RNA sequencing unravels novel L cell constituents and mechanisms of GLP-1 secretion in human gastric bypass-operated intestine.

AIMS/HYPOTHESIS: Roux-en-Y gastric bypass surgery (RYGB) frequently results in remission of type 2 diabetes as well as exaggerated secretion of glucagon-like peptide-1 (GLP-1). Here, we assessed RYGB-induced transcriptomic alterations in the small intestine and investigated how they were related to the regulation of GLP-1 production and secretion in vitro and in vivo. METHODS: Human jejunal samples taken perisurgically and 1 year post RYGB (n=13) were analysed by RNA-seq. Guided by bioinformatics analysis we targeted four genes involved in cholesterol biosynthesis, which we confirmed to be expressed in human L cells, for potential involvement in GLP-1 regulation using siRNAs in GLUTag and STC-1 cells. Gene expression analyses, GLP-1 secretion measurements, intracellular calcium imaging and RNA-seq were performed in vitro. OGTTs were performed in C57BL/6j and iScd1-/- mice and immunohistochemistry and gene expression analyses were performed ex vivo. RESULTS: Gene Ontology (GO) analysis identified cholesterol biosynthesis as being most affected by RYGB. Silencing or chemical inhibition of stearoyl-CoA desaturase 1 (SCD1), a key enzyme in the synthesis of monounsaturated fatty acids, was found to reduce Gcg expression and secretion of GLP-1 by GLUTag and STC-1 cells. Scd1 knockdown also reduced intracellular Ca2+ signalling and membrane depolarisation. Furthermore, Scd1 mRNA expression was found to be regulated by NEFAs but not glucose. RNA-seq of SCD1 inhibitor-treated GLUTag cells identified altered expression of genes implicated in ATP generation and glycolysis. Finally, gene expression and immunohistochemical analysis of the jejunum of the intestine-specific Scd1 knockout mouse model, iScd1-/-, revealed a twofold higher L cell density and a twofold increase in Gcg mRNA expression. CONCLUSIONS/INTERPRETATION: RYGB caused robust alterations in the jejunal transcriptome, with genes involved in cholesterol biosynthesis being most affected. Our data highlight SCD as an RYGB-regulated L cell constituent that regulates the production and secretion of GLP-1.

Lessons from single-cell RNA sequencing of human islets.

Islet dysfunction is central in type 2 diabetes and full-blown type 2 diabetes develops first when the beta cells lose their ability to secrete adequate amounts of insulin in response to raised plasma glucose. Several mechanisms behind beta cell dysfunction have been put forward but many important questions still remain. Furthermore, our understanding of the contribution of each islet cell type in type 2 diabetes pathophysiology has been limited by technical boundaries. Closing this knowledge gap will lead to a leap forward in our understanding of the islet as an organ and potentially lead to improved treatments. The development of single-cell RNA sequencing (scRNAseq) has led to a breakthrough for characterising the transcriptome of each islet cell type and several important observations on the regulation of cell-type-specific gene expression have been made. When it comes to identifying type 2 diabetes disease mechanisms, the outcome is still limited. Several studies have identified differentially expressed genes, although there is very limited consensus between the studies. As with all new techniques, scRNAseq has limitations; in addition to being extremely expensive, genes expressed at low levels may not be detected, noise may not be appropriately filtered and selection biases for certain cell types are at hand. Furthermore, recent advances suggest that commonly used computational tools may be suboptimal for analysis of scRNAseq data in small-scale studies. Fortunately, development of new computational tools holds promise for harnessing the full potential of scRNAseq data. Here we summarise how scRNAseq has contributed to increasing the understanding of various aspects of islet biology as well as type 2 diabetes disease mechanisms. We also focus on challenges that remain and propose steps to promote the utilisation of the full potential of scRNAseq in this area.

Postprandial triglyceride levels rather than fat distribution may reflect early signs of disturbed fat metabolism in Iraqi immigrants.

PURPOSE: Previous studies have shown that at a similar body mass index, Middle Eastern immigrants are more insulin resistant and at higher risk for type 2 diabetes (T2D) than native Europeans. Insulin resistance is strongly associated with disturbed fat metabolism and cardiovascular disease (CVD). However, fat metabolism is poorly investigated comparing Middle Eastern and European ethnicities. METHODS: This observational study included 26 Iraqi and 16 Swedish-born men without T2D or clinical risk factors for CVD. An oral fat tolerance test (OFTT) was performed, where plasma triglycerides (p-TG) were measured for 6 h. mRNA expression and adipocyte size were measured in subcutaneous adipose tissue biopsies collected prior to OFTT, and magnetic resonance imaging was conducted to assess body fat distribution. RESULTS: The median p-TG accumulation was higher and the clearance slower among Iraqis than Swedes. None of the groups reached their fasting p-TG (Iraqis 1.55 mmol/l; Swedes 0.95 mmol/l) after 6 h (Iraqis p-TG 3.10 mmol/l; Swedes p-TG 1.50 mmol/l). Adipocyte size, mRNA expression, and fat accumulation in the liver, muscle and abdomen were similar in both groups. CONCLUSION: Postprandial p-TG levels rather than fat distribution may reflect early signs of disturbed fat metabolism in Iraqi immigrants without CVD risk factors.

No direct effect of SGLT2 activity on glucagon secretion.

AIMS/HYPOTHESIS: Sodium-glucose cotransporter (SGLT) 2 inhibitors constitute a new class of glucose-lowering drugs, but they increase glucagon secretion, which may counteract their glucose-lowering effect. Previous studies using static incubation of isolated human islets or the glucagon-secreting cell line α-TC1 suggested that this results from direct inhibition of alpha cell SGLT1/2-activity. The aim of this study was to test whether the effects of SGLT2 on glucagon secretion demonstrated in vitro could be reproduced in a more physiological setting. METHODS: We explored the effect of SGLT2 activity on glucagon secretion using isolated perfused rat pancreas, a physiological model for glucagon secretion. Furthermore, we investigated Slc5a2 (the gene encoding SGLT2) expression in rat islets as well as in mouse and human islets and in mouse and human alpha, beta and delta cells to test for potential inter-species variations. SGLT2 protein content was also investigated in mouse, rat and human islets. RESULTS: Glucagon output decreased three- to fivefold within minutes of shifting from low (3.5 mmol/l) to high (10 mmol/l) glucose (4.0 ± 0.5 pmol/15 min vs 1.3 ± 0.3 pmol/15 min, p < 0.05). The output was unaffected by inhibition of SGLT1/2 with dapagliflozin or phloridzin or by addition of the SGLT1/2 substrate α-methylglucopyranoside, whether at low or high glucose concentrations (p = 0.29-0.99). Insulin and somatostatin secretion (potential paracrine regulators) was also unaffected. Slc5a2 expression and SGLT2 protein were marginal or below detection limit in rat, mouse and human islets and in mouse and human alpha, beta and delta cells. CONCLUSIONS/INTERPRETATION: Our combined data show that increased plasma glucagon during SGLT2 inhibitor treatment is unlikely to result from direct inhibition of SGLT2 in alpha cells, but instead may occur downstream of their blood glucose-lowering effects.

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.

A central role for GRB10 in regulation of islet function in man.

Variants in the growth factor receptor-bound protein 10 (GRB10) gene were in a GWAS meta-analysis associated with reduced glucose-stimulated insulin secretion and increased risk of type 2 diabetes (T2D) if inherited from the father, but inexplicably reduced fasting glucose when inherited from the mother. GRB10 is a negative regulator of insulin signaling and imprinted in a parent-of-origin fashion in different tissues. GRB10 knock-down in human pancreatic islets showed reduced insulin and glucagon secretion, which together with changes in insulin sensitivity may explain the paradoxical reduction of glucose despite a decrease in insulin secretion. Together, these findings suggest that tissue-specific methylation and possibly imprinting of GRB10 can influence glucose metabolism and contribute to T2D pathogenesis. The data also emphasize the need in genetic studies to consider whether risk alleles are inherited from the mother or the father.

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.

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.

Loss of TFB1M results in mitochondrial dysfunction that leads to impaired insulin secretion and diabetes.

We have previously identified transcription factor B1 mitochondrial (TFB1M) as a type 2 diabetes (T2D) risk gene, using human and mouse genetics. To further understand the function of TFB1M and how it is associated with T2D, we created a ß-cell-specific knockout of Tfb1m, which gradually developed diabetes. Prior to the onset of diabetes, ß-Tfb1m(-/-) mice exhibited retarded glucose clearance owing to impaired insulin secretion. ß-Tfb1m(-/-) islets released less insulin in response to fuels, contained less insulin and secretory granules and displayed reduced ß-cell mass. Moreover, mitochondria in Tfb1m-deficient ß-cells were more abundant with disrupted architecture. TFB1M is known to control mitochondrial protein translation by adenine dimethylation of 12S ribosomal RNA (rRNA). Here, we found that the levels of TFB1M and mitochondrial-encoded proteins, mitochondrial 12S rRNA methylation, ATP production and oxygen consumption were reduced in ß-Tfb1m(-/-) islets. Furthermore, the levels of reactive oxygen species (ROS) in response to cellular stress were increased whereas induction of defense mechanisms was attenuated. We also show increased apoptosis and necrosis as well as infiltration of macrophages and CD4(+) cells in the islets. Taken together, our findings demonstrate that Tfb1m-deficiency in ß-cells caused mitochondrial dysfunction and subsequently diabetes owing to combined loss of ß-cell function and mass. These observations reflect pathogenetic processes in human islets: using RNA sequencing, we found that the TFB1M risk variant exhibited a negative gene-dosage effect on islet TFB1M mRNA levels, as well as insulin secretion. Our findings highlight the role of mitochondrial dysfunction in impairments of ß-cell function and mass, the hallmarks of 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.

Decreased insulin secretion and glucose clearance in exocrine pancreas-insufficient pigs.

The effect of exocrine pancreatic function on the glucose-mediated insulin response and glucose utilization were studied in an exocrine pancreas-insufficient (EPI) pig model. Five 10-week-old EPI pigs after pancreatic duct ligation and 6 age-matched, non-operated control pigs were used in the study. Blood glucose, plasma insulin and C-peptide concentrations were monitored during meal (MGTT), oral (OGTT) and intravenous (IVGTT) glucose tolerance tests. Upon post-mortem examination, the pancreatic remnants of the EPI pigs showed acinar fibrotic atrophy but normal islets and ß-cell morphology. The EPI pigs displayed increased fasting glucose concentrations compared with control animals (6.4 ± 0.4 versus 4.8 ± 0.1 mmol l(-1) , P < 0.0001) but unchanged insulin concentrations (2.4 ± 0.6 versus 2.1 ± 0.2 pmol l(-1) ). During the OGTT and IVGTT, the EPI pigs showed slower, impaired glucose utilization, with the disruption of a well-timed insulin response. Plasma C-peptide concentrations confirmed the delayed insulin response during the IVGTT in EPI pigs. Oral pancreatic enzyme supplementation (PES) of EPI pigs improved glucose clearance during IVGTT [AUC(glucose) 1295 ± 70 mmol l(-1) × (120 min) in EPI versus 1044 ± 32 mmol l(-1) × (120 min) in EPI + PES, P < 0.0001] without reinforcing the release of insulin [AUC(C-peptide) 14.4 ± 3.8 nmol l(-1) × (120 min) in EPI versus 6.4 ± 1.3 nmol l(-1) × (120 min) in EPI + PES, P < 0.002]. The results suggest the existence of an acino-insular axis regulatory communication. The presence of pancreatic enzymes in the gut facilitates glucose utilization in an insulin-independent manner, indicating the existence of a gut-derived pancreatic enzyme-dependent mechanism involved in peripheral glucose utilization.

Cocaine- and amphetamine-regulated transcript (CART) protects beta cells against glucotoxicity and increases cell proliferation.

Cocaine- and amphetamine-regulated transcript (CART) is an islet peptide that promotes glucose-stimulated insulin secretion in beta cells via cAMP/PKA-dependent pathways. In addition, CART is a regulator of neuronal survival. In this study, we examined the effect of exogenous CART 55-102 on beta cell viability and dissected its signaling mechanisms. Evaluation of DNA fragmentation and chromatin condensation revealed that CART 55-102 reduced glucotoxicity-induced apoptosis in both INS-1 (832/13) cells and isolated rat islets. Glucotoxicity in INS-1 (832/13) cells also caused a 50% reduction of endogenous CART protein. We show that CART increased proliferation in INS-1 (832/13) cells, an effect that was blocked by PKA, PKB, and MEK1 inhibitors. In addition, CART induced phosphorylation of CREB, IRS, PKB, FoxO1, p44/42 MAPK, and p90RSK in INS-1 (832/13) cells and isolated rat islets, all key mediators of cell survival and proliferation. Thus, we demonstrate that CART 55-102 protects beta cells against glucotoxicity and promotes proliferation. Taken together our data point to the potential use of CART in therapeutic interventions targeted at enhancing functional beta cell mass and long-term insulin secretion in 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.

The effects of Calorie restriction and Bariatric surgery on Circulating Proneurotensin levels.

CONTEXT: Proneurotensin (pNT) is associated with obesity and T2D, but the effects of Roux-en-Y gastric bypass (RYGB) on postprandial pNT levels are not well studied. OBJECTIVE: Assess effects of RYGB versus very low-energy diet (VLED) on pNT levels in response to mixed-meal tests (MMT), and long-term effects of RYGB on fasting pNT.Study participants: Cohort 1: Nine normoglycemic (NG) and ten T2D patients underwent MMT before and after VLED, immediately post-RYGB and six weeks post-RYGB. Cohort 2: Ten controls with normal weight and ten patients with obesity and T2D, who underwent RYGB or vertical sleeve gastrectomy (VSG), were subjected to MMTs and GIP infusions pre-surgery and three months post-surgery. GLP-1 infusions were performed in normal weight participants. Cohort 3: Fasting pNT was assessed pre-RYGB (n=161), two months post-RYGB (n=92) and 1-year post-RYGB (n=118) in NG and T2D patients. pNT levels were measured using ELISA. RESULTS: Reduced fasting and postprandial pNT were evident after VLED and immediately following RYGB. Reintroduction of solid food post-RYGB increased fasting and postprandial pNT. Prior to RYGB, all patients lacked a meal response in pNT, but this was evident post-RYGB/VSG. GIP- or GLP-1 infusion had no effect on pNT levels. Fasting pNT were higher 1-year post-RYGB regardless of glycemic status. CONCLUSION: RYGB causes a transient reduction in pNT as a consequence of caloric restriction. The RYGB/VSG-induced rise in postprandial pNT is independent of GIP and GLP-1 and higher fasting pNT are maintained one year post-surgically.

Metabolic remission precedes possible weight regain after gastric bypass surgery.

OBJECTIVE: Some patients regain weight to a variable extent from 1 year after Roux-en-Y gastric bypass surgery (RYGB), though rarely reaching preoperative values. The aim of the present study was to investigate whether, when, and to what extent metabolic remission occurs. METHODS: Fasting metabolite and lipid profiles were determined in blood plasma collected from a nonrandomized intervention study involving 148 patients before RYGB and at 2, 12, and 60 months post RYGB. Both short-term and long-term alterations in metabolism were assessed. Anthropometric and clinical variables were assessed at all study visits. RESULTS: This study found that the vast majority of changes in metabolite levels occurred during the first 2 months post RYGB. Notably, thereafter the metabolome started to return toward the presurgical state. Consequently, a close-to-presurgical metabolome was observed at the time when patients reached their lowest weight and glucose level. Lipids with longer acyl chains and a higher degree of unsaturation were altered more dramatically compared with shorter and more saturated lipids, suggesting a systematic and reversible lipid remodeling. CONCLUSIONS: Remission of the metabolic state was observed prior to notable weight regain. Further and more long-term studies are required to assess whether the extent of metabolic remission predicts future weight regain and glycemic deterioration.

GPR162 is a beta cell CART receptor.

Cocaine and amphetamine-regulated transcript (CART) is expressed in pancreatic islet cells and neuronal elements. We have previously established insulinotropic actions of CART in human and rodent islets. The receptor for CART in the pancreatic beta cells is unidentified. We used RNA sequencing of Cartpt knockdown (KD) INS-1 832/13 cells and identified GPR162 as the most Cartpt-regulated receptor. We therefore tested if GPR162 mediates the effects of CART in beta cells. Binding of CART to GPR162 was established using proximity ligation assay, radioactive binding, and co-immunoprecipitation, and KD of Gpr162 mRNA caused reduced binding. Gpr162 KD cells had blunted CARTp-induced exocytosis, and reduced CARTp-induced insulin secretion. Furthermore, we identified a hitherto undescribed GPR162-dependent role of CART as a regulator of cytoskeletal arrangement. Thus, our findings provide mechanistic insight into the effect of CART on insulin secretion and show that GPR162 is the CART receptor in beta cells.

Type 2 diabetes candidate genes, including PAX5, cause impaired insulin secretion in human pancreatic islets.

Type 2 diabetes (T2D) is caused by insufficient insulin secretion from pancreatic ß cells. To identify candidate genes contributing to T2D pathophysiology, we studied human pancreatic islets from approximately 300 individuals. We found 395 differentially expressed genes (DEGs) in islets from individuals with T2D, including, to our knowledge, novel (OPRD1, PAX5, TET1) and previously identified (CHL1, GLRA1, IAPP) candidates. A third of the identified expression changes in islets may predispose to diabetes, as expression of these genes associated with HbA1c in individuals not previously diagnosed with T2D. Most DEGs were expressed in human ß cells, based on single-cell RNA-Seq data. Additionally, DEGs displayed alterations in open chromatin and associated with T2D SNPs. Mouse KO strains demonstrated that the identified T2D-associated candidate genes regulate glucose homeostasis and body composition in vivo. Functional validation showed that mimicking T2D-associated changes for OPRD1, PAX5, and SLC2A2 impaired insulin secretion. Impairments in Pax5-overexpressing ß cells were due to severe mitochondrial dysfunction. Finally, we discovered PAX5 as a potential transcriptional regulator of many T2D-associated DEGs in human islets. Overall, we have identified molecular alterations in human pancreatic islets that contribute to ß cell dysfunction in T2D pathophysiology.

The role of CART in islet biology.

Cocaine- and amphetamine-regulated transcript (CART) is mostly known for its appetite regulating effects in the central nervous system. However, CART is also highly expressed in the peripheral nervous system as well as in certain endocrine cells. Our group has dedicated more than 20 years to understand the role of CART in the pancreatic islets and in this review we summarize what is known to date about CART expression and function in the islets. CART is expressed in both islet cells and nerve fibers innervating the islets. Large species differences are at hand and CART expression is highly dynamic and increased during development, as well as in Type 2 Diabetes and certain endocrine tumors. In the human islets CART is expressed in alpha cells and beta cells and the expression is increased in T2D patients. CART increases insulin secretion, reduces glucagon secretion, and protects against beta cell death by reducing apoptosis and increasing proliferation. It is still not fully understood how CART mediates its effects or which receptors that are involved. Nevertheless, CART is endowed with several properties that are beneficial in a T2D perspective. Many of the described effects of CART resemble those of GLP-1, and interestingly CART has been found to potentiate some of the effects of GLP-1, paving the way for CART-based treatments in combination with GLP-1-based drugs.

Overexpressed beta cell CART increases insulin secretion in mouse models of insulin resistance and diabetes.

Impaired beta cell function and beta cell death are key features of type 2 diabetes (T2D). Cocaine- and amphetamine-regulated transcript (CART) is necessary for normal islet function in mice. CART increases glucose-stimulated insulin secretion in vivo in mice and in vitro in human islets and CART protects beta cells against glucotoxicity-induced cell death in vitro in rats. Furthermore, beta cell CART is upregulated in T2D patients and in diabetic rodent models as a consequence of hyperglycaemia. The aim of this study was to assess the impact of upregulated beta cell CART on islet hormone secretion and glucose homeostasis in a transgenic mouse model. To this end, mice with beta cell-specific overexpression of CART (CARTtg mice) were generated. CARTtg mice challenged by aging, high fat diet feeding or streptozotocin treatment were phenotyped with respect to in vivo and in vitro insulin and glucagon secretion, glucose homeostasis, and beta cell mass. In addition, the impact of adenoviral overexpression of CART on insulin secretion was studied in INS-1 832/13 cells. CARTtg mice had a normal metabolic phenotype under basal conditions. On the other hand, with age CARTtg mice displayed increased insulin secretion and improved glucose elimination, compared with age-matched WT mice. Furthermore, compared with WT controls, CARTtg mice had increased insulin secretion after feeding a high fat diet, as well as lower glucose levels and higher insulin secretion after streptozotocin treatment. Viral overexpression of CART in INS-1 832/13 cells resulted in increased glucose-stimulated insulin secretion. Together, these results imply that beta cell CART acts to increase insulin secretion when beta cell function is challenged. We propose that the increase in beta cell CART is part of a compensatory mechanisms trying to counteract the hyperglycaemia in T2D.

Obesity-associated Blunted Subcutaneous Adipose Tissue Blood Flow After Meal Improves After Bariatric Surgery.

CONTEXT: Glucose-dependent insulinotropic peptide (GIP) and meal ingestion increase subcutaneous adipose tissue (SAT) perfusion in healthy individuals. The effects of GIP and a meal on visceral adipose tissue (VAT) perfusion are unclear. OBJECTIVE: Our aim was to investigate the effects of meal and GIP on VAT and SAT perfusion in obese individuals with type 2 diabetes mellitus (T2DM) before and after bariatric surgery. METHODS: We recruited 10 obese individuals with T2DM scheduled for bariatric surgery and 10 control individuals. Participants were studied under 2 stimulations: meal ingestion and GIP infusion. SAT and VAT perfusion was measured using 15O-H2O positron emission tomography-magnetic resonance imaging at 3 time points: baseline, 20 minutes, and 50 minutes after the start of stimulation. Obese individuals were studied before and after bariatric surgery. RESULTS: Before bariatric surgery the responses of SAT perfusion to meal (P = .04) and GIP-infusion (P = .002) were blunted in the obese participants compared to controls. VAT perfusion response did not differ between obese and control individuals after a meal or GIP infusion. After bariatric surgery SAT perfusion response to a meal was similar to that of controls. SAT perfusion response to GIP administration remained lower in the operated-on than control participants. There was no change in VAT perfusion response after bariatric surgery. CONCLUSION: The vasodilating effects of GIP and meal are blunted in SAT but not in VAT in obese individuals with T2DM. Bariatric surgery improves the effects of a meal on SAT perfusion, but not the effects of GIP. Postprandial increase in SAT perfusion after bariatric surgery seems to be regulated in a GIP-independent manner.