HDAC7 is overexpressed in human diabetic islets and impairs insulin secretion in rat islets and clonal beta cells.

AIMS/HYPOTHESIS: Pancreatic beta cell dysfunction is a prerequisite for the development of type 2 diabetes. Histone deacetylases (HDACs) may affect pancreatic endocrine function and glucose homeostasis through alterations in gene regulation. Our aim was to investigate the role of HDAC7 in human and rat pancreatic islets and clonal INS-1 beta cells (INS-1 832/13). METHODS: To explore the role of HDAC7 in pancreatic islets and clonal beta cells, we used RNA sequencing, mitochondrial functional analyses, microarray techniques, and HDAC inhibitors MC1568 and trichostatin A. RESULTS: Using RNA sequencing, we found increased HDAC7 expression in human pancreatic islets from type 2 diabetic compared with non-diabetic donors. HDAC7 expression correlated negatively with insulin secretion in human islets. To mimic the situation in type 2 diabetic islets, we overexpressed Hdac7 in rat islets and clonal beta cells. In both, Hdac7 overexpression resulted in impaired glucose-stimulated insulin secretion. Furthermore, it reduced insulin content, mitochondrial respiration and cellular ATP levels in clonal beta cells. Overexpression of Hdac7 also led to changes in the genome-wide gene expression pattern, including increased expression of Tcf7l2 and decreased expression of gene sets regulating DNA replication and repair as well as nucleotide metabolism. In accordance, Hdac7 overexpression reduced the number of beta cells owing to enhanced apoptosis. Finally, we found that inhibiting HDAC7 activity with pharmacological inhibitors or small interfering RNA-mediated knockdown restored glucose-stimulated insulin secretion in beta cells that were overexpressing Hdac7. CONCLUSIONS/INTERPRETATION: Taken together, these results indicate that increased HDAC7 levels caused beta cell dysfunction and may thereby contribute to defects seen in type 2 diabetic islets. Our study supports HDAC7 inhibitors as a therapeutic option for the treatment of type 2 diabetes.

Identification of novel genes for glucose metabolism based upon expression pattern in human islets and effect on insulin secretion and glycemia.

Normal glucose homeostasis is characterized by appropriate insulin secretion and low HbA1c. Gene expression signatures associated with these two phenotypes could be essential for islet function and pathophysiology of type 2 diabetes (T2D). Herein, we employed a novel approach to identify candidate genes involved in T2D by correlating islet microarray gene expression data (78 donors) with insulin secretion and HbA1c level. The expression of 649 genes (P < 0.05) was correlated with insulin secretion and HbA1c. Of them, five genes (GLR1A, PPP1R1A, PLCDXD3, FAM105A and ENO2) correlated positively with insulin secretion/negatively with HbA1c and one gene (GNG5) correlated negatively with insulin secretion/positively with HbA1c were followed up. The five positively correlated genes have lower expression levels in diabetic islets, whereas GNG5 expression is higher. Exposure of human islets to high glucose for 24 h resulted in up-regulation of GNG5 and PPP1R1A expression, whereas the expression of ENO2 and GLRA1 was down-regulated. No effect was seen on the expression of FAM105A and PLCXD3. siRNA silencing in INS-1 832/13 cells showed reduction in insulin secretion for PPP1R1A, PLXCD3, ENO2, FAM105A and GNG5 but not GLRA1. Although no SNP in these gene loci passed the genome-wide significance for association with T2D in DIAGRAM+ database, four SNPs influenced gene expression in cis in human islets. In conclusion, we identified and confirmed PPP1R1A, FAM105A, ENO2, PLCDX3 and GNG5 as potential regulators of islet function. We provide a list of candidate genes as a resource for exploring their role in the pathogenesis of T2D.

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

AIMS/HYPOTHESIS: The 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. METHODS: Expression 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, Ca(2+) response and oxygen consumption rate were determined. RESULTS: Immunohistochemistry 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 Ca(2+) 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 Ca(2+) changes induced by AMS coincided with an increase in oxygen consumption in INS-1(832/13) cells. CONCLUSIONS/INTERPRETATION: Activation of 5-HT2B receptors stimulates GSIS in beta cells by triggering downstream changes in cellular Ca(2+) flux that enhance mitochondrial metabolism. Our findings suggest that serotonin and the 5-HT2B receptor stimulate insulin release.

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.

Complement inhibitor CD55 governs the integrity of membrane rafts in pancreatic beta cells, but plays no role in insulin secretion.

CD55 is a glycosylphosphatidylinositol-anchored protein, which inhibits complement activation by acting on the complement C3 convertases. CD55 is widely localized in the cholesterol rich regions of the cell plasma membrane termed membrane rafts. CD55 is attached to these specialized regions via a GPI link on the outer leaflet of the plasma membrane. Membrane rafts anchor many important signaling proteins, which control several cellular functions within the cell. For example, we recently demonstrated that the membrane raft protein and complement inhibitor CD59 also controls insulin secretion by an intracellular mechanism. Therefore, we have in this study aimed at addressing the expression and function of CD55 in pancreatic beta cells. To this end, we observe that CD55 is highly expressed in INS1 832/13 beta cells as well as human pancreatic islets. Diabetic human islets show a tendency for increased expression of CD55 when compared to the healthy controls. Importantly, silencing of CD55 in INS1 832/13 cells does not affect their insulin secretory capacity. On the other hand, silencing of CD55 diminished the intensity of membrane rafts as determined by Atto-SM staining. We hence conclude that CD55 expression is affected by glycemic status in human islets and plays a critical role in maintaining the conserved structure of rafts in pancreatic islets, which is similar to that of the related complement inhibitor CD59. However CD55 does not interfere with insulin secretion in beta cells, which is in sharp contrast to the action of the complement inhibitor CD59.

Islet Gene View-a tool to facilitate islet research.

Characterization of gene expression in pancreatic islets and its alteration in type 2 diabetes (T2D) are vital in understanding islet function and T2D pathogenesis. We leveraged RNA sequencing and genome-wide genotyping in islets from 188 donors to create the Islet Gene View (IGW) platform to make this information easily accessible to the scientific community. Expression data were related to islet phenotypes, diabetes status, other islet-expressed genes, islet hormone-encoding genes and for expression in insulin target tissues. The IGW web application produces output graphs for a particular gene of interest. In IGW, 284 differentially expressed genes (DEGs) were identified in T2D donor islets compared with controls. Forty percent of DEGs showed cell-type enrichment and a large proportion significantly co-expressed with islet hormone-encoding genes; glucagon (<i>GCG</i>, 56%), amylin (<i>IAPP</i>, 52%), insulin (<i>INS</i>, 44%), and somatostatin (<i>SST</i>, 24%). Inhibition of two DEGs, <i>UNC5D</i> and <i>SERPINE2</i>, impaired glucose-stimulated insulin secretion and impacted cell survival in a human ß-cell model. The exploratory use of IGW could help designing more comprehensive functional follow-up studies and serve to identify therapeutic targets in T2D.

Glucose-Dependent Granule Docking Limits Insulin Secretion and Is Decreased in Human Type 2 Diabetes.

Glucose-stimulated insulin secretion is biphasic, with a rapid first phase and a slowly developing sustained second phase; both are disturbed in type 2 diabetes (T2D). Biphasic secretion results from vastly different release probabilities of individual insulin granules, but the morphological and molecular basis for this is unclear. Here, we show that human insulin secretion and exocytosis critically depend on the availability of membrane-docked granules and that T2D is associated with a strong reduction in granule docking. Glucose accelerated granule docking, and this effect was absent in T2D. Newly docked granules only slowly acquired release competence; this was regulated by major signaling pathways, but not glucose. Gene expression analysis indicated that key proteins involved in granule docking are downregulated in T2D, and overexpression of these proteins increased granule docking. The findings establish granule docking as an important glucose-dependent step in human insulin secretion that is dysregulated in T2D.

Glucose-Induced Changes in Gene Expression in Human Pancreatic Islets: Causes or Consequences of Chronic Hyperglycemia.

Dysregulation of gene expression in islets from patients with type 2 diabetes (T2D) might be causally involved in the development of hyperglycemia, or it could develop as a consequence of hyperglycemia (i.e., glucotoxicity). To separate the genes that could be causally involved in pathogenesis from those likely to be secondary to hyperglycemia, we exposed islets from human donors to normal or high glucose concentrations for 24 h and analyzed gene expression. We compared these findings with gene expression in islets from donors with normal glucose tolerance and hyperglycemia (including T2D). The genes whose expression changed in the same direction after short-term glucose exposure, as in T2D, were considered most likely to be a consequence of hyperglycemia. Genes whose expression changed in hyperglycemia but not after short-term glucose exposure, particularly those that also correlated with insulin secretion, were considered the strongest candidates for causal involvement in T2D. For example, ERO1LB, DOCK10, IGSF11, and PRR14L were downregulated in donors with hyperglycemia and correlated positively with insulin secretion, suggesting a protective role, whereas TMEM132C was upregulated in hyperglycemia and correlated negatively with insulin secretion, suggesting a potential pathogenic role. This study provides a catalog of gene expression changes in human pancreatic islets after exposure to glucose.

Genetic determinants of circulating GIP and GLP-1 concentrations.

The secretion of insulin and glucagon from the pancreas and the incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) from the gastrointestinal tract is essential for glucose homeostasis. Several novel treatment strategies for type 2 diabetes (T2D) mimic GLP-1 actions or inhibit incretin degradation (DPP4 inhibitors), but none is thus far aimed at increasing the secretion of endogenous incretins. In order to identify new potential therapeutic targets for treatment of T2D, we performed a meta-analysis of a GWAS and an exome-wide association study of circulating insulin, glucagon, GIP, and GLP-1 concentrations measured during an oral glucose tolerance test in up to 7,828 individuals. We identified 6 genome-wide significant functional loci associated with plasma incretin concentrations in or near the SLC5A1 (encoding SGLT1), GIPR, ABO, GLP2R, F13A1, and HOXD1 genes and studied the effect of these variants on mRNA expression in pancreatic islet and on metabolic phenotypes. Immunohistochemistry showed expression of GIPR, ABO, and HOXD1 in human enteroendocrine cells and expression of ABO in pancreatic islets, supporting a role in hormone secretion. This study thus provides candidate genes and insight into mechanisms by which secretion and breakdown of GIP and GLP-1 are regulated.

Influence of Familial Renal Glycosuria Due to Mutations in the SLC5A2 Gene on Changes in Glucose Tolerance over Time.

Familial renal glycosuria is an inherited disorder resulting in glucose excretion in the urine despite normal blood glucose concentrations. It is most commonly due to mutations in the SLC5A2 gene coding for the glucose transporter SGLT2 in the proximal tubule. Several drugs have been introduced as means to lower glucose in patients with type 2 diabetes targeting SGLT2 resulting in renal glycosuria, but no studies have addressed the potential effects of decreased renal glucose reabsorption and chronic glycosuria on the prevention of glucose intolerance. Here we present data on a large pedigree with renal glycosuria due to two mutations (c.300-303+2del and p.A343V) in the SLC5A2 gene. The mutations, which in vitro affected glucose transport in a cell line model, and the ensuing glycosuria were not associated with better glycemic control during a follow-up period of more than 10 years. One individual, who was compound heterozygous for mutations in the SLC5A2 gene suffered from severe urogenital candida infections and postprandial hypoglycemia. In conclusion, in this family with familial glycosuria we did not find any evidence that chronic loss of glucose in the urine would protect from deterioration of the glucose tolerance over time.

The complement inhibitor CD59 regulates insulin secretion by modulating exocytotic events.

Type 2 diabetes is triggered by reduced insulin production, caused by genetic and environmental factors such as inflammation originating from the innate immune system. Complement proteins are a component of innate immunity and kill non-self cells by perforating the plasma membrane, a reaction prevented by CD59. Human pancreatic islets express CD59 at very high levels. CD59 is primarily known as a plasma membrane protein in membrane rafts, but most CD59 protein in pancreatic ß cells is intracellular. Removing extracellular CD59 disrupts membrane rafts and moderately stimulates insulin secretion, whereas silencing intracellular CD59 markedly suppresses regulated secretion by exocytosis, as demonstrated by TIRF imaging. CD59 interacts with the exocytotic proteins VAMP2 and Syntaxin-1. CD59 expression is reduced by glucose and in rodent diabetes models but upregulated in human diabetic islets, potentially reflecting compensatory reactions. This unconventional action of CD59 broadens the established view of innate immunity in type 2 diabetes.