Malin Fex

Common variant in MTNR1B associated with increased risk of type 2 diabetes and impaired early insulin secretion

Genome-wide association studies have shown that variation in MTNR1B (melatonin receptor 1B) is associated with insulin and glucose concentrations. Here we show that the risk genotype of this SNP predicts future type 2 diabetes (T2D) in two large prospective studies. Specifically, the risk genotype was associated with impairment of early insulin response to both oral and intravenous glucose and with faster deterioration of insulin secretion over time. We also show that the MTNR1B mRNA is expressed in human islets, and immunocytochemistry confirms that it is primarily localized in β cells in islets. Nondiabetic individuals carrying the risk allele and individuals with T2D showed increased expression of the receptor in islets. Insulin release from clonal β cells in response to glucose was inhibited in the presence of melatonin. These data suggest that the circulating hormone melatonin, which is predominantly released from the pineal gland in the brain, is involved in the pathogenesis of T2D. Given the increased expression of MTNR1B in individuals at risk of T2D, the pathogenic effects are likely exerted via a direct inhibitory effect on β cells. In view of these results, blocking the melatonin ligand-receptor system could be a therapeutic avenue in T2D.

Frataxin deficiency in pancreatic islets causes diabetes due to loss of beta cell mass

Diabetes is caused by an absolute (type 1) or relative (type 2) deficiency of insulin-producing beta cells. We have disrupted expression of the mitochondrial protein frataxin selectively in pancreatic beta cells. Mice were born healthy but subsequently developed impaired glucose tolerance progressing to overt diabetes mellitus. These observations were explained by impairment of insulin secretion due to a loss of beta cell mass in knockout animals. This phenotype was preceded by elevated levels of reactive oxygen species in knockout islets, an increased frequency of apoptosis, and a decreased number of proliferating beta cells. Hence, disruption of the frataxin gene in pancreatic beta cells causes diabetes following cellular growth arrest and apoptosis, paralleled by an increase in reactive oxygen species in islets. These observations might provide insight into the deterioration of beta cell function observed in different subtypes of diabetes in humans.

Common variant in MTNR1B associated with increased risk of type 2 diabetes and impaired early insulin secretion RID A-4476-2009

Genome-wide association studies have shown that variation in MTNR1B (melatonin receptor 1B) is associated with insulin and glucose concentrations. Here we show that the risk genotype of this SNP predicts future type 2 diabetes (T2D) in two large prospective studies. Specifically, the risk genotype was associated with impairment of early insulin response to both oral and intravenous glucose and with faster deterioration of insulin secretion over time. We also show that the MTNR1B mRNA is expressed in human islets, and immunocytochemistry confirms that it is primarily localized in β cells in islets. Nondiabetic individuals carrying the risk allele and individuals with T2D showed increased expression of the receptor in islets. Insulin release from clonal β cells in response to glucose was inhibited in the presence of melatonin. These data suggest that the circulating hormone melatonin, which is predominantly released from the pineal gland in the brain, is involved in the pathogenesis of T2D. Given the increased expression of MTNR1B in individuals at risk of T2D, the pathogenic effects are likely exerted via a direct inhibitory effect on β cells. In view of these results, blocking the melatonin ligand-receptor system could be a therapeutic avenue in T2D.

TCF7L2 is a master regulator of insulin production and processing

Genome-wide association studies have revealed >60 loci associated with type 2 diabetes (T2D), but the underlying causal variants and functional mechanisms remain largely elusive. Although variants in TCF7L2 confer the strongest risk of T2D among common variants by presumed effects on islet function, the molecular mechanisms are not yet well understood. Using RNA-sequencing, we have identified a TCF7L2-regulated transcriptional network responsible for its effect on insulin secretion in rodent and human pancreatic islets. ISL1 is a primary target of TCF7L2 and regulates proinsulin production and processing via MAFA, PDX1, NKX6.1, PCSK1, PCSK2 and SLC30A8, thereby providing evidence for a coordinated regulation of insulin production and processing. The risk T-allele of rs7903146 was associated with increased TCF7L2 expression, and decreased insulin content and secretion. Using gene expression profiles of 66 human pancreatic islets donors’, we also show that the identified TCF7L2-ISL1 transcriptional network is regulated in a genotype-dependent manner. Taken together, these results demonstrate that not only synthesis of proinsulin is regulated by TCF7L2 but also processing and possibly clearance of proinsulin and insulin. These multiple targets in key pathways may explain why TCF7L2 has emerged as the gene showing one of the strongest associations with T2D.

Apelin is a novel islet peptide

Apelin, a recently discovered peptide with wide tissue distribution, regulates feeding behavior, improves glucose utilization, and inhibits insulin secretion. We examined whether apelin is expressed in human islets, as well as in normal and type 2 diabetic (T2D) animal islets. Further, we studied islet apelin regulation and the effect of apelin on insulin secretion. Apelin expression and regulation was examined in human and animal specimens using immunocytochemistry, in situ hybridization, and real-time PCR. Insulin secretion was studied in INS-1 (832/13) clonal beta cells. APJ-receptor expression was studied using real-time PCR. In human and murine islets apelin was predominantly expressed in beta cells and alpha cells; a subpopulation of the PP cells in human islets also harbored apelin. In porcine and feline islets apelin was mainly expressed in beta cells. APJ-receptor expression was detected in INS-1 (832/13) cells, and in human and mouse islets. A high dose (1microM) of apelin-36 caused a moderate increase in glucose-stimulated insulin secretion (30%; p<0.001), while lower concentrations (10-100nM) of apelin robustly reduced insulin secretion by 50% (p<0.001). Apelin was upregulated in beta cells of T2D db/db mice (47% vs. controls; p<0.02) and GK-rats (74% vs. controls; p<0.002), but human islet apelin expression was unaffected by glucose. On the other hand, human islet apelin expression was diminished after culture in glucocorticoids (16% vs. controls; p<0.01). We conclude that apelin is a novel insulin-regulating islet peptide in humans and several laboratory animals. Islet apelin expression is negatively regulated by glucocorticoids, and upregulated in T2D animals. The presence of apelin receptors in islets suggests a role for apelin as a paracrine or autocrine messenger within the islets.

Rat insulin promoter 2-Cre recombinase mice bred onto a pure C57BL/6J background exhibit unaltered glucose tolerance

Beta-cell-specific gene targeting is a widely used tool when studying genes involved in beta-cell function. For this purpose, several conditional beta-cell knockouts have been generated using the rat insulin promoter 2-Cre recombinase (RIP2-Cre) mouse. However, it was recently observed that expression of Cre alone in beta-cells may affect whole body glucose homeostasis. Therefore, we investigated glucose homeostasis, insulin secretion, and beta-cell mass in our line of RIP2-Cre mice bred onto the C57BL/6J genetic background. We used 12- and 28-week-old female RIP2-Cre mice for analyses of insulin secretion in vitro, glucose homeostasis in vivo and beta-cell mass. Our mouse line has been backcrossed for 14 generations to yield a near 100% pure C57BL/6J background. We found that fasting plasma glucose and insulin levels were similar in both genotypes. An i.v. glucose tolerance test revealed no differences in glucose clearance and insulin secretion between 12-week-old RIP2-Cre and WT mice. Moreover, insulin secretion in vitro in islets isolated from 28-week-old RIP2-Cre mice and controls was similar. In addition, beta-cell mass was not different between the two genotypes at 28 weeks of age. In our experiments, we observed no differences in glucose tolerance, insulin secretion in vivo and in vitro, or in beta-cell mass between the genotypes. As our RIP2-Cre mice are on a near 100% pure genetic background (C57BL/6J), we suggest that the perturbations in glucose homeostasis previously reported in RIP2-Cre mouse lines can be accounted for by differences in genetic background.

Altered serotonin (5-HT) 1D and 2A receptor expression may contribute to defective insulin and glucagon secretion in human type 2 diabetes.

Islet produced 5-hydroxy tryptamine (5-HT) is suggested to regulate islet hormone secretion in a paracrine and autocrine manner in rodents. Hitherto, no studies demonstrate a role for this amine in human islet function, nor is it known if 5-HT signaling is involved in the development of beta cell dysfunction in type 2 diabetes (T2D). To clarify this, we performed a complete transcriptional mapping of 5-HT receptors and processing enzymes in human islets and investigated differential expression of these genes in non-diabetic and T2D human islet donors. We show the expression of fourteen 5-HT receptors as well as processing enzymes involved in the biosynthesis of 5-HT at the mRNA level in human islets. Two 5-HT receptors (HTR1D and HTR2A) were over-expressed in T2D islet donors. Both receptors (5-HT1d and 5-HT2a) were localized to human alpha, beta and delta cells. 5-HT inhibited both insulin and glucagon secretion in non-diabetic islet donors. In islets isolated from T2D donors the amine significantly increased release of insulin in response to glucose. Our results suggest that 5-HT signaling participates in regulation of overall islet hormone secretion in non- diabetic individuals and over-expression of HTR1D and HTR2A may either contribute to islet dysfunction in T2D or arise as a consequence of an already dysfunctional islet.

{beta}-Cell Lipases and Insulin Secretion.

Lipids have been implicated in β-cell stimulus-secretion coupling. Thus, lipases in β-cells would be required to generate coupling factors from intracellular lipids. Indeed, we found that glucose stimulates lipolysis in rodent islets and clonal β-cells. Lipolysis and diglyceride lipase activity in islets are abolished by orlistat, a pan-lipase inhibitor. Moreover, orlistat dose-dependently inhibits glucose- and forskolin-stimulated insulin secretion, while leaving glucose oxidation and the rise in ATP-to-ADP ratio intact. In an effort to identify β-cell lipase(s), we found that hormone-sensitive lipase (HSL), the rate-limiting enzyme for acylglyceride hydrolysis in adipocytes, is active in rodent β-cells. To further address the role of HSL, a global and β-cell–specific inactivation, respectively, of the lipase has been created in mice. Whereas our line of HSL null mice is moderately glucose intolerant due to reduced peripheral insulin sensitivity, it exhibits normal islet metabolism and insulin secretion. Preliminary analysis of the β-cell–specific HSL knockout has revealed no evidence for disturbed islet function. Thus, studies of ours and others indicate that there is a complex lipid regulatory component in β-cell stimulus-secretion coupling. The role of HSL and other lipases needs to be further clarified to provide a balanced view of the role of lipids and lipolysis in β-cells.

Autoimmunity against INS-IGF2 expressed in human pancreatic islets

Background: Islet INS-IGF2 was examined as a possible autoantigen in type 1 diabetes. Results: INS-IGF2 expression was inversely related to donor HbA1c and glucose-stimulated insulin release. Autoantibodies doubly reactive with INS-IGF2 and insulin were more common in patients with type 1 diabetes than controls. Conclusion: INS-IGF2 is recognized by autoantibodies in type 1 diabetes. Significance: Autoantibodies doubly reactive with both INS-IGF2 and insulin may contribute to type 1 diabetes. Insulin is a major autoantigen in islet autoimmunity and progression to type 1 diabetes. It has been suggested that the insulin B-chain may be critical to insulin autoimmunity in type 1 diabetes. INS-IGF2 consists of the preproinsulin signal peptide, the insulin B-chain, and eight amino acids of the C-peptide in addition to 138 amino acids from the IGF2 gene. We aimed to determine the expression of INS-IGF2 in human pancreatic islets and autoantibodies in newly diagnosed children with type 1 diabetes and controls. INS-IGF2, expressed primarily in beta cells, showed higher levels of expression in islets from normal compared with donors with either type 2 diabetes (p = 0.006) or high HbA1c levels (p < 0.001). INS-IGF2 autoantibody levels were increased in newly diagnosed patients with type 1 diabetes (n = 304) compared with healthy controls (n = 355; p < 0.001). Displacement with cold insulin and INS-IGF2 revealed that more patients than controls had doubly reactive insulin-INS-IGF2 autoantibodies. These data suggest that INS-IGF2, which contains the preproinsulin signal peptide, the B-chain, and eight amino acids of the C-peptide may be an autoantigen in type 1 diabetes. INS-IGF2 and insulin may share autoantibody-binding sites, thus complicating the notion that insulin is the primary autoantigen in type 1 diabetes.

Serotonin (5-HT) receptor 2b activation augments glucose-stimulated insulin secretion in human and mouse islets of Langerhans.

Aims/hypothesisThe Gq-coupled 5-hydroxytryptamine 2B (5-HT2B) receptor is known to regulate the proliferation of islet beta cells during pregnancy. However, the role of serotonin in the control of insulin release is still controversial. The aim of the present study was to explore the role of the 5-HT2B receptor in the regulation of insulin secretion in mouse and human islets, as well as in clonal INS-1(832/13) cells.MethodsExpression of HTR2B mRNA and 5-HT2B protein was examined with quantitative real-time PCR, RNA sequencing and immunohistochemistry. α-Methyl serotonin maleate salt (AMS), a serotonin receptor agonist, was employed for robust 5-HT2B receptor activation. Htr2b was silenced with small interfering RNA in INS-1(832/13) cells. Insulin secretion, Ca2+ response and oxygen consumption rate were determined.ResultsImmunohistochemistry revealed that 5-HT2B is expressed in human and mouse islet beta cells. Activation of 5-HT2B receptors by AMS enhanced glucose-stimulated insulin secretion (GSIS) in human and mouse islets as well as in INS-1(832/13) cells. Silencing Htr2b in INS-1(832/13) cells led to a 30% reduction in GSIS. 5-HT2B receptor activation produced robust, regular and sustained Ca2+ oscillations in mouse islets with an increase in both peak distance (period) and time in the active phase as compared with control. Enhanced insulin secretion and Ca2+ changes induced by AMS coincided with an increase in oxygen consumption in INS-1(832/13) cells.Conclusions/interpretationActivation of 5-HT2B receptors stimulates GSIS in beta cells by triggering downstream changes in cellular Ca2+ flux that enhance mitochondrial metabolism. Our findings suggest that serotonin and the 5-HT2B receptor stimulate insulin release.