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In obesity, increased mitochondrial metabolism with the accumulation of oxidative stress leads to mitochondrial damage and β-cell dysfunction. In particular, β-cells express antioxidant enzymes at relatively low levels and are highly vulnerable to oxidative stress. Early in the development of obesity, β-cells exhibit increased glucose-stimulated insulin secretion in order to compensate for insulin resistance. This increase in β-cell function under the condition of enhanced metabolic stress suggests that β-cells possess a defense mechanism against increased oxidative damage, which may become insufficient or decline at the onset of type 2 diabetes. Here, we show that metabolic stress induces β-cell hypoxia inducible factor 2α (HIF-2α), which stimulates antioxidant gene expression (e.g., Sod2 and Cat) and protects against mitochondrial reactive oxygen species (ROS) and subsequent mitochondrial damage. Knockdown of HIF-2α in Min6 cells exaggerated chronic high glucose–induced mitochondrial damage and β-cell dysfunction by increasing mitochondrial ROS levels. Moreover, inducible β-cell HIF-2α knockout mice developed more severe β-cell dysfunction and glucose intolerance on a high-fat diet, along with increased ROS levels and decreased islet mitochondrial mass. Our results provide a previously unknown mechanism through which β-cells defend against increased metabolic stress to promote β-cell compensation in obesity.

In obesity, increased mitochondrial metabolism with the accumulation of oxidative stress leads to mitochondrial damage and beta cell dysfunction. In particular, beta cells express anti-oxidant enzymes at relatively low levels and are highly vulnerable to oxidative stress. Early in the development of obesity, beta cells exhibit increased GSIS, in order to compensate for insulin resistance. This increase in beta cell function under the condition of enhanced metabolic stress suggests that beta cells possess a defense mechanism(s) against increased oxidative damage, which may become insufficient or decline at the onset of type 2 diabetes. Here, we show that metabolic stress induces beta cell HIF-2a, which stimulates anti-oxidant gene expression (e.g. Sod2 and Cat) and protects against mitochondrial ROS and subsequent mitochondrial damage. Knockdown of HIF-2a in Min6 cells exaggerated chronic high glucose-induced mitochondrial damage and beta cell dysfunction by increasing mitochondrial ROS levels. Moreover, inducible beta cell HIF-2a KO mice developed more severe beta cell dysfunction and glucose intolerance on high fat diet, along with increased ROS levels and decreased islet mitochondrial mass. Our results provide a previously unknown mechanism for how beta cell defends against increased metabolic stress to promote beta cell compensation in obesity.

Objective Atherosclerosis is a major cause of cardiovascular disease. Monocyte-endothelial cell interactions are partly mediated by expression of monocyte CX3CR1 and endothelial cell fractalkine (CX3CL1). Interrupting the interaction between this ligand–receptor pair should reduce monocyte binding to the endothelial wall and reduce atherosclerosis. We sought to reduce atherosclerosis by preventing monocyte-endothelial cell interactions through use of a long-acting CX3CR1 agonist. Methods In this study, the chemokine domain of CX3CL1 was fused to the mouse Fc region to generate a long-acting soluble form of CX3CL1 suitable for chronic studies. CX3CL1-Fc or saline was injected twice a week (30 mg/kg) for 4 months into Ldlr knockout (KO) mice on an atherogenic western diet. Results CX3CL1-Fc-treated Ldlr KO mice showed decreased en face aortic lesion surface area and reduced aortic root lesion size with decreased necrotic core area. Flow cytometry analyses of CX3CL1-Fc-treated aortic wall cell digests revealed a decrease in M1-like polarized macrophages and T cells. Moreover, CX3CL1-Fc administration reduced diet-induced atherosclerosis after switching from an atherogenic to a normal chow diet. In vitro monocyte adhesion studies revealed that CX3CL1-Fc treatment caused fewer monocytes to adhere to a human umbilical vein endothelial cell monolayer. Furthermore, a dorsal window chamber model demonstrated that CX3CL1-Fc treatment decreased in vivo leukocyte adhesion and rolling in live capillaries after short-term ischemia-reperfusion. Conclusion These results indicate that CX3CL1-Fc can inhibit monocyte/endothelial cell adhesion as well as reduce atherosclerosis., Highlights • Long-acting FKN-Fc reduced atherosclerosis in LDLR KO mice in a prevention model. • FKN-Fc administration accelerated diet-induced regression of established atherosclerosis. • Monocyte adhesion in vitro and in vivo is reduced in the presence of FKN-Fc.

Objective Recent evidence indicates that inhibition of prolyl hydroxylase domain (PHD) proteins can exert beneficial effects to improve metabolic abnormalities in mice and humans. However, the underlying mechanisms are not clearly understood. This study was designed to address this question. Methods A pan-PHD inhibitor compound was injected into WT and liver-specific hypoxia-inducible factor (HIF)-2α KO mice, after onset of obesity and glucose intolerance, and changes in glucose and glucagon tolerance were measured. Tissue-specific changes in basal glucose flux and insulin sensitivity were also measured by hyperinsulinemic euglycemic clamp studies. Molecular and cellular mechanisms were assessed in normal and type 2 diabetic human hepatocytes, as well as in mouse hepatocytes. Results Administration of a PHD inhibitor compound (PHDi) after the onset of obesity and insulin resistance improved glycemic control by increasing insulin and decreasing glucagon sensitivity in mice, independent of body weight change. Hyperinsulinemic euglycemic clamp studies revealed that these effects of PHDi treatment were mainly due to decreased basal hepatic glucose output and increased liver insulin sensitivity. Hepatocyte-specific deletion of HIF-2α markedly attenuated these effects of PHDi treatment, showing PHDi effects are HIF-2α dependent. At the molecular level, HIF-2α induced increased Irs2 and cyclic AMP-specific phosphodiesterase gene expression, leading to increased and decreased insulin and glucagon signaling, respectively. These effects of PHDi treatment were conserved in human and mouse hepatocytes. Conclusions Our results elucidate unknown mechanisms for how PHD inhibition improves glycemic control through HIF-2α-dependent regulation of hepatic insulin and glucagon sensitivity., Highlights • PHD inhibitor treatment improves glycemic control in obese glucose-intolerant mice. • PHD inhibitor treatment decreases liver glucagon sensitivity in obese mice. • The effects of PHD inhibition on glycemic control is hepatocyte HIF-2α-dependent. • PHD inhibitor treatment stimulates HIF-2α-dependent cAMP-specific PDE expression. • In human and mouse hepatocytes, PHD inhibitor treatment suppresses glucagon signaling.

Summary Insulin resistance is a major factor in obesity-linked type 2 diabetes. PPARγ is a master regulator of adipogenesis, and small molecule agonists, termed thiazolidinediones, are potent therapeutic insulin sensitizers. Here, we studied the role of transcriptional co-activator with PDZ-binding motif (TAZ) as a transcriptional co-repressor of PPARγ. We found that adipocyte-specific TAZ knockout (TAZ AKO) mice demonstrate a constitutively active PPARγ state. Obese TAZ AKO mice show improved glucose tolerance and insulin sensitivity compared to littermate controls. PPARγ response genes are upregulated in adipose tissue from TAZ AKO mice and adipose tissue inflammation was also decreased. In vitro and in vivo mechanistic studies revealed that the TAZ-PPARγ interaction is partially dependent on ERK-mediated Ser112 PPARγ phosphorylation. As adipocyte PPARγ Ser112 phosphorylation is increased in obesity, repression of PPARγ activity by TAZ could contribute to insulin resistance. These results identify TAZ as a new factor in the development of obesity-induced insulin resistance.

Decreased adipose tissue oxygen tension and increased HIF-1α expression can trigger adipose tissue inflammation and dysfunction in obesity. Our current understanding of obesity-associated decreased adipose tissue oxygen tension is mainly focused on changes in oxygen supply and angiogenesis. Here, we demonstrate that increased adipocyte O2 demand, mediated by ANT2 activity, is the dominant cause of adipocyte hypoxia. Deletion of adipocyte Ant2 improves obesity-induced intracellular adipocyte hypoxia by decreasing obesity-induced adipocyte oxygen demand, without effects on mitochondrial number or mass, or oligomycin-sensitive respiration. This led to decreased adipose tissue HIF-1α expression and inflammation with improved glucose tolerance and insulin resistance in both a preventative or therapeutic setting. Our results suggest that ANT2 may be a target for the development of insulin sensitizing drugs and that ANT2 inhibition might have clinical utility.

// Jason Peart 1, 2 , Jinming Li 1, 3 , Hojun Lee 1, 3 , Matthew Riopel 1 , Zhi-Chao Feng 1 and Rennian Wang 1, 3 1 Children's Health Research Institute, London, Ontario, N6C 2V5, Canada 2 Department of Pathology, University of Western Ontario, London, Ontario, N6A 3K7, Canada 3 Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario, N6A 3K7, Canada Correspondence to: Rennian Wang, email: rwang@uwo.ca Keywords: β1 integrin, mouse insulin promoter (MIP), glucose tolerance test, Cre recombinase, beta-cell mass Received: November 01, 2016 Accepted: April 21, 2017 Published: May 18, 2017 ABSTRACT β1 integrin is essential for pancreatic beta-cell development and maintenance in rodents and humans. However, the effects of a temporal beta-cell specific β1 integrin knockout on adult islet function are unknown. We utilized a mouse insulin 1 promoter driven tamoxifen-inducible Cre-recombinase β1 integrin knockout mouse model (MIPβ1KO) to investigate β1 integrin function in adult pancreatic beta-cells. Adult male MIPβ1KO mice were significantly glucose intolerant due to impaired glucose-stimulated insulin secretion in vivo and ex vivo at 8 weeks post-tamoxifen. The expression of Insulin and Pancreatic and duodenal homeobox-1 mRNA was significantly reduced in MIPβ1KO islets, along with reductions in insulin exocytotic proteins. Morphological analyses demonstrated that beta-cell mass, islet density, and the number of large-sized islets was significantly reduced in male MIPβ1KO mice. Significant reductions in the phosphorylation of signaling molecules focal adhesion kinase, extracellular signal-regulated kinases 1 and 2, and v-Akt murine thymoma viral oncogene were observed in male MIPβ1KO islets when compared to controls. MIPβ1KO islets displayed a significant increase in protein levels of the apoptotic marker cleaved-Poly (ADP-ribose) polymerase and a reduction of the cell cycle marker cyclin D1. Female MIPβ1KO mice did not develop glucose intolerance or reduced beta-cell mass until 16 weeks post-tamoxifen. Glucose intolerance remained in both genders of aged MIPβ1KO mice. This data demonstrates that β1 integrin is required for the maintenance of glucose homeostasis through postnatal beta-cell function and expansion.

Decreased adipose tissue oxygen tension and increased HIF-1α expression can trigger adipose tissue inflammation and dysfunction in obesity. Our current understanding of obesity-associated decreased adipose tissue oxygen tension is mainly focused on changes in oxygen supply and angiogenesis. Here, we demonstrate that increased adipocyte O2 demand, mediated by ANT2 activity, is the dominant cause of adipocyte hypoxia. Deletion of adipocyte Ant2 improves obesity-induced intracellular adipocyte hypoxia by decreasing obesity-induced adipocyte oxygen demand, without effects on mitochondrial number or mass, or oligomycin-sensitive respiration. This led to decreased adipose tissue HIF-1α expression and inflammation with improved glucose tolerance and insulin resistance in both a preventative or therapeutic setting. Our results suggest that ANT2 may be a target for the development of insulin sensitizing drugs and that ANT2 inhibition might have clinical utility.

Adipose tissue dysfunction leads to increased hepatocyte HIF-1α expression, promoting first-pass incretin hormone degradation., The decrease in incretin effects is an important etiologic component of type 2 diabetes with unknown mechanisms. In an attempt to understand obesity-induced changes in liver oxygen homeostasis, we found that liver HIF-1α expression was increased mainly by soluble factors released from obese adipocytes, leading to decreased incretin effects. Deletion of hepatocyte HIF-1α protected obesity-induced glucose intolerance without changes in body weight, liver steatosis, or insulin resistance. In-depth mouse metabolic phenotyping revealed that obesity increased first-pass degradation of an incretin hormone GLP-1 with increased liver DPP4 expression and decreased sinusoidal blood flow rate, reducing active GLP-1 levels in peripheral circulation. Hepatocyte HIF-1α KO blocked these changes induced by obesity. Deletion of hepatocyte HIF-2α did not change liver DPP4 expression but improved hepatic steatosis. Our results identify a previously unknown pathway for obesity-induced impaired beta cell glucose response (incretin effects) and the development of glucose intolerance through inter-organ communications.

The composition of the gastrointestinal microbiota and associated metabolites changes dramatically with diet and the development of obesity. Although many correlations have been described, specific mechanistic links between these changes and glucose homeostasis remain to be defined. Here we show that blood and intestinal levels of the microbiota-produced N-formyl peptide, formyl-methionyl-leucyl-phenylalanine, are elevated in high-fat diet–induced obese mice. Genetic or pharmacological inhibition of the N-formyl peptide receptor Fpr1 leads to increased insulin levels and improved glucose tolerance, dependent upon glucagon-like peptide 1. Obese Fpr1 knockout mice also display an altered microbiome, exemplifying the dynamic relationship between host metabolism and microbiota. Overall, we describe a new mechanism by which the gut microbiota can modulate glucose metabolism, providing a potential approach for the treatment of metabolic disease.

The nature of obesity-associated islet inflammation and its impact on β cell abnormalities remains poorly defined. Here, we explore immune cell components of islet inflammation and define their roles in regulating β cell function and proliferation. Islet inflammation in obese mice is dominated by macrophages. We identify two islet-resident macrophage populations, characterized by their anatomical distributions, distinct phenotypes, and functional properties. Obesity induces the local expansion of resident intra-islet macrophages, independent of recruitment from circulating monocytes. Functionally, intra-islet macrophages impair β cell function in a cell-cell contact-dependent manner. Increased engulfment of βcell insulin secretory granules by intra-islet macrophages in obese mice may contribute to restricting insulin secretion. In contrast, both intra- and peri-islet macrophage populations from obese mice promote β cell proliferation in a PDGFR signaling-dependent manner. Together, these data define distinct roles and mechanisms for islet macrophages in the regulation of islet β cells.

// Mark Trinder 1,2,* , Liangyi Zhou 1,3,* , Amanda Oakie 1,3 , Matthew Riopel 1 and Rennian Wang 1,2,4 1 Children’s Health Research Institute, London, Ontario, Canada 2 Departments of Physiology & Pharmacology, University of Western Ontario, London, Ontario, Canada 3 Department of Pathology, University of Western Ontario, London, Ontario, Canada 4 Department of Medicine, University of Western Ontario, London, Ontario, Canada * These authors have contributed equally to this study Correspondence to: Rennian Wang, email: // Keywords : inducible MIP-βIRKO, fetal pancreas, β-cell proliferation, islet vasculature, Pathology Section Received : October 28, 2015 Accepted : June 12, 2016 Published : June 30, 2016 Abstract The presence of insulin receptor (IR) on β-cells suggests that insulin has an autocrine/paracrine role in the regulation of β-cell function. It has previously been reported that the β-cell specific loss of IR (βIRKO) leads to the development of impaired glycemic regulation and β-cell death in mice. However, temporally controlled βIRKO induced during the distinct transitions of fetal pancreas development has yet to be investigated. We hypothesized that the presence of IR on β-cells during the 2 nd transition phase of the fetal murine pancreas is required for maintaining normal islet development.We utilized a mouse insulin 1 promoter driven tamoxifen-inducible Cre-recombinase IR knockout (MIP-βIRKO) mouse model to investigate the loss of β-cell IR during pancreatic development at embryonic day (e) 13, a phase of endocrine proliferation and β-cell fate determination. Fetal pancreata examined at e19-20 showed significantly reduced IR levels in the β-cells of MIP-βIRKO mice. Morphologically, MIP-βIRKO pancreata exhibited significantly enlarged islet size with increased β-cell area and proliferation. MIP-βIRKO pancreata also displayed significantly increased Igf-2 protein level and Akt activity with a reduction in phospho-p53 when compared to control littermates. Islet vascular formation and Vegf-a protein level was significantly increased in MIP-βIRKO pancreata.Our results demonstrate a developmental role for the β-cell IR, whereby its loss leads to an islet compensatory overgrowth, and contributes further information towards elucidating the temporally sensitive signaling during β-cell commitment.

We have previously reported that the fractalkine (FKN)/CX3CR1 system represents a novel regulatory mechanism for insulin secretion and β cell function. Here, we demonstrate that chronic administration of a long-acting form of FKN, FKN-Fc, can exert durable effects to improve glucose tolerance with increased glucose-stimulated insulin secretion and decreased β cell apoptosis in obese rodent models. Unexpectedly, chronic FKN-Fc administration also led to decreased α cell glucagon secretion. In islet cells, FKN inhibited ATP-sensitive potassium channel conductance by an ERK-dependent mechanism, which triggered β cell action potential (AP) firing and decreased α cell AP amplitude. This results in increased glucose-stimulated insulin secretion and decreased glucagon secretion. Beyond its islet effects, FKN-Fc also exerted peripheral effects to enhance hepatic insulin sensitivity due to inhibition of glucagon action. In hepatocytes, FKN treatment reduced glucagon-stimulated cAMP production and CREB phosphorylation in a pertussis toxin-sensitive manner. Together, these results raise the possibility of use of FKN-based therapy to improve type 2 diabetes by increasing both insulin secretion and insulin sensitivity.

The human fetal pancreas expresses a variety of extracellular matrix (ECM) binding receptors known as integrins. A provisional ECM protein found in blood clots that can bind to integrin receptors and promote β cell function and survival is fibrin. However, its role in support of human fetal pancreatic cells is unknown. We investigated how fibrin promotes human fetal pancreatic cell differentiation in vitro and in vivo. Human fetal pancreata were collected from 15 to 21 weeks of gestation and collagenase digested. Cells were then plated on tissue-culture polystyrene, or with 2D or 3D fibrin gels up to 2 weeks, or subcutaneously transplanted in 3D fibrin gels. The human fetal pancreas contained rich ECM proteins and expressed integrin αVβ3. Fibrin-cultured human fetal pancreatic cells had significantly increased expression of PDX-1, glucagon, insulin, and VEGF-A, along with increased integrin αVβ3 and phosphorylated FAK and p70(s6k). Fibrin-cultured cells treated with rapamycin, the mTOR pathway inhibitor, had significantly decreased phospho-p70(s6k) and PDX-1 expression. Transplanting fibrin-mixed cells into nude mice improved vascularization compared with collagen controls. These results suggest that fibrin supports islet cell differentiation via p70(s6k) and promotes vascularization in human fetal islet-epithelial clusters in vivo.

The interactions between c-Kit and its ligand, stem cell factor (SCF), play an important role in haematopoiesis, pigmentation and gametogenesis. c-Kit is also found in the pancreas, and recent studies have revealed that c-Kit marks a subpopulation of highly proliferative pancreatic endocrine cells that may harbour islet precursors. c-Kit governs and maintains pancreatic endocrine cell maturation and function via multiple signalling pathways. In this review we address the importance of c-Kit signalling within the pancreas, including its profound role in islet morphogenesis, islet vascularisation, and beta cell survival and function. We also discuss the impact of c-Kit signalling in pancreatic disease and the use of c-Kit as a potential target for the development of cell-based and novel drug therapies in the treatment of diabetes.

Fibrin is derived from fibrinogen during injury to produce a blood clot and thus promote wound repair. Composed of different domains, including Arg-Gly-Asp amino acid motifs, fibrin is used extensively as a hydrogel and sealant in the clinic. By binding to cell surface receptors like integrins and acting as a supportive 3D scaffold, fibrin has been useful in promoting cell differentiation, proliferation, function, and survival. In particular, fibrin has been able to maintain islet cell architecture, promote beta cell insulin secretion, and islet angiogenesis, as well as inducing a protective effect against cell death. During islet transplantation, fibrin improved neovascularization and islet function. These improvements resulted in reduced number of transplanted islets necessary to reverse diabetes. Therefore, fibrin, as a biocompatible and biodegradable scaffold, should be considered during subcutaneous islet transplantation and beta cell expansion in vitro to ensure maintenance of islet cell function, proliferation, and survival to develop effective cell-based therapies for the treatment of diabetes.

Chromogranin A (CgA) is a prohormone and a granulogenic factor that regulates secretory pathways in neuroendocrine tissues. In β-cells of the endocrine pancreas, CgA is a major cargo in insulin secretory vesicles. The impact of CgA deficiency on the formation and exocytosis of insulin vesicles is yet to be investigated. In addition, no literature exists on the impact of CgA on mitochondrial function in β-cells. Using three different antibodies, we demonstrate that CgA is processed to vasostatin- and catestatin-containing fragments in pancreatic islet cells. CgA deficiency in Chga-KO islets leads to compensatory overexpression of chromogranin B, secretogranin II, SNARE proteins and insulin genes, as well as increased insulin protein content. Ultrastructural studies of pancreatic islets revealed that Chga-KO β-cells contain fewer immature secretory granules than wild-type (WT) control but increased numbers of mature secretory granules and plasma membrane-docked vesicles. Compared to WT control, CgA-deficient β-cells exhibited increases in mitochondrial volume, numerical densities and fusion, as well as increased expression of nuclear encoded genes (Ndufa9, Ndufs8, Cyc1 and Atp5o). These changes in secretory vesicles and the mitochondria likely contribute to the increased glucose-stimulated insulin secretion observed in Chga-KO mice. We conclude that CgA is an important regulator for coordination of mitochondrial dynamics, secretory vesicular quanta and GSIS for optimal secretory functioning of β-cells, suggesting a strong, CgA-dependent positive link between mitochondrial fusion and GSIS.

c-Kit and its ligand stem cell factor (SCF) are important for β-cell survival and maturation; meanwhile, interactions between the Fas receptor (Fas) and Fas ligand are capable of triggering β-cell apoptosis. Disruption of c-Kit signaling leads to severe loss of β-cell mass and function with upregulation of Fas expression in c-Kit Wv/+ mouse islets, suggesting that there is a critical balance between c-Kit and Fas activation in β-cells. In the present study, we investigated the interrelationship between c-Kit and Fas activation that mediates β-cell survival and function. We generated double mutant, c-Kit Wv/+ ;Fas lpr/lpr ( Wv −/−), mice to study the physiological and functional role of Fas with respect to β-cell function in c-Kit Wv/+ mice. Isolated islets from these mice and the INS-1 cell line were used. We observed that islets in c-Kit Wv/+ mice showed a significant increase in β-cell apoptosis along with upregulated p53 and Fas expression. These results were verified in vitro in INS-1 cells treated with SCF or c-Kit siRNA combined with a p53 inhibitor and Fas siRNA. In vivo, Wv −/− mice displayed improved β-cell function, with significantly enhanced insulin secretion and increased β-cell mass and proliferation compared with Wv +/+ mice. This improvement was associated with downregulation of the Fas-mediated caspase-dependent apoptotic pathway and upregulation of the cFlip/NF-κB pathway. These findings demonstrate that a balance between the c-Kit and Fas signaling pathways is critical in the regulation of β-cell survival and function.

The receptor tyrosine kinase, c-Kit, and its ligand, stem cell factor, control a variety of cellular processes, including pancreatic beta cell survival and differentiation as revealed in c-Kit ( Wv ) mice, which have a point mutation in the c-Kit allele leading to loss of kinase activity and develop diabetes. The present study further investigated the intrinsic role of c-Kit in beta cells, especially the underlying mechanisms that influence beta cell function.We generated a novel transgenic mouse model with c-KIT overexpression specifically in beta cells (c-KitβTg) to further examine the physiological and functional roles of c-Kit in beta cells. Isolated islets from these mice were used to investigate the underlying molecular pathway of c-Kit in beta cells. We also characterised the ability of c-Kit to protect animals from high-fat-diet-induced diabetes, as well as to rescue c-Kit ( Wv ) mice from early onset of diabetes.c-KitβTg mice exhibited improved beta cell function, with significantly improved insulin secretion, and increased beta cell mass and proliferation in response to high-fat-diet-induced diabetes. c-KitβTg islets exhibited upregulation of: (1) insulin receptor and IRSs; (2) Akt and glycogen synthase kinase 3β phosphorylation; and (3) transcription factors important for islet function. c-KIT overexpression in beta cells also rescued diabetes observed in c-Kit ( Wv ) mice.These findings demonstrate that c-Kit plays a direct protective role in beta cells, by regulating glucose metabolism and beta cell function. c-Kit may therefore represent a novel target for treating diabetes.

Previous studies have shown that the stem cell marker, c-Kit, is involved in glucose homeostasis. We recently reported that c-KitWv/+ male mice displayed the onset of diabetes at 8 weeks of age; however, the mechanisms by which c-Kit regulates β-cell proliferation and function are unknown. The purpose of this study is to examine if c-KitWv/+ mutation-induced β-cell dysfunction is associated with downregulation of the phospho-Akt/Gsk3β pathway in c-KitWv/+ male mice. Histology and cell signaling were examined in C57BL/6J/KitWv/+ (c-Kit Wv/+) and wild-type (c-Kit+/+) mice using immunofluorescence and western blotting approaches. The Gsk3β inhibitor, 1-azakenpaullone (1-AKP), was administered to c-KitWv/+ and c-Kit+/+ mice for 2 weeks, whereby alterations in glucose metabolism were examined and morphometric analyses were performed. A significant reduction in phosphorylated Akt was observed in the islets of c-KitWv/+ mice (P

β1-Integrin, a critical regulator of β cell survival and function, has been shown to protect against cell death and promote insulin expression and secretion in rat and human islet cells in vitro. The aim of the present study was to examine whether the knockout of β1-integrin in collagen I-producing cells would have physiological and functional implications in pancreatic endocrine cells in vivo. Using adult mice with a conditional knockout of β1-integrin in collagen I-producing cells, the effects of β1-integrin deficiency on glucose metabolism and pancreatic endocrine cells were examined. Male β1-integrin-deficient mice display impaired glucose tolerance, with a significant reduction in pancreatic insulin content (p < 0.01). Morphometric analysis revealed a significant reduction in β cell mass (p < 0.001) in β1-integrin-deficient mice, along with a significant decrease in β cell proliferation, Pdx-1 and Nkx6.1 expression when compared with controls. Interestingly, these physiological and morphometric alterations in female β1-integrin-deficient mice were less significant. Furthermore, β1-integrin-deficient mice displayed decreased FAK (p < 0.05) and ERK1/2 (p < 0.001) phosphorylation, reduced cyclin D1 levels (p < 0.001) and increased caspase 3 cleavage (p < 0.01), while no changes in Akt phosphorylation were observed, indicating that the β1-integrin signals through the FAK-MAPK-ERK pathway in vivo. Our results demonstrate that β1-integrin is involved in the regulation of glucose metabolism and contributes to the maintenance of β cell survival and function in vivo.

MiRNAs are regulatory molecules that can be packaged into exosomes and secreted from cells. Here, we show that adipose tissue macrophages (ATMs) in obese mice secrete miRNA-containing exosomes (Exos), which cause glucose intolerance and insulin resistance when administered to lean mice. Conversely, ATM Exos obtained from lean mice improve glucose tolerance and insulin sensitivity when administered to obese recipients. miR-155 is one of the miRNAs overexpressed in obese ATM Exos, and earlier studies have shown that PPARγ is a miR-155 target. Our results show that miR-155KO animals are insulin sensitive and glucose tolerant compared to controls. Furthermore, transplantation of WT bone marrow into miR-155KO mice mitigated this phenotype. Taken together, these studies show that ATMs secrete exosomes containing miRNA cargo. These miRNAs can be transferred to insulin target cell types through mechanisms of paracrine or endocrine regulation with robust effects on cellular insulin action, in vivo insulin sensitivity, and overall glucose homeostasis.

Development of the human pancreas is well-known to involve tightly controlled differentiation of pancreatic precursors to mature cells that express endocrine- or exocrine-specific protein products. However, details of human pancreatic development at the ultrastructural level are limited. The present study analyzed 8–20 week fetal age human pancreata using scanning and transmission electron microscopy (TEM), TEM immunogold and double or triple immunofluorescence staining. Primary organization of islets and acini occurred during the developmental period examined. Differentiating endocrine and exocrine cells developed from the ductal tubules and subsequently formed isolated small clusters. Extracellular matrix fibers and proteins accumulated around newly differentiated cells during their migration and cluster formation. Glycogen expression was robust in ductal cells of the pancreas from 8–15 weeks of fetal age; however, this became markedly reduced at 20 weeks, with a concomitant increase in acinar cell glycogen content. Insulin secretory granules transformed from being dense and round at 8 weeks to distinct geometric (multilobular, crystalline) structures by 14–20 weeks. Initially many of the differentiating endocrine cells were multihormonal and contained polyhormonal granules; by 20 weeks, monohormonal cells were in the majority. Interestingly, certain secretory granules in the early human fetal pancreatic cells showed positivity for both exocrine (amylase) and endocrine proteins. This combined ultrastructural and immunohistochemical study showed that, during early developmental stages, the human pancreas contains differentiating epithelial cells that associate closely with the extracellular matrix, have dynamic glycogen expression patterns and contain polyhormonal as well as mixed endocrine/ exocrine granules.

Extracellular matrix (ECM)-integrin stimulation can promote beta cell differentiation, proliferation and function. However, beta cells lose their insulin secretion function in response to glucose stimulation, and senesce when cultured with ECM proteins for a long time. Fibrin is a provisional ECM protein that is capable of maintaining beta cell function, yet the mechanisms by which this occurs is unknown. The present study examined how fibrin interacts with integrin receptors to promote beta cell cluster formation, proliferation and function. The rat insulinoma cell line, INS-1, was cultured on tissue-culture polystyrene, or with 2-D or 3-D fibrin gels for up to 4 weeks. Cells cultured with fibrin formed islet-like clusters and showed direct contacts with fibrin determined by scanning electron microscopy. Fibrin-cultured INS-1 cells also had significantly increased glucose-stimulated insulin secretion. A significant increase in integrin αvβ3 protein and phosphorylated FAK, Erk1/2 and Akt levels was observed in fibrin-cultured INS-1 cells, which was associated with significantly increased cell proliferation and decreased cell apoptosis. Integrin αvβ3 blockade affected INS-1 cell spreading on fibrin gels, and resulted in significantly decreased FAK phosphorylation and increased cleaved caspase-3 levels. These results show that fibrin promotes beta cell function, proliferation and survival via integrin αvβ3 interactions.

Integrin receptors are responsible for integrating extracellular matrix signals inside the cell. The most prominent integrin receptor, β1 integrin, has a role in cell function, survival and differentiation. Recently, we demonstrated a profound in vivo role of β1 integrin expression in the pancreas on glucose homeostasis and islet function. Here, we extend these results by examining the role of β1 integrin in exocrine pancreatic structure and function. Adult C57Bl/6 mice hemizygous for a collagen type Iα2 (Col1a2) promoter-controlled tamoxifen-inducible Cre recombinase gene and homozygous for loxP-β1 integrin were injected with tamoxifen or corn oil to generate mice deleted or not for β1 integrin. Pancreata derived from these male mice were analyzed by quantitative reverse transcriptase-polymerase chain reaction, western blot and immunofluorescence. Our results showed that β1 integrin-deficient mice displayed a significant decrease in pancreas weight with a significant reduction of amylase, regenerating islet-derived protein II and carboxypeptidase-A expression (P

Diabetes mellitus is a chronic condition resulting from insufficient β-cell mass, which leads to improper glycemia regulation. Research efforts have focused on expanding islets and β-cells in vitro for use in cell-based therapies to cure diabetes. Collagens are triple-helix extracellular matrix proteins with widespread expression in mammals. With multiple functions, collagen can provide structural integrity in addition to mediating cellular signaling. In the pancreas, collagens I and IV are abundant and support cell structures while also stimulating cell surface receptors to influence pancreatic cell processes. Collagen-based materials and scaffolds have also been used to assist in the maintenance and expansion of islet cells in vitro, primarily through integrin and discoindin domain receptors. Islet transplantation using collagen-based scaffolds may improve long-term glycemic control, but progressive research efforts are required to realize this potential in humans. This review will outline the critical role played by native collagens I and IV and their receptors in maintaining islet function. The advantages of using collagens I and IV as culture gels/scaffolds and islet encapsulation vehicles for transplantation will be described.