3 mice increased IFNγ expression by CD8+ T cells, which primarily recognise an islet autoantigen. A peptide corresponding to RRV VP7 amino acids 5–13, with sequence similarity to this islet autoantigen, did not induce activation or proliferation of NOD8.3 mouse T cells. RRV infection of NOD mice elevated B cell MHC I expression in PLN and MLN, and increased the B cell-mediated proliferation of islet antigen-specific CD8+ T cells. These studies demonstrate that RRV infection of NOD mice activates APC, T cells and B cells at sites where autoreactive lymphocytes accumulate, in association with proinflammatory cytokine expression and an increased capacity to present antigen. Taken together with previous findings, these data support a possible role for bystander activation in type 1 diabetes acceleration by RRV.

High Glucose Represses β-Klotho Expression and Impairs Fibroblast Growth Factor 21 Action in Mouse Pancreatic Islets

Circulating fibroblast growth factor 21 (FGF21) levels are elevated in diabetic subjects and correlate directly with abnormal glucose metabolism, while pharmacologically administered FGF21 can ameliorate hyperglycemia. The pancreatic islet is an FGF21 target, yet the actions of FGF21 in the islet under normal and diabetic conditions are not fully understood. This study investigated the effects of high glucose on islet FGF21 actions in a diabetic mouse model by investigating db/db mouse islet responses to exogenous FGF21, the direct effects of glucose on FGF21 signaling, and the involvement of peroxisome proliferator–activated receptor γ (PPARγ) in FGF21 pathway activation. Results showed that both adult db/db mouse islets and normal islets treated with high glucose ex vivo displayed reduced β-klotho expression, resistance to FGF21, and decreased PPARγ expression. Rosiglitazone, an antidiabetic PPARγ ligand, ameliorated these effects. Our data indicate that hyperglycemia in type 2 diabetes mellitus may lead to FGF21 resistance in pancreatic islets, probably through reduction of PPARγ expression, which provides a novel mechanism for glucose-mediated islet dysfunction.

The possible mechanisms by which maternal hypothyroidism impairs insulin secretion in adult male offspring in rats

Previous studies have recently shown that maternal hypothyroidism leads to impaired glucose metabolism and reduced insulin secretion in adult offspring in rats. The aim of this study was to locate the defect in the insulin secretion pathway induced by maternal hypothyroidism. Pregnant Wistar rats were divided into two groups; the control group consumed water, while the hypothyroid (FH) group received water containing 0.025% 6-propyl-2-thiouracil during gestation. An intravenous glucose tolerance test was carried out on 5-month-old male offspring. In in vitro studies, the effects of various secretagogues and inhibitors acting at different levels of the insulin secretion cascade were investigated, and insulin content, insulin secretion and glucokinase activity of the islets were compared. Although insulin content of the FH islets did not differ from that of control islets, insulin secretion from FH islets was reduced when it was challenged by glucose or arginine. Compared with control islets, activities of both hexokinase and glucokinase were also significantly decreased in the FH islets. Although, in both groups, increasing glibenclamide and nifedipine concentrations in the presence of 16.7 mmol l(-1) glucose increased and decreased insulin secretion, respectively, the percentage of changes in secretion of FH islets was significantly lower compared with control islets. The response of FH islets to high extracellular potassium concentration and diazoxide was also significantly lower than that of the control islets. These findings demonstrate that impaired insulin secretion in the FH group is probably related to alterations in different steps of the insulin secretion pathway and not in the insulin pool of β-cells.

ER stress in rodent islets of Langerhans is concomitant with obesity and β-cell compensation but not with β-cell dysfunction and diabetes

Objective:The objective of this study was to determine whether ER stress correlates with β-cell dysfunction in obesity-associated diabetes.Methods:Quantitative RT-PCR and western blot analysis were used to investigate changes in the expression of markers of ER stress, the unfolded protein response (UPR) and β-cell function in islets isolated from (1) non-diabetic Zucker obese (ZO) and obese female Zucker diabetic fatty (fZDF) rats compared with their lean littermates and from (2) high-fat-diet-fed fZDF rats (HF-fZDF), to induce diabetes, compared with age-matched non-diabetic obese fZDF rats.Results:Markers of an adaptive ER stress/UPR and β-cell function are elevated in islets isolated from ZO and fZDF rats compared with their lean littermates. In islets isolated from HF-fZDF rats, there was no significant change in the expression of markers of ER stress compared with age matched, obese, non-diabetic fZDF rats.Conclusions:These results provide evidence that obesity-induced activation of the UPR is an adaptive response for increasing the ER folding capacity to meet the increased demand for insulin. As ER stress is not exacerbated in high-fat-diet-induced diabetes, we suggest that failure of the islet to mount an effective adaptive UPR in response to an additional increase in insulin demand, rather than chronic ER stress, may ultimately lead to β-cell failure and hence diabetes.

Signaling in Insulin-Secreting MIN6 Pseudoislets and Monolayer Cells

Cell-cell interactions are of fundamental importance for cellular function. In islets of Langerhans, which control blood glucose levels by secreting insulin in response to the blood glucose concentration, the secretory response of intact islets is higher than that of insulin-producing beta-cells not arranged in the islet architecture. The objective was to define mechanisms by which cellular performance is enhanced when cells are arranged in three-dimensional space. The task was addressed by making a comprehensive analysis based on protein expression patterns generated from insulin-secreting MIN6 cells grown as islet-like clusters, so-called pseudoislets, and in monolayers. After culture, glucose-stimulated insulin secretion (GSIS) was measured from monolayers and pseudoislets. GSIS rose 6-fold in pseudoislets but only 3-fold in monolayers when the glucose concentration was increased from 2 to 20 mmol/L. Proteins from pseudoislets and monolayers were extracted and analyzed by liquid-chromatography mass spectrometry, and differentially expressed proteins were mapped onto KEGG pathways. Protein profiling identified 1576 proteins, which were common to pseudoislets and monolayers. When mapped onto KEGG pathways, 11 highly enriched pathways were identified. On the basis of differences in expression of proteins belonging to the pathways in pseudoislets and monolayers, predictions of differential pathway activation were performed. Mechanisms enhancing insulin secretory capacity of the beta-cell, when situated in the islet, include pathways regulating glucose metabolism, cell interaction, and translational regulation.

Impaired Ca2+ Signaling in β-Cells Lacking Leptin Receptors by Cre-loxP Recombination

Obesity is a major risk factor for diabetes and is typically associated with hyperleptinemia and a state of leptin resistance. The impact of chronically elevated leptin levels on the function of insulin-secreting β-cells has not been elucidated. We previously generated mice lacking leptin signaling in β-cells by using the Cre-loxP strategy and showed that these animals develop increased body weight and adiposity, hyperinsulinemia, impaired glucose-stimulated insulin secretion and insulin resistance. Here, we performed several in vitro studies and observed that β-cells lacking leptin signaling in this model are capable of properly metabolizing glucose, but show impaired intracellular Ca2+ oscillations and lack of synchrony within the islets in response to glucose, display reduced response to tolbutamide and exhibit morphological abnormalities including increased autophagy. Defects in intracellular Ca2+ signaling were observed even in neonatal islets, ruling out the possible contribution of obesity to the β-cell irregularities observed in adults. In parallel, we also detected a disrupted intracellular Ca2+ pattern in response to glucose and tolbutamide in control islets from adult transgenic mice expressing Cre recombinase under the rat insulin promoter, despite these animals being glucose tolerant and secreting normal levels of insulin in response to glucose. This unexpected observation impeded us from discerning the consequences of impaired leptin signaling as opposed to long-term Cre expression in the function of insulin-secreting cells. These findings highlight the need to generate improved Cre-driver mouse models or new tools to induce Cre recombination in β-cells.

GPR40 protein levels are crucial to the regulation of stimulated hormone secretion in pancreatic islets. Lessons from spontaneous obesity-prone and non-obese type 2 diabetes in rats

The role of islet GPR40 protein in the pathogenesis of diabetes is unclear. We explored the influence of GPR40 protein levels on hormone secretion in islets from two rat models of spontaneous type 2 diabetes displaying either hyperlipidaemia or hyperglycaemia.GPR40 expression was analysed by confocal microscopy, Western blot and qPCR in islets from preobese Zucker (fa/fa) rats, diabetic Goto-Kakizaki (GK) rats, and controls.Confocal microscopy of control islets showed expression of GPR40 protein in insulin, glucagon and somatostatin cells. GPR40 expression was strongly increased in islets of hyperlipidaemic fa/fa rats and coincided with a concentration-related increase in palmitate-induced release of insulin and glucagon and its inhibition of somatostatin release. Conversely, hyperglycaemic GK islets displayed an extremely faint expression of GPR40 as did high-glucose-cultured control islets. This was reflected in abolished palmitate-induced hormone response in GK islets and high-glucose-cultured control islets. The palmitate antagonist rosiglitazone promoted reappearance of GPR40 in high-glucose-cultured islets and served as partial agonist in glucose-stimulated insulin release.GPR40 protein is abundantly expressed in pancreatic islets and modulates stimulated hormone secretion. Mild hyperlipidaemia in obesity-prone diabetes creates increased GPR40 expression and increased risk for an exaggerated palmitate-induced insulin response and lipotoxicity, a metabolic situation suitable for GPR40 antagonist treatment. Chronic hyperglycaemia creates abrogated GPR40 expression and downregulated insulin release, a metabolic situation suitable for GPR40 agonist treatment to avoid glucotoxicity. GPR40 protein is interactively modulated by both free fatty acids and glucose and is a promising target for pharmacotherapy in different variants of type 2 diabetes.

Swim training restores glucagon-like peptide-1 insulinotropic action in pancreatic islets from monosodium glutamate-obese rats

Glucagon-like peptide-1 (GLP-1) is an important modulator of insulin secretion by endocrine pancreas. In the present study, we investigated the effect of swim training on GLP-1 insulinotropic action in pancreatic islets from monosodium glutamate (MSG)-obese rats. Obesity was induced by neonatal MSG administration. MSG-obese and control (CON) exercised rats swam for 30 min (3 times week(-1) ) for 10 weeks. Pancreatic islets were isolated by colagenase technique and incubated with low (5.6 mM) or high (16.7 mM) glucose concentrations in the presence or absence of GLP-1 (10 nM). In addition, GLP-1 gene expression in ileum was quantified in fasting and glucose conditions. Exercise reduced obesity and hyperinsulinemia in MSG-obese rats. Swim training also inhibited glucose-induced insulin secretion in islets from both groups. Islets from MSG-obese rats maintained GLP-1 insulinotropic response in low glucose concentration. In contrast, in the presence of high glucose concentration, GLP-1 insulinotropic action was absent in islets from MSG-obese rats. Islets from MSG-exercised rats showed reduced GLP-1 insulinotropic action in the presence of low glucose. However, in high glucose concentration swim training restored GLP-1 insulinotropic response in islets from MSG-obese rats. In all groups, glucose intake increased GLP-1 immunoreactivity and gene expression in ileum cells in relation to fasting conditions. Swim training reduced these parameters only in ileum cells from CON-exercised rats. Neither MSG treatment nor exercise affected GLP-1 expression in the ileum. Exercise avoids insulin hypersecretion restoring GLP-1's insulinotropic action in pancreatic islets from MSG-obese rats.

Chronic glucolipotoxic conditions in pancreatic islets impair insulin secretion due to dysregulated calcium dynamics, glucose responsiveness and mitochondrial activity

BackgroundIn the progression towards diabetes, glucolipotoxicity is one of the main causes of pancreatic beta cell pathology. The aim of this study was to examine the in vitro effects of chronic glucolipotoxic conditions on cellular responses in pancreatic islets, including glucose and fat metabolism, Calcium mobilization, insulin secretion and insulin content.ResultsExposure of islets to chronic glucolipotoxic conditions decreased glucose stimulated insulin secretion in vitro. Reduced protein levels of Glut2/slc2a2, and decreased glucokinase and pyruvate carboxylase mRNA levels indicated a significant lowering in glucose sensing. Concomitantly, both fatty acid uptake and triglyceride accumulation increased significantly while fatty acid oxidation decreased. This general suppression in glucose metabolism correlated well with a decrease in mitochondrial number and activity, reduction in cellular ATP content and dampening of the TCA cycle. Further, we also observed a decrease in IP3 levels and lower Calcium mobilization in response to glucose. Importantly, chronic glucolipotoxic conditions in vitro decreased insulin gene expression, insulin content, insulin granule docking (to the plasma membrane) and insulin secretion.ConclusionsOur results present an integrated view of the effects of chronic glucolipotoxic conditions on known and novel signaling events, in vitro, that results in reduced glucose responsiveness and insulin secretion.

Induction of an antiviral state and attenuated coxsackievirus replication in type III interferon-treated primary human pancreatic islets.

Type III interferons (IFNs), also called lambda interferons (IFN-λ), comprise three isoforms, IFN-λ1 (interleukin-29 [IL-29]), IFN-λ2 (IL-28A), and IFN-λ3 (IL-28B). Only limited information is available on their expression and biological functions in humans. Type I and type II IFNs protect human pancreatic islets against coxsackievirus infection, and this is important since such viruses have been proposed to play a role in the development of human type 1 diabetes. Here we investigated whether type III IFN is expressed during infection of human islet cells with coxsackievirus and if type III IFN regulates permissiveness to such infections. We show that human islets respond to a coxsackievirus serotype B3 (CVB3) infection by inducing the expression of type III IFNs. We also demonstrate that islet endocrine cells from nondiabetic individuals express the type III IFN receptor subunits IFN-λR1 and IL-10R2. Pancreatic alpha cells express both receptor subunits, while pancreatic beta cells express only IL-10R2. Type III IFN stimulation elicited a biological response in human islets as indicated by the upregulated expression of antiviral genes as well as pattern recognition receptors. We also show that type III IFN significantly reduces CVB3 replication. Our studies reveal that type III IFNs are expressed during CVB3 infection and that the expression of the type III IFN receptor by the human pancreatic islet allows this group of IFNs to regulate the islets' permissiveness to infection. Our novel observations suggest that type III IFNs may regulate viral replication and thereby contribute to reduced tissue damage and promote islet cell survival during coxsackievirus infection.

Failure of the Adaptive Unfolded Protein Response in Islets of Obese Mice Is Linked With Abnormalities in β-Cell Gene Expression and Progression to Diabetes

The normal β-cell response to obesity-associated insulin resistance is hypersecretion of insulin. Type 2 diabetes develops in subjects with β-cells that are susceptible to failure. Here, we investigated the time-dependent gene expression changes in islets of diabetes-prone db/db and diabetes-resistant ob/ob mice. The expressions of adaptive unfolded protein response (UPR) genes were progressively induced in islets of ob/ob mice, whereas they declined in diabetic db/db mice. Genes important for β-cell function and maintenance of the islet phenotype were reduced with time in db/db mice, whereas they were preserved in ob/ob mice. Inflammation and antioxidant genes displayed time-dependent upregulation in db/db islets but were unchanged in ob/ob islets. Treatment of db/db mouse islets with the chemical chaperone 4-phenylbutyric acid partially restored the changes in several β-cell function genes and transcription factors but did not affect inflammation or antioxidant gene expression. These data suggest that the maintenance (or suppression) of the adaptive UPR is associated with β-cell compensation (or failure) in obese mice. Inflammation, oxidative stress, and a progressive loss of β-cell differentiation accompany diabetes progression. The ability to maintain the adaptive UPR in islets may protect against the gene expression changes that underlie diabetes development in obese mice.

The complex effects of cannabinoids on insulin secretion from rat isolated islets of Langerhans

Recent interest in the endocrine pancreas has revealed the presence of a functional endocannabinoid system in pancreatic islets, however, the effects of endocannabinoids and cannabinoid CB receptor activation on downstream signalling and on insulin release still remains unclear. In the current study, a variety of purported cannabinoid CB receptor agonists and antagonists were evaluated for their effects on insulin secretion. In fresh rat isolated islets, the endocannabinoid anandamide caused a glucose-dependent, concentration-dependent inhibition of insulin release, with two populations of islets being identified based on their sensitivity to anandamide. Methanandamide (a non-hydrolysable analogue of anandamide) elicited similar inhibition of insulin secretion, comparable to the responses obtained with anandamide-sensitive islets, suggesting that the islet responsiveness may be due to differences in local metabolism of anandamide. The antagonists O-2050 (CB1) and AM630 (CB2) failed to reveal the involvement of cannabinoid receptors in the inhibitory activity of anandamide on insulin release. Inhibition of fatty acid amide hydrolase (FAAH) with URB597 did not alter basal or glucose-induced insulin secretion, suggesting that endogenous islet endocannabinoids do not affect insulin release, or that islet FAAH content is low. URB597 also failed to affect the inhibitory actions of anandamide on insulin release in fresh isolated islets. However, in islets following overnight culture, anandamide caused augmentation of basal and glucose-mediated insulin release. The effects of cannabinoid agents on insulin secretion described in this study does not identify a precise mode of action but points to important modulation which may be dependent on local metabolism and prevailing cellular conditions.