Early deficits in insulin secretion, beta cell mass and islet blood perfusion precede onset of autoimmune type 1 diabetes in BioBreeding rats.

AIMS/HYPOTHESIS: Genetic studies show coupling of genes affecting beta cell function to type 1 diabetes, but hitherto no studies on whether beta cell dysfunction could precede insulitis and clinical onset of type 1 diabetes are available. METHODS: We used 40-day-old BioBreeding (BB) DRLyp/Lyp rats (a model of spontaneous autoimmune type 1 diabetes) and diabetes-resistant DRLyp/+ and DR+/+ littermates (controls) to investigate beta cell function in vivo, and insulin and glucagon secretion in vitro. Beta cell mass was assessed by optical projection tomography (OPT) and morphometry. Additionally, measurements of intra-islet blood flow were performed using microsphere injections. We also assessed immune cell infiltration, cytokine expression in islets (by immunohistochemistry and qPCR), as well as islet Glut2 expression and ATP/ADP ratio to determine effects on glucose uptake and metabolism in beta cells. RESULTS: DRLyp/Lyp rats were normoglycaemic and without traces of immune cell infiltrates. However, IVGTTs revealed a significant decrease in the acute insulin response to glucose compared with control rats (1685.3 ± 121.3 vs 633.3 ± 148.7; p < 0.0001). In agreement, insulin secretion was severely perturbed in isolated islets, and both first- and second-phase insulin release were lowered compared with control rats, while glucagon secretion was similar in both groups. Interestingly, after 5-7 days of culture of islets from DRLyp/Lyp rats in normal media, glucose-stimulated insulin secretion (GSIS) was improved; although, a significant decrease in GSIS was still evident compared with islets from control rats at this time (7393.9 ± 1593.7 vs 4416.8 ± 1230.5 pg islet-1 h-1; p < 0.0001). Compared with controls, OPT of whole pancreas from DRLyp/Lyp rats revealed significant reductions in medium (4.1 × 109 ± 9.5 × 107 vs 3.8 × 109 ± 5.8 × 107 µm3; p = 0.044) and small sized islets (1.6 × 109 ± 5.1 × 107 vs 1.4 × 109 ± 4.5 × 107 µm3; p = 0.035). Finally, we found lower intra-islet blood perfusion in vivo (113.1 ± 16.8 vs 76.9 ± 11.8 µl min-1 [g pancreas]-1; p = 0.023) and alterations in the beta cell ATP/ADP ratio in DRLyp/Lyp rats vs control rats. CONCLUSIONS/INTERPRETATION: The present study identifies a deterioration of beta cell function and mass, and intra-islet blood flow that precedes insulitis and diabetes development in animals prone to autoimmune type 1 diabetes. These underlying changes in islet function may be previously unrecognised factors of importance in type 1 diabetes development.

The effects of a HTR2B stop codon and testosterone on energy metabolism and beta cell function among antisocial Finnish males.

Herein, we examined insulin resistance (IR), insulin sensitivity (IS), beta cell activity, and glucose metabolism in subjects with antisocial personality disorder (ASPD), and whether the serotonin 2B (5-HT2B) receptor and testosterone have a role in energy metabolism. A cohort of subjects belonging to a founder population that included 98 ASPD males, aged 25-30, was divided into groups based on the presence of a heterozygous 5-HT2B receptor loss-of-function gene mutation (HTR2B Q20*; n = 9) or not (n = 89). Serum glucose and insulin levels were measured in a 5 h oral glucose tolerance test (75 g) and indices describing IR, IS, and beta cell activity were calculated. Body mass index (BMI) was also determined. Concentrations of the serotonin metabolite 5-hydroxyindoleacetic acid were measured in cerebrospinal fluid, and testosterone levels from serum. An IR-like state comprising high IR, low IS, and high beta cell activity indices was observed among ASPD subjects without the HTR2B Q20* allele. By contrast, being an ASPD HTR2B Q20* carrier appeared to be preventive of these pathophysiologies. The HTR2B Q20* allele and testosterone predicted lower BMI independently, but an interaction between HTR2B Q20* and testosterone lead to increased insulin sensitivity among HTR2B Q20* carriers with low testosterone levels. The HTR2B Q20* allele also predicted reduced beta cell activity and enhanced glucose metabolism. Reduced 5-HT2B receptor function at low or normal testosterone levels may be protective of obesity. Results were observed among Finnish males having an antisocial personality disorder, which limits the generality.

CART is overexpressed in human type 2 diabetic islets and inhibits glucagon secretion and increases insulin secretion.

AIMS/HYPOTHESIS: Insufficient insulin release and hyperglucagonaemia are culprits in type 2 diabetes. Cocaine- and amphetamine-regulated transcript (CART, encoded by Cartpt) affects islet hormone secretion and beta cell survival in vitro in rats, and Cart (-/-) mice have diminished insulin secretion. We aimed to test if CART is differentially regulated in human type 2 diabetic islets and if CART affects insulin and glucagon secretion in vitro in humans and in vivo in mice. METHODS: CART expression was assessed in human type 2 diabetic and non-diabetic control pancreases and rodent models of diabetes. Insulin and glucagon secretion was examined in isolated islets and in vivo in mice. Ca(2+) oscillation patterns and exocytosis were studied in mouse islets. RESULTS: We report an important role of CART in human islet function and glucose homeostasis in mice. CART was found to be expressed in human alpha and beta cells and in a subpopulation of mouse beta cells. Notably, CART expression was several fold higher in islets of type 2 diabetic humans and rodents. CART increased insulin secretion in vivo in mice and in human and mouse islets. Furthermore, CART increased beta cell exocytosis, altered the glucose-induced Ca(2+) signalling pattern in mouse islets from fast to slow oscillations and improved synchronisation of the oscillations between different islet regions. Finally, CART reduced glucagon secretion in human and mouse islets, as well as in vivo in mice via diminished alpha cell exocytosis. CONCLUSIONS/INTERPRETATION: We conclude that CART is a regulator of glucose homeostasis and could play an important role in the pathophysiology of type 2 diabetes. Based on the ability of CART to increase insulin secretion and reduce glucagon secretion, CART-based agents could be a therapeutic modality in type 2 diabetes.

Increased Melatonin Signaling Is a Risk Factor for Type 2 Diabetes.

Type 2 diabetes (T2D) is a global pandemic. Genome-wide association studies (GWASs) have identified >100 genetic variants associated with the disease, including a common variant in the melatonin receptor 1 b gene (MTNR1B). Here, we demonstrate increased MTNR1B expression in human islets from risk G-allele carriers, which likely leads to a reduction in insulin release, increasing T2D risk. Accordingly, in insulin-secreting cells, melatonin reduced cAMP levels, and MTNR1B overexpression exaggerated the inhibition of insulin release exerted by melatonin. Conversely, mice with a disruption of the receptor secreted more insulin. Melatonin treatment in a human recall-by-genotype study reduced insulin secretion and raised glucose levels more extensively in risk G-allele carriers. Thus, our data support a model where enhanced melatonin signaling in islets reduces insulin secretion, leading to hyperglycemia and greater future risk of T2D. The findings also imply that melatonin physiologically serves to inhibit nocturnal insulin release.

MafA-Controlled Nicotinic Receptor Expression Is Essential for Insulin Secretion and Is Impaired in Patients with Type 2 Diabetes.

Monoamine and acetylcholine neurotransmitters from the autonomic nervous system (ANS) regulate insulin secretion in pancreatic islets. The molecular mechanisms controlling neurotransmitter signaling in islet ß cells and their impact on diabetes development are only partially understood. Using a glucose-intolerant, MafA-deficient mouse model, we demonstrate that MAFA controls ANS-mediated insulin secretion by activating the transcription of nicotinic (ChrnB2 and ChrnB4) and adrenergic (Adra2A) receptor genes, which are integral parts of acetylcholine- and monoamine-signaling pathways. We show that acetylcholine-mediated insulin secretion requires nicotinic signaling and that nicotinic receptor expression is positively correlated with insulin secretion and glycemic control in human donor islets. Moreover, polymorphisms spanning MAFA-binding regions within the human CHRNB4 gene are associated with type 2 diabetes. Our data show that MAFA transcriptional activity is required for establishing ß cell sensitivity to neurotransmitter signaling and identify nicotinic signaling as a modulator of insulin secretion impaired in type 2 diabetes.

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.

Islet-specific monoamine oxidase A and B expression depends on MafA transcriptional activity and is compromised in type 2 diabetes.

Lack or dysfunction of insulin producing ß cells results in the development of type 1 and type 2 diabetes mellitus, respectively. Insulin secretion is controlled by metabolic stimuli (glucose, fatty acids), but also by monoamine neurotransmitters, like dopamine, serotonin, and norepinephrine. Intracellular monoamine levels are controlled by monoamine oxidases (Mao) A and B. Here we show that MaoA and MaoB are expressed in mouse islet ß cells and that inhibition of Mao activity reduces insulin secretion in response to metabolic stimuli. Moreover, analysis of MaoA and MaoB protein expression in mouse and human type 2 diabetic islets shows a significant reduction of MaoB in type 2 diabetic ß cells suggesting that loss of Mao contributes to ß cell dysfunction. MaoB expression was also reduced in ß cells of MafA-deficient mice, a mouse model for ß cell dysfunction, and biochemical studies showed that MafA directly binds to and activates MaoA and MaoB transcriptional control sequences. Taken together, our results show that MaoA and MaoB expression in pancreatic islets is required for physiological insulin secretion and lost in type 2 diabetic mouse and human ß cells. These findings demonstrate that regulation of monoamine levels by Mao activity in ß cells is pivotal for physiological insulin secretion and that loss of MaoB expression may contribute to the ß cell dysfunction in type 2 diabetes.

Photocoagulation of human retinal pigment epithelium in vitro: unravelling the effects on ARPE-19 by transcriptomics and proteomics.

PURPOSE: Despite the extensive use of retinal photocoagulation for ischaemia and vascular leakage in retinal vascular disease, the molecular mechanisms behind its clinical beneficial effects are still poorly understood. One important target of laser irradiation is the retinal pigment epithelium (RPE). In this study, we aimed at identifying the isolated effects of photocoagulation of RPE at both the mRNA and protein expression levels. METHODS: Human ARPE-19 cells were exposed to photocoagulation. Gene expression and protein expression were compared to untreated cells using microarray and liquid chromatography-mass spectrometry analysis. Genes and proteins queried by microarray and mass spectrometry were subjected to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database pathway analyses. RESULTS: Laser irradiation resulted in an induction of the cytoprotective heat-shock protein subfamily Hsp70 as well as in a suppression of the vascular permeability factor carbonic anhydrase 9 (CA9). These expression patterns were evident at both the mRNA and protein levels. KEGG pathway analyses revealed genes and proteins involved in cellular turnover, repair and inflammation. CONCLUSIONS: By characterizing the transcriptional and translational effects of laser coagulation on the RPE cells in culture, we have revealed responses, which might contribute to some of the beneficial effects obtained by photocoagulation for ischaemia and vascular leakage in retinal vascular disease.

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.

Autoimmunity against INS-IGF2 protein expressed in human pancreatic islets.

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.

Microphthalmia transcription factor regulates pancreatic ß-cell function.

Precise regulation of ß-cell function is crucial for maintaining blood glucose homeostasis. Pax6 is an essential regulator of ß-cell-specific factors like insulin and Glut2. Studies in the developing eye suggest that Pax6 interacts with Mitf to regulate pigment cell differentiation. Here, we show that Mitf, like Pax6, is expressed in all pancreatic endocrine cells during mouse postnatal development and in the adult islet. A Mitf loss-of-function mutation results in improved glucose tolerance and enhanced insulin secretion but no increase in ß-cell mass in adult mice. Mutant ß-cells secrete more insulin in response to glucose than wild-type cells, suggesting that Mitf is involved in regulating ß-cell function. In fact, the transcription of genes critical for maintaining glucose homeostasis (insulin and Glut2) and ß-cell formation and function (Pax4 and Pax6) is significantly upregulated in Mitf mutant islets. The increased Pax6 expression may cause the improved ß-cell function observed in Mitf mutant animals, as it activates insulin and Glut2 transcription. Chromatin immunoprecipitation analysis shows that Mitf binds to Pax4 and Pax6 regulatory regions, suggesting that Mitf represses their transcription in wild-type ß-cells. We demonstrate that Mitf directly regulates Pax6 transcription and controls ß-cell function.

HTR1A a novel type 1 diabetes susceptibility gene on chromosome 5p13-q13.

BACKGROUND: We have previously performed a genome-wide linkage study in Scandinavian Type 1 diabetes (T1D) families. In the Swedish families, we detected suggestive linkage (LOD≤2.2) to the chromosome 5p13-q13 region. The aim of our study was to investigate the linked region in search for possible T1D susceptibility genes. METHODOLOGY/PRINCIPAL FINDINGS: Microsatellites were genotyped in the Scandinavian families to fine-map the previously linked region. Further, SNPs were genotyped in Swedish and Danish families as well as Swedish sporadic cases. In the Swedish families we detected genome-wide significant linkage to the 5-hydroxytryptamine receptor 1A (HTR1A) gene (LOD 3.98, p<9.8×10(-6)). Markers tagging two separate genes; the ring finger protein 180 (RNF180) and HTR1A showed association to T1D in the Swedish and Danish families (p<0.002, p<0.001 respectively). The association was not confirmed in sporadic cases. Conditional analysis indicates that the primary association was to HTR1A. Quantitative PCR show that transcripts of both HTR1A and RNF180 are present in human islets of Langerhans. Moreover, immunohistochemical analysis confirmed the presence of the 5-HTR1A protein in isolated human islets of Langerhans as well as in sections of human pancreas. CONCLUSIONS: We have identified and confirmed the association of both HTR1A and RFN180, two genes in high linkage disequilibrium (LD) to T1D in two separate family materials. As both HTR1A and RFN180 were expressed at the mRNA level and HTR1A as protein in human islets of Langerhans, we suggest that HTR1A may affect T1D susceptibility by modulating the initial autoimmune attack or either islet regeneration, insulin release, or both.

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.