MT1 and MT2 melatonin receptors are expressed in nonoverlapping neuronal populations.

Melatonin (MLT) exerts its physiological effects principally through two high-affinity membrane receptors MT1 and MT2. Understanding the exact mechanism of MLT action necessitates the use of highly selective agonists/antagonists to stimulate/inhibit a given MLT receptor. The respective distribution of MT1 and MT2 within the CNS and elsewhere is controversial, and here we used a "knock-in" strategy replacing MT1 or MT2 coding sequences with a LacZ reporter. The data show striking differences in the distribution of MT1 and MT2 receptors in the mouse brain: whereas the MT1 subtype was expressed in very few structures (notably including the suprachiasmatic nucleus and pars tuberalis), MT2 subtype receptors were identified within numerous brain regions including the olfactory bulb, forebrain, hippocampus, amygdala and superior colliculus. Co-expression of the two subtypes was observed in very few structures, and even within these areas they were rarely present in the same individual cell. In conclusion, the expression and distribution of MT2 receptors are much more widespread than previously thought, and there is virtually no correspondence between MT1 and MT2 cellular expression. The precise phenotyping of cells/neurons containing MT1 or MT2 receptor subtypes opens new perspectives for the characterization of links between MLT brain targets, MLT actions and specific MLT receptor subtypes.

TLR4 triggered complex inflammation in human pancreatic islets.

Type 2 Diabetes (T2D) is strongly associated with obesity and inflammation. Toll-like receptor-4 (TLR-4) is the major pro-inflammatory pathway with its ligands and downstream products increased systemically in T2D and in at-risk individuals. Detailed mechanisms of the complex proinflammatory response in pancreatic islets remain unknown. In isolated human islets LPS induced IL-1ß, IL-6, IL-8 and TNF production in a TLR4-dependent manner and severely impaired ß-cell survival and function. IL-6 antagonism improved ß-cell function. IL-8, which was identified specifically in α-cells, initiated monocyte migration, a process fully blocked by IL-8 neutralization. The TLR4 response was potentiated in obese donors; with higher IL-1ß, IL-6 and IL-8 expression than in non-obese donors. TLR4 activation leads to a complex multi-cellular inflammatory response in human islets, which involves ß-cell failure, cytokine production and macrophage recruitment to islets. In obesity, the amplified TLR4 response may potentiate ß-cell damage and accelerate diabetes progression.

Associations between hepatic miRNA expression, liver triacylglycerols and gut microbiota during metabolic adaptation to high-fat diet in mice.

AIMS/HYPOTHESIS: Despite the current pandemic of metabolic diseases, our understanding of the diverse nature of the development of metabolic alterations in people who eat a high-fat diet (HFD) is still poor. We recently demonstrated a cardio-metabolic adaptation in mice fed an HFD, which was characterised by a specific gut and periodontal microbiota profile. Since the severity of hepatic disease is characterised by specific microRNA (miRNA) signatures and the gut microbiota is a key driver of both hepatic disease and miRNA expression, we analysed the expression of three hepatic miRNA and studied their correlation with hepatic triacylglycerol content and gut microbiota. METHODS: Two cohorts of C57BL/6 4-week-old wild-type (WT) male mice (n = 62 and n = 96) were fed an HFD for 3 months to provide a model of metabolic adaptation. Additionally 8-week-old C57BL/6 mice, either WT or of different genotypes, with diverse gut microbiota (ob/ob, Nod1, Cd14 knockout [Cd14KO] and Nod2) or without gut microbiota (axenic mice) were fed a normal chow diet. Following which, glycaemic index, body weight, blood glucose levels and hepatic triacylglycerol levels were measured. Gut (caecum) microbiota taxa were analysed by pyrosequencing. To analyse hepatic miRNA expression, real-time PCR was performed on total extracted miRNA samples. Data were analysed using two-way ANOVA followed by the Dunnett's post hoc test, or by the unpaired Student's t test. A cluster analysis and multivariate analyses were also performed. RESULTS: Our results demonstrated that the expression of miR-181a, miR-666 and miR-21 in primary murine hepatocytes is controlled by lipopolysaccharide in a dose-dependent manner. Of the gut microbiota, Firmicutes were positively correlated and Proteobacteria and Bacteroides acidifaciens were negatively correlated with liver triacylglycerol levels. Furthermore, the relative abundance of Firmicutes was negatively correlated with hepatic expression of miR-666 and miR-21. In contrast, the relative abundance of B. acidifaciens was positively correlated with miR-21. CONCLUSIONS/INTERPRETATION: We propose the involvement of hepatic miRNA, liver triacylglycerols and gut microbiota as a new triad that underlies the molecular mechanisms by which gut microbiota governs hepatic pathophysiology during metabolic adaptation to HFD.

Deletion of apoptosis signal-regulating kinase 1 (ASK1) protects pancreatic beta-cells from stress-induced death but not from glucose homeostasis alterations under pro-inflammatory conditions.

BACKGROUND: Type 2 diabetes is characterized by pancreatic beta-cell dysfunction and is associated with low-grade inflammation. Recent observations suggest that apoptosis signal-regulating kinase 1 (ASK1) is involved in beta-cell death in response to different stressors. In this study, we tested whether ASK1 deficiency protects beta-cells from glucolipotoxic conditions and cytokines treatment or from glucose homeostasis alteration induced by endotoxemia. METHODOLOGY/PRINCIPAL FINDINGS: Insulin secretion was neither affected upon shRNA-mediated downregulation of ASK1 in MIN6 cells nor in islets from ASK1-deficient mice. ASK1 silencing in MIN6 cells and deletion in islets did not prevent the deleterious effect of glucolipotoxic conditions or cytokines on insulin secretion. However, it protected MIN6 cells from death induced by ER stress or palmitate and islets from short term caspase activation in response to cytokines. Moreover, endotoxemia induced by LPS infusion increased insulin secretion during hyperglycemic clamps but the response was similar in wild-type and ASK1-deficient mice. Finally, insulin sensitivity in the presence of LPS was not affected by ASK1-deficiency. CONCLUSIONS/SIGNIFICANCE: Our study demonstrates that ASK1 is not involved in beta-cell function and dysfunction but controls stress-induced beta-cell death.

TLR2/6 and TLR4-activated macrophages contribute to islet inflammation and impair beta cell insulin gene expression via IL-1 and IL-6.

AIMS/HYPOTHESIS: Inflammation contributes to pancreatic beta cell dysfunction in type 2 diabetes. Toll-like receptor (TLR)-2 and -4 ligands are increased systemically in recently diagnosed type 2 diabetes patients, and TLR2- and TLR4-deficient mice are protected from the metabolic consequences of a high-fat diet. Here we investigated the role of macrophages in TLR2/6- and TLR4-mediated effects on islet inflammation and beta cell function. METHODS: Genetic and pharmacological approaches were used to determine the effects of TLR2/6 and TLR4 ligands on mouse islets, human islets and purified rat beta cells. Islet macrophages were depleted and sorted by flow cytometry and the effects of TLR2/6- and TLR4-activated bone-marrow-derived macrophages (BMDMs) on beta cell function were assessed. RESULTS: Macrophages contributed to TLR2/6- and TLR4-induced islet Il1a/IL1A and Il1b/IL1B mRNA expression in mouse and human islets and IL-1ß secretion from human islets. TLR2/6 and TLR4 ligands also reduced insulin gene expression; however, this occurred in a non-beta cell autonomous manner. TLR2/6- and TLR4-activated BMDMs reduced beta cell insulin secretion partly via reducing Ins1, Ins2, and Pdx1 mRNA expression. Antagonism of the IL-1 receptor and neutralisation of IL-6 completely reversed the effects of activated macrophages on beta cell gene expression. CONCLUSIONS/INTERPRETATION: We conclude that islet macrophages are major contributors to islet IL-1ß secretion in response to TLR2/6 and TLR4 ligands. BMDMs stimulated with TLR2/6 and TLR4 ligands reduce insulin secretion from pancreatic beta cells, partly via IL-1ß- and IL-6-mediated decreased insulin gene expression.