Evidence against a role for NLRP3-driven islet inflammation in db/db mice.

OBJECTIVES: Type 2 diabetes (T2D) is associated with chronic, low grade inflammation. Activation of the NLRP3 inflammasome and secretion of its target interleukin-1ß (IL-1ß) have been implicated in pancreatic ß cell failure in T2D. Specific targeting of the NLRP3 inflammasome to prevent pancreatic ß cell death could allow for selective T2D treatment without compromising all IL-1ß-associated immune responses. We hypothesized that treating a mouse model of T2D with MCC950, a compound that specifically inhibits NLRP3, would prevent pancreatic ß cell death, thereby preventing the onset of T2D. METHODS: Diabetic db/db mice were treated with MCC950 via drinking water for 8 weeks from 6 to 14 weeks of age, a period over which they developed pancreatic ß cell failure. We assessed metabolic parameters such as body composition, glucose tolerance, or insulin secretion over the course of the intervention. RESULTS: MCC950 was a potent inhibitor of NLRP3-induced IL-1ß in vitro and was detected at high levels in the plasma of treated db/db mice. Treatment of pre-diabetic db/db mice with MCC950, however, did not prevent pancreatic dysfunction and full onset of the T2D pathology. When examining the NLRP3 pathway in the pancreas of db/db mice, we could not detect an activation of this pathway nor increased levels of its target IL-1ß. CONCLUSIONS: NLRP3 driven-pancreatic IL-1ß inflammation does not play a key role in the pathogenesis of the db/db murine model of T2D.

Glucose regulation of islet stress responses and beta-cell failure in type 2 diabetes.

Pancreatic beta-cells exposed to high glucose concentrations display altered gene expression, function, survival and growth that may contribute to the slow deterioration of the functional beta-cell mass in type 2 diabetes. These glucotoxic alterations may result from various types of stress imposed by the hyperglycaemic environment, including oxidative stress, endoplasmic reticulum stress, cytokine-induced apoptosis and hypoxia. The glucose regulation of oxidative stress-response and integrated stress-response genes in cultured rat islets follows an asymmetric V-shaped profile parallel to that of beta-cell apoptosis, with a large increase at low glucose and a moderate increase at high vs. intermediate glucose concentrations. These observations suggest that both types of stress could play a role in the alteration of the functional beta-cell mass under states of prolonged hypoglycaemia and hyperglycaemia. In addition, beta-cell demise under glucotoxic conditions may also result from beta-cell hypoxia and, in vivo, from their exposure to inflammatory cytokines released locally by non-endocrine islet cells. A better understanding of the relative contribution of each type of stress to beta-cell glucotoxicity and of their pathophysiological cause in vivo may lead to new therapeutic strategies to prevent the slow deterioration of the functional beta-cell mass in glucose intolerant and type 2 diabetic patients.

Cluster analysis of rat pancreatic islet gene mRNA levels after culture in low-, intermediate- and high-glucose concentrations.

AIMS/HYPOTHESIS: Survival and function of insulin-secreting pancreatic beta cells are markedly altered by changes in nutrient availability. In vitro, culture in 10 rather than 2 mmol/l glucose improves rodent beta cell survival and function, whereas glucose concentrations above 10 mmol/l are deleterious. METHODS: To identify the mechanisms of such beta cell plasticity, we tested the effects of 18 h culture at 2, 5, 10 and 30 mmol/l glucose on the transcriptome of rat islets pre-cultured for 1 week at 10 mmol/l glucose using Affymetrix Rat 230 2.0 arrays. RESULTS: Culture in either 2-5 or 30 mmol/l instead of 10 mmol/l glucose markedly impaired beta cell function, while little affecting cell survival. Of about 16,000 probe-sets reliably detected in islets, some 5,000 were significantly up- or downregulated at least 1.4-fold by glucose. Analysis of these probe-sets with GeneCluster software identified ten mRNA profiles with unidirectional up- or downregulation between 2 and 10, 2 and 30, 5 and 10, 5 and 30 or 10 and 30 mmol/l glucose. It also identified eight complex V-shaped or inverse V-shaped profiles with a nadir or peak level of expression in 5 or 10 mmol/l glucose. Analysis of genes belonging to these various clusters using Onto-express and GenMAPP software revealed several signalling and metabolic pathways that may contribute to induction of beta cell dysfunction and apoptosis after culture in low- or high- vs intermediate-glucose concentration. CONCLUSIONS/INTERPRETATION: We have identified 18 distinct mRNA profiles of glucose-induced changes in islet gene mRNA levels that should help understand the mechanisms by which glucose affects beta cell survival and function under states of chronic hypo- or hyperglycaemia.

Acute nutrient regulation of the unfolded protein response and integrated stress response in cultured rat pancreatic islets.

AIMS/HYPOTHESIS: Inadequate chaperone function relative to client protein load in the endoplasmic reticulum triggers an adaptive unfolded protein response (UPR), including the integrated stress response (ISR), the latter being also activated by other types of stresses. It is well established that pancreatic beta cells, which synthesise and secrete insulin upon nutrient stimulation, are markedly affected by pathological disruption or excessive activation of the UPR. However, whether and how physiological nutrient stimulation affects the beta cell UPR has been little investigated. MATERIALS AND METHODS: We compared the effects of increasing glucose concentrations and of endoplasmic reticulum Ca(2+) emptying with thapsigargin on the UPR (X-box binding protein [Xbp1] mRNA splicing and XBP1/activating transcription factor [ATF] 6-target gene expression) and ISR (eukaryotic translation initiation factor 2A phosphorylation, ATF4 protein levels and target gene expression) in isolated rat islets. RESULTS: Thapsigargin strongly increased both UPR and ISR. In comparison, glucose moderately increased the UPR between 5 and 30 mmol/l, but exerted complex effects on the ISR as follows: (1) marked reduction between 2 and 10 mmol/l; (2) moderate increase parallel to the UPR between 10 and 30 mmol/l. These glucose effects occurred within 2 h, were mimicked by other metabolic substrates, but were independent of changes in Ca(2+) influx or insulin secretion. Remarkably, attenuating the glucose stimulation of protein synthesis with a low concentration of cycloheximide prevented UPR activation but not ISR reduction by high glucose. CONCLUSIONS/INTERPRETATION: Nutrient stimulation acutely activates rat islet UPR in a manner dependent on protein synthesis, while exerting complex effects on the ISR. These effects may contribute to nutrient-induced maintenance of the beta cell phenotype.