Search

A variety of islet autoantibodies (AAbs) can predict and possibly dictate eventual type 1 diabetes (T1D) diagnosis. Upwards of 75% of those with T1D are positive for AAbs against glutamic acid decarboxylase (GAD65 or GAD), a producer of gamma-aminobutyric acid (GABA) in human pancreatic beta cells. Interestingly, bacterial populations within the human gut also express GAD and produce GABA. Evidence suggests that dysbiosis of the microbiome may correlate with T1D pathogenesis and physiology. Therefore, autoimmune linkages between the gut microbiome and islets susceptible to autoimmune attack need to be further elucidated. Utilizing in silico analyses, we show that 25 GAD sequences from human gut bacterial sources show sequence and motif similarities to human beta cell GAD65. Our motif analyses determined that most gut GAD sequences contain the pyroxical dependent decarboxylase (PDD) domain of human GAD65, which is important for its enzymatic activity. Additionally, we showed overlap with known human GAD65 T cell receptor epitopes, which may implicate the immune destruction of beta cells. Thus, we propose a physiological hypothesis in which changes in the gut microbiome in those with T1D result in a release of bacterial GAD, thus causing miseducation of the host immune system. Due to the notable similarities we found between human and bacterial GAD, these deputized immune cells may then target human beta cells leading to the development of T1D., Competing Interests: The authors have declared that no competing interests exist.

The liberation of energy stores from adipocytes is critical to support survival in times of energy deficit; however, uncontrolled or chronic lipolysis associated with insulin resistance and/or insulin insufficiency disrupts metabolic homeostasis 1,2 . Coupled to lipolysis is the release of a recently identified hormone, fatty-acid-binding protein 4 (FABP4) 3 . Although circulating FABP4 levels have been strongly associated with cardiometabolic diseases in both preclinical models and humans 4-7 , no mechanism of action has yet been described 8-10 . Here we show that hormonal FABP4 forms a functional hormone complex with adenosine kinase (ADK) and nucleoside diphosphate kinase (NDPK) to regulate extracellular ATP and ADP levels. We identify a substantial effect of this hormone on beta cells and given the central role of beta-cell function in both the control of lipolysis and development of diabetes, postulate that hormonal FABP4 is a key regulator of an adipose-beta-cell endocrine axis. Antibody-mediated targeting of this hormone complex improves metabolic outcomes, enhances beta-cell function and preserves beta-cell integrity to prevent both type 1 and type 2 diabetes. Thus, the FABP4-ADK-NDPK complex, Fabkin, represents a previously unknown hormone and mechanism of action that integrates energy status with the function of metabolic organs, and represents a promising target against metabolic disease., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Understanding mechanisms of immune regulation is key to developing immunotherapies for autoimmunity and cancer. We examined the role of mononuclear phagocytes during peripheral T cell regulation in type 1 diabetes and melanoma. MERTK expression and activity in mononuclear phagocytes in the pancreatic islets promoted islet T cell regulation, resulting in reduced sensitivity of T cell scanning for cognate antigen in prediabetic islets. MERTK-dependent regulation led to reduced T cell activation and effector function at the disease site in islets and prevented rapid progression of type 1 diabetes. In human islets, MERTK-expressing cells were increased in remaining insulin-containing islets of type 1 diabetic patients, suggesting that MERTK protects islets from autoimmune destruction. MERTK also regulated T cell arrest in melanoma tumors. These data indicate that MERTK signaling in mononuclear phagocytes drives T cell regulation at inflammatory disease sites in peripheral tissues through a mechanism that reduces the sensitivity of scanning for antigen leading to reduced responsiveness to antigen., Competing Interests: Disclosures: The authors declare no competing interests exist., (© 2021 Lindsay et al.)

Proteasomal spliced peptides (PSPs) have been identified in the class I major histocompatibility complex (MHC) peptidomes of several tumors and have emerged as novel neoantigens that can stimulate highly specific T cells. Much debate has surrounded the percentage of PSPs in the immunopeptidome; reported numbers have ranged from <1-5% to 12-45%. Recently, our laboratory demonstrated in nonobese diabetic (NOD) mice that hybrid insulin peptides (HIPs), a special class of spliced peptides, are formed during insulin granule degradation in crinosomes of the pancreatic β cells and that modified peptides comprised a significant source of false positive HIP assignments. Herein, this study is extended to crinosomes isolated from other mouse strains and to two recent MHC class I studies, to see if modified peptides explained discrepancies in reported percentages of PSPs. This analysis revealed that both MHC-I peptidomes contained many spectra erroneously assigned as PSPs. While many false positive PSPs did arise from modified peptides, others arose from probable data processing errors. Thus, the reported numbers of PSPs in the literature are likely elevated due to errors associated with data processing and analysis., (© 2021 Wiley-VCH GmbH.)

Mitogen-activated protein kinase phosphatase-5 (MKP-5) is a regulator of extracellular signaling that is known to regulate lipid metabolism. In this study, we found that obesity caused by a high-fat diet (HFD) decreased the expression of MKP-5 in the pancreas and primary islet cells derived from mice. Then, we further investigated the role of MKP-5 in the protection of islet cells from lipotoxicity by modulating MKP-5 expression. As a critical inducer of lipotoxicity, palmitic acid (PA) was used to treat islet β-cells. We found that MKP-5 overexpression restored PA-mediated autophagy inhibition in Rin-m5f cells and protected these cells from PA-induced apoptosis and dysfunction. Consistently, a lack of MKP-5 aggravated the adverse effects of lipotoxicity. Islet cells from HFD-fed mice were infected using recombinant adenovirus expressing MKP-5 (Ad-MKP-5), and we found that Ad-MKP-5 was able to alleviate HFD-induced apoptotic protein activation and relieve the HFD-mediated inhibition of functional proteins. Notably, HFD-mediated impairments in autophagic flux were restored by Ad-MKP-5 transduction. Furthermore, the autophagy inhibitor 3-methyladenine (3-MA) was used to treat Rin-m5f cells, confirming that the MKP-5 overexpression suppressed apoptosis, dysfunction, inflammatory response, and oxidative stress induced by PA via improving autophagic signaling. Lastly, employing c-Jun amino-terminal kinas (JNK), P38, or extracellular-regulated kinase (ERK) inhibitors, we established that the JNK and P38 MAPK pathways were involved in the MKP-5-mediated apoptosis, dysfunction, and autophagic inhibition observed in islet β cells in response to lipotoxicity.

Type 1 diabetes is an autoimmune disease resulted from self-destruction of insulin-producing pancreatic β cells. However, the pathological pathways that trigger the autoimmune destruction remain poorly understood. Clinical studies have demonstrated close associations of neutrophils and neutrophil elastase (NE) with β-cell autoimmunity in patients with Type 1 diabetes. The present study aims to investigate the impact of NE inhibition on development of autoimmune diabetes in NOD mice. NE pharmacological inhibitor (sivelestat) or biological inhibitor (elafin) was supplemented into NOD mice to evaluate their effects on islet inflammation and diabetogenesis. The impact of NE inhibition on innate and adaptive immune cells was measured with flow cytometry and immunohistochemistry. A significant but transient increase in neutrophil infiltration accompanied with elevated NE activity was observed in the neonatal period of NOD mice. Treatment of NOD mice with sivelestat or elafin at the early age led to a marked reduction in spontaneous development of insulitis and autoimmune diabetes. Mechanistically, inhibition of NE significantly attenuated infiltration of macrophages and islet inflammation, thus ameliorating cytotoxic T cell-mediated autoimmune attack of pancreatic β cells. In vitro studies showed that NE directly induced inflammatory responses in both min6 β cells and RAW264.7 macrophages, and promoted macrophage migration. These findings support an important role of NE in triggering the onset and progression of β-cell autoimmunity, and suggest that pharmacological inhibition of NE may represent a promising therapeutic strategy for treatment of autoimmune diabetes., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Chronic exposure of pancreatic β-cells to elevated nutrient levels impairs their function and potentially induces apoptosis. Like in other cell types, AMPK is activated in β-cells under conditions of nutrient deprivation, while little is known on AMPK responses to metabolic stresses. Here, we first reviewed recent studies on the role of AMPK activation in β-cells. Then, we investigated the expression profile of AMPK pathways in β-cells following metabolic stresses. INS-1E β-cells and human islets were exposed for 3 days to glucose (5.5-25 mM), palmitate or oleate (0.4 mM), and fructose (5.5 mM). Following these treatments, we analyzed transcript levels of INS-1E β-cells by qRT-PCR and of human islets by RNA-Seq; with a special focus on AMPK-associated genes, such as the AMPK catalytic subunits α1 ( Prkaa1 ) and α2 ( Prkaa2 ). AMPKα and pAMPKα were also evaluated at the protein level by immunoblotting. Chronic exposure to the different metabolic stresses, known to alter glucose-stimulated insulin secretion, did not change AMPK expression, either in insulinoma cells or in human islets. Expression profile of the six AMPK subunits was marginally modified by the different diabetogenic conditions. However, the expression of some upstream kinases and downstream AMPK targets, including K-ATP channel subunits, exhibited stress-specific signatures. Interestingly, at the protein level, chronic fructose treatment favored fasting-like phenotype in human islets, as witnessed by AMPK activation. Collectively, previously published and present data indicate that, in the β-cell, AMPK activation might be implicated in the pre-diabetic state, potentially as a protective mechanism.

Type 1 diabetes (T1D) results from autoimmune destruction of insulin-producing beta cells in pancreatic islets. The degradation of the glycosaminoglycan heparan sulfate (HS) by the endo-β-D-glycosidase heparanase plays a critical role in multiple stages of the disease process. Heparanase aids (i) migration of inflammatory leukocytes from the vasculature to the islets, (ii) intra-islet invasion by insulitis leukocytes, and (iii) selective destruction of beta cells. These disease stages are marked by the solubilization of HS in the subendothelial basement membrane (BM), HS breakdown in the peri-islet BM, and the degradation of HS inside beta cells, respectively. Significantly, healthy islet beta cells are enriched in highly sulfated HS which is essential for their viability, protection from damage by reactive oxygen species (ROS), beta cell function and differentiation. Consequently, mouse and human beta cells but not glucagon-producing alpha cells (which contain less-sulfated HS) are exquisitely vulnerable to heparanase-mediated damage. In vitro, the death of HS-depleted mouse and human beta cells can be prevented by HS replacement using highly sulfated HS mimetics or analogues. T1D progression in NOD mice and recent-onset T1D in humans correlate with increased expression of heparanase by circulating leukocytes of myeloid origin and heparanase-expressing insulitis leukocytes. Treatment of NOD mice with the heparanase inhibitor and HS replacer, PI-88, significantly reduced T1D incidence by 50%, impaired the development of insulitis and preserved beta cell HS. These outcomes identified heparanase as a novel destructive tool in T1D, distinct from the conventional cytotoxic and apoptosis-inducing mechanisms of autoreactive T cells. In contrast to exogenous catalytically active heparanase, endogenous heparanase may function in HS homeostasis, gene expression and insulin secretion in normal beta cells and immune gene expression in leukocytes. In established diabetes, the interplay between hyperglycemia, local inflammatory cells (e.g. macrophages) and heparanase contributes to secondary micro- and macro-vascular disease. We have identified dual activity heparanase inhibitors/HS replacers as a novel class of therapeutic for preventing T1D progression and potentially for mitigating secondary vascular disease that develops with long-term T1D.

Understanding how cell subpopulations in a tissue impact overall system function is challenging. There is extensive heterogeneity among insulin-secreting β-cells within islets of Langerhans, including their insulin secretory response and gene expression profile, and this heterogeneity can be altered in diabetes. Several studies have identified variations in nutrient sensing between β-cells, including glucokinase (GK) levels, mitochondrial function, or expression of genes important for glucose metabolism. Subpopulations of β-cells with defined electrical properties can disproportionately influence islet-wide free-calcium activity ([Ca 2+ ]) and insulin secretion via gap-junction electrical coupling. However, it is poorly understood how subpopulations of β-cells with altered glucose metabolism may impact islet function. To address this, we utilized a multicellular computational model of the islet in which a population of cells deficient in GK activity and glucose metabolism was imposed on the islet or in which β-cells were heterogeneous in glucose metabolism and GK kinetics were altered. This included simulating GK gene (GCK) mutations that cause monogenic diabetes. We combined these approaches with experimental models in which gck was genetically deleted in a population of cells or GK was pharmacologically inhibited. In each case, we modulated gap-junction electrical coupling. Both the simulated islet and the experimental system required 30-50% of the cells to have near-normal glucose metabolism, fewer than cells with normal K ATP conductance. Below this number, the islet lacked any glucose-stimulated [Ca 2+ ] elevations. In the absence of electrical coupling, the change in [Ca 2+ ] was more gradual. As such, electrical coupling allows a large minority of cells with normal glucose metabolism to promote glucose-stimulated [Ca 2+ ]. If insufficient numbers of cells are present, which we predict can be caused by a subset of GCK mutations that cause monogenic diabetes, electrical coupling exacerbates [Ca 2+ ] suppression. This demonstrates precisely how metabolically heterogeneous β-cell populations interact to impact islet function., (Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Background/aims: This study was performed to determine whether adding coenzyme Q10 (CoQ10) to metformin (MET) has a beneficial effect as a treatment for sirolimus (SRL)-induced diabetes mellitus (DM)., Methods: DM was induced in rats by daily treatment with SRL (0.3 mg/kg, subcutaneous) for 28 days, and animals were treated with CoQ10 (20 mg/kg, oral) and MET (250 mg/kg, oral) alone or in combination for the latter 14 days of SRL treatment. The effects of CoQ10 and MET on SRL-induced DM were assessed with the intraperitoneal glucose tolerance test (IPGTT) and by determining plasma insulin concentration and the homeostatic model assessment of insulin resistance (HOMA-R) index. We also evaluated the effect of CoQ10 on pancreatic islet size, apoptosis, oxidative stress, and mitochondria morphology., Results: IPGTT revealed overt DM in SRL-treated rats. The addition of CoQ10 to MET further improved hyperglycemia, decreased HOMA-R index, and increased plasma insulin concentration compared with the SRL group than MET alone therapy. While SRL treatment induced smaller islets with decreased insulin staining intensity, the combination of CoQ10 and MET significantly improved insulin staining intensity, which was accompanied by a reduction in oxidative stress and apoptosis. In addition, co-treatment of CoQ10 and MET significantly increased the levels of antiperoxidative enzymes in the pancreas islet cells compared with MET. At the subcellular level, addition of CoQ10 to MET improved the average mitochondrial area and insulin granule number., Conclusion: Addition of CoQ10 to MET has a beneficial effect on SRL-induced DM compared to MET alone.

Viral infection of the insulin-producing cells in the pancreas has been proposed in the etiology of type 1 diabetes. Protein kinase R (PKR) is a cytoplasmic protein activated through phosphorylation in response to cellular stress and particularly viral infection. As PKR expression in pancreatic beta-cells has been interpreted as a viral footprint, this cross-sectional study aimed at characterizing the PKR expression in non-diabetic human pancreases. PKR expression was evaluated in pancreas tissue from 16 non-diabetic organ donors, using immunohistochemistry, qPCR, and western blot. Immunohistochemistry and western blot showed readily detectable PKR expression in the pancreatic parenchyma. The qPCR detected PKR mRNA in both endocrine and exocrine samples, with a slightly higher expression in the islets. In conclusion, PKR is constitutively expressed in both endocrine and exocrine parts of the pancreas and its expression should not be interpreted as a viral footprint in pancreatic beta cells.

Diabetes mellitus is characterized by hyperglycemia which causes oxidative stress. Propolis has been reported to have antihyperglycemic and antioxidant potentials. The present study therefore examined the anti-hyperglycemic, antioxidant and anti-inflammatory activities of Malaysian propolis (MP) using streptozotocin-induced diabetic rats. Ethanol extract of MP showed in vitro antioxidant (DPPH, FRAP and H2O2 radical scavenging) and α-glucosidase inhibition activities. Male Sprague Dawley rats were either treated with distilled water (normal control and diabetic control), MP (300 mg/kg b. w.), metformin (Met) (300 mg/kg b. w.) or both. After four weeks, fasting blood glucose decreased, while body weight change and serum insulin level increased significantly in MP, Met and MP + Met treated diabetic groups compared to diabetic control (DC) group. Furthermore, pancreatic antioxidant enzymes, total antioxidant capacity, interleukin (IL)-10 and proliferating cell nuclear antigen increased, while malondialdehyde, nuclear factor-kappa B (p65), tumor necrosis factor alpha, IL-1β and cleaved caspase-3 decreased significantly in the treated diabetic groups compared to DC group. Histopathology of the pancreas showed increased islet area and number of beta cells in the treated groups, compared to DC group, with D + MP + Met group comparable to normal control. We conclude that MP has anti-hyperglycemic, antioxidant, anti-inflammatory and antiapoptotic potentials, and exhibits synergistic effect with metformin., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The regulatory mechanisms that ensure an accurate control of gene transcription are central to cellular function, development and disease. Such mechanisms rely largely on noncoding regulatory sequences that allow the establishment and maintenance of cell identity and tissue-specific cellular functions.The study of chromatin structure and nucleosome positioning allowed revealing transcription factor accessible genomic sites with regulatory potential, facilitating the comprehension of tissue-specific cis-regulatory networks. Recently a new technique coupled with high-throughput sequencing named Assay for Transposase Accessible Chromatin (ATAC-seq) emerged as an efficient method to chart open chromatin genome wide. The application of such technique to different cell types allowed unmasking tissue-specific regulatory elements and characterizing cis-regulatory networks. Herein we describe the implementation of the ATAC-seq method to human pancreatic islets, a tissue playing a central role in the control of glucose metabolism.

Vaspin (visceral adipose tissue-derived serine protease inhibitor) is a recently discovered adipokine that has been implicated in diabetes mellitus and other metabolic disorders. However, the effects of vaspin on pancreatic β cell function and related mechanisms are not fully understood. Thus, the present study was performed to investigate the effects of vaspin on pancreatic β cell function and the potential underlying mechanisms. Both in vitro (rat insulinoma cells, INS-1) and in vivo (high fat diet fed rats) experiments were conducted. The results showed that vaspin significantly increased INS-1 cell secretory function. Potential mechanisms were explored using inhibitors, western blot and real-time PCR techniques. We found that vaspin increased the levels of IRS-2 mRNA and IRS-2 total protein, while decreased the serine phosphorylation level of IRS-2 protein. Moreover, vaspin increased the Akt phosphorylation protein level which was reversed by PI3K inhibitor ly294002. In addition, vaspin increased the phosphorylation levels of mTOR and p70S6K, which was inhibited by rapamycin. Meanwhile, we found that the NF-κB mRNA and protein levels were reduced after vaspin treatment, similar to the effect of NF-κB inhibitor TPCK. Furthermore, vaspin increased the glucose stimulated insulin secretion (GSIS) level, lowered blood glucose level and improved the glucose tolerance and insulin sensitivity of high fat diet fed rats. Hyperglycemic clamp test manifested that vaspin improved islet β cell function. Together, these findings provide a new understanding of the function of vaspin on pancreatic β cell and suggest that it may serve as a potential agent for the prevention and treatment of type 2 diabetes.

Increasing evidence suggests a crucial role of inflammation in cytokine-mediated β-cell dysfunction and death in type 1 diabetes mellitus, although the mechanisms are incompletely understood. Sphingosine 1-phosphate (S1P) is a multifunctional bioactive sphingolipid involved in the development of many autoimmune and inflammatory diseases. Here, we investigated the role of intracellular S1P in insulin-secreting INS1E cells by genetically manipulating the S1P-metabolizing enzyme S1P lyase (SPL). The expression of spl was down-regulated by cytokines in INS1E cells and rat islets. Overexpression of SPL protected against cytokine toxicity. Interestingly, the SPL overexpression did not suppress the cytokine-induced NFκB-iNOS-NO pathway but attenuated calcium leakage from endoplasmic reticulum (ER) stores as manifested by lower cytosolic calcium levels, higher expression of the ER protein Sec61a, decreased dephosphorylation of Bcl-2-associated death promoter (Bad) protein, and weaker caspase-3 activation in cytokine-treated (IL-1β, TNFα, and IFNγ) cells. This coincided with reduced cytokine-mediated ER stress, indicated by measurements of CCAAT/enhancer-binding protein homologous protein ( chop ) and immunoglobulin heavy chain binding protein ( bip ) levels. Moreover, cytokine-treated SPL-overexpressing cells exhibited increased expression of prohibitin 2 (Phb2), involved in the regulation of mitochondrial assembly and respiration. SPL-overexpressing cells were partially protected against cytokine-mediated ATP reduction and inhibition of glucose-induced insulin secretion. siRNA-mediated spl suppression resulted in effects opposite to those observed for SPL overexpression. Knockdown of phb2 partially reversed beneficial effects of SPL overexpression. In conclusion, the relatively low endogenous Spl expression level in insulin-secreting cells contributes to their extraordinary vulnerability to proinflammatory cytokine toxicity and may therefore represent a promising target for β-cell protection in type 1 diabetes mellitus., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Oxidative stress is a major cause of islet injury and dysfunction during isolation and transplantation procedures. Cyanidin-3-O-glucoside (C3G), which is present in various fruits and vegetables especially in Chinese bayberry, shows a potent antioxidant property. In this study, we determined whether C3G could protect neonatal porcine islets (NPI) from reactive oxygen species (H 2 O 2 )-induced injury in vitro and promote the function of NPI in diabetic mice. We found that C3G had no deleterious effect on NPI and that C3G protected NPI from damage induced by H 2 O 2 Significantly higher hemeoxygenase-1 ( HO1 ) gene expression was detected in C3G-treated NPI compared to untreated islets before and after transplantation ( P  < 0.05). Western blot analysis showed a significant increase in the levels of phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphatidylinositol 3-kinase (PI3K/Akt) proteins in C3G-treated NPI compared to untreated islets. C3G induced the nuclear translocation of nuclear erythroid 2-related factor 2 (NRF2) and the significant elevation of HO1 protein. Recipients of C3G-treated NPI with or without C3G-supplemented drinking water achieved normoglycemia earlier compared to recipients of untreated islets. Mice that received C3G-treated islets with or without C3G-supplemented water displayed significantly lower blood glucose levels at 5-10 weeks post-transplantation compared to mice that received untreated islets. Mice that received C3G-treated NPI and C3G-supplemented drinking water had significantly ( P  < 0.05) lower blood glucose levels at 7 and 8 weeks post-transplantation compared to mice that received C3G-treated islets. These findings suggest that C3G has a beneficial effect on NPI through the activation of ERK1/2- and PI3K/AKT-induced NRF2-mediated HO1 signaling pathway., (© 2017 Society for Endocrinology.)

Efficient islet release from the pancreas requires the combination of collagenase, neutral protease (cNP), or thermolysin (TL). Recently, it has been shown that clostripain (CP) may also contribute to efficient islet release from the human pancreas. The aim of this study was to evaluate the impact of these proteases on human islet integrity in a prospective approach. Islets were isolated from the pancreas of 10 brain-dead human organ donors. Purified islets were precultured for 3 to 4 d at 37 °C to ensure that preparations were cleared of predamaged islets, and only integral islets were subjected to 90 min of incubation at 37 °C in Hank's balanced salt solution supplemented with cNP, TL, or CP. The protease concentrations were calculated for a pancreas of 100 g trimmed weight utilizing 120 dimethyl-casein units of cNP, 70,000 caseinase units of TL, or 200 benzoyl-l-arginine-ethyl-ester units of CP (1×). These activities were then increased both 5× and 10×. After subsequent 24-h culture in enzyme-free culture medium, treated islets were assessed and normalized to sham-treated controls. Compared with controls and CP, islet yield was significantly reduced by using the 5× activity of cNP and TL, inducing also fragmentation and DNA release. Viability significantly decreased not until adding the 1× activity of cNP, 5× activity of TL, or 10× activity of CP. Although mitochondrial function was significantly lowered by 1× cNP and 5× TL, CP did not affect mitochondria at any concentration. cNP- and TL-incubated islets significantly lost intracellular insulin already at 1× activity, while the 10× activity of CP had to be added to observe a similar effect. cNP and TL have a similar toxic potency regarding islet integrity. CP also induces adverse effects on islets, but the toxic threshold is generally higher. We hypothesize that CP can serve as supplementary protease to minimize cNP or TL activity for efficient pancreas digestion.

Background: Islet transplantation is an effective therapy in type 1 diabetes and recalcitrant hypoglycemia. However, there is an ongoing need to circumvent islet loss posttransplant. We explore herein the potential of the pan-caspase inhibitor F573 to mitigate early apoptosis-mediated islet death within portal and extrahepatic portal sites in mice., Methods: Mouse or human islets were cultured in standard media ±100 μM F573 and subsequently assessed for viability and apoptosis via terminal deoxynucleotidyl transferase dUTP nick end labeling staining and caspase-3 activation. Diabetic mice were transplanted with syngeneic islets placed under the kidney capsule (KC) or into the subcutaneous deviceless (DL) site at a marginal islet dose (150 islets), or into the portal vein (PV) at a full dose (500 islets). Human islets were transplanted under the KC of diabetic immunodeficient mice at a marginal dose (500 islet equivalents). Islets were cultured in the presence of F573, and F573 was administered subcutaneously on days 0 to 5 posttransplant. Control mice were transplanted with nontreated islets and were injected with saline. Graft function was measured by nonfasting blood glucose and glucose tolerance testing., Results: F573 markedly reduced human and mouse islet apoptosis after in vitro culture (P < 0.05 and P < 0.05, respectively). Furthermore, F573 improved human islet function when transplanted under the KC (P < 0.05); whereas F573 did not enhance murine islet marginal KC transplants. Conversely, F573 significantly improved mouse islet engraftment in the PV and DL site (P < 0.05 and P < 0.05, respectively)., Conclusions: The pan-caspase inhibitor F573 markedly reduces human and mouse islet apoptosis and improves engraftment most effectively in the portal and DL subcutaneous sites.

After multiple decades of investigation, the precise mechanisms involved in fuel-stimulated insulin secretion are still being revealed. One avenue for gaining deeper knowledge is to apply emergent tools of "metabolomics," involving mass spectrometry and nuclear magnetic resonance-based profiling of islet cells in their fuel-stimulated compared with basal states. The current article summarizes recent insights gained from application of metabolomics tools to the specific process of glucose-stimulated insulin secretion, revealing 2 new mechanisms that may provide targets for improving insulin secretion in diabetes., (© 2017 John Wiley & Sons Ltd.)

Ca 2+ is the key intracellular regulator of insulin secretion, acting in the β-cell as the ultimate trigger for exocytosis. In response to high glucose, ATP-sensitive K + channel closure and plasma membrane depolarization engage a sophisticated machinery to drive pulsatile cytosolic Ca 2+ changes. Voltage-gated Ca 2+ channels, Ca 2+ -activated K + channels and Na + /Ca 2+ exchange all play important roles. The use of targeted Ca 2+ probes has revealed that during each cytosolic Ca 2+ pulse, uptake of Ca 2+ by mitochondria, endoplasmic reticulum (ER), secretory granules and lysosomes fine-tune cytosolic Ca 2+ dynamics and control organellar function. For example, changes in the expression of the Ca 2+ -binding protein Sorcin appear to provide a link between ER Ca 2+ levels and ER stress, affecting β-cell function and survival. Across the islet, intercellular communication between highly interconnected "hubs," which act as pacemaker β-cells, and subservient "followers," ensures efficient insulin secretion. Loss of connectivity is seen after the deletion of genes associated with type 2 diabetes (T2D) and follows metabolic and inflammatory insults that characterize this disease. Hubs, which typically comprise ~1%-10% of total β-cells, are repurposed for their specialized role by expression of high glucokinase (Gck) but lower Pdx1 and Nkx6.1 levels. Single cell-omics are poised to provide a deeper understanding of the nature of these cells and of the networks through which they communicate. New insights into the control of both the intra- and intercellular Ca 2+ dynamics may thus shed light on T2D pathology and provide novel opportunities for therapy., (© 2017 John Wiley & Sons Ltd.)

Glucose-stimulated insulin secretion (GSIS) involves interplay between metabolic and cationic events. Seminal contributions from multiple laboratories affirm essential roles for small G-proteins (Rac1, Cdc42, Arf6, Rab27A) in GSIS. Activation of these signalling proteins promotes cytoskeletal remodeling, transport and docking of insulin granules on the plasma membrane for exocytotic secretion of insulin. Evidence in rodent and human islets suggests key roles for lipidation (farnesylation and geranylgeranylation) of these G-proteins for their targeting to appropriate cellular compartments for optimal regulation of effectors leading to GSIS. Interestingly, however, inhibition of prenylation appears to cause mislocalization of non-prenylated, but (paradoxically) activated G-proteins, in "inappropriate" compartments leading to activation of stress kinases and onset of mitochondrial defects, loss in GSIS and apoptosis of the islet β-cell. This review highlights our current understanding of roles of G-proteins and their post-translational lipidation (prenylation) signalling networks in islet function in normal health, metabolic stress (glucolipotoxicity and ER stress) and diabetes. Critical knowledge gaps that need to be addressed for the development of therapeutics to halt defects in these signalling steps in β-cells in models of impaired insulin secretion and diabetes are also highlighted and discussed., (© 2017 John Wiley & Sons Ltd.)

In addition to degrading misfolded and damaged proteins, the proteasome regulates the fate of cells in response to stress. The role of the proteasome in pro-inflammatory cytokine-induced human beta-cell apoptosis is unknown. Using INS-1, INS-1E and human islets exposed to combinations of IFNγ, IL-1β and TNFα with or without addition of small molecules, we assessed the role of the immunoproteasome in pancreatic beta-cell demise. Here, we show that cytokines induce the expression and activity of the immuno-proteasome in INS-1E cells and human islets. Cytokine-induced expression of immuno-proteasome subunits, but not activity, depended upon histone deacetylase 3 activation. Inhibition of JAK1/STAT1 signaling did not affect proteasomal activity. Inhibition of the immuno-proteasome subunit PSMB8 aggravated cytokine-induced human beta-cell apoptosis while reducing intracellular levels of oxidized proteins in INS-1 cells. While cytokines increased total cellular NFκB subunit P50 and P52 levels and reduced the cytosolic NFκB subunit P65 and IκB levels, these effects were unaffected by PSMB8 inhibition. We conclude that beta cells upregulate immuno-proteasome expression and activity in response to IFNγ, likely as a protective response to confine inflammatory signaling., (© 2017 Society for Endocrinology.)

This study investigated the abnormal expression of ATP synthase β-subunit (ATPsyn-β) in pancreas islets of rat model of polycystic ovary syndrome (PCOS) with type 2 diabetes mellitus (T2DM), and the secretion function changes after up-regulation of ATP5b. Sixty female SD rats were divided into three groups randomly and equally. The rat model of PCOS with T2DM was established by free access to the high-carbohydrate/high-fat diet, subcutaneous injections of DHEA, and a single injection of streptozotocin. The pancreas was removed for the detection of the ATPsyn-β expression by immunohistochemical staining, Western blotting and reverse transcription-PCR (RT-PCR). The pancreas islets of the rats were cultured, isolated with collagenase V and purified by gradient centrifugation, and the insulin secretion after treatment with different glucose concentrations was tested. Lentivirus ATP5b was successfully constructed with the vector of GV208 and transfected into the pancreas islets for the over-expression of ATPsyn-β. The insulin secretion and intracellular ATP content were determined after transfection of the PCOS-T2DM pancreas islets with Lenti-ATP5b. The results showed that the expression of ATPsyn-β protein and mRNA was significantly decreased in the pancreas of PCOS-T2DM rats. The ATP content in the pancreas islets was greatly increased and the insulin secretion was improved after the up-regulation of ATPsyn-β in the pancreas islets transfected with lenti-ATP5b. These results indicated that for PCOS, the ATPsyn-β might be one of the key factors for the attack of T2DM.

CMPF (2-(2-carboxyethyl)-4-methyl-5-propylfuran-3-carboxylic acid) is a metabolite that circulates at high concentrations in type 2 and gestational diabetes patients. Further, human clinical studies suggest it might have a causal role in these diseases. CMPF inhibits insulin secretion in mouse and human islets in vitro and in vivo in rodents. However, the metabolic fate of CMPF and the relationship of structure to effects on insulin secretion have not been significantly studied. The syntheses of CMPF and analogues are described. These include isotopically labeled molecules. Study of these materials in vivo has led to the first observation of a metabolite of CMPF. In addition, a wide range of CMPF analogues have been prepared and characterized in insulin secretion assays using both mouse and human islets. Several molecules that influence insulin secretion in vitro were identified. The molecules described should serve as interesting probes to further study the biology of CMPF.

Di(2-ethyhexyl) phthalate (DEHP) is commonly used as a plasticizer, which loosely binds to plastic materials and easily leaches out of these products and enters into the environment. Exposure to DEHP can impair pancreatic beta cells (INS-1 cells)function, which is associated with Insulin Resistance (IR) and type 2 diabetes. However, the mechanism of how DEHP leads to Insulin Resistance is unknown. Our results showed that the cell viability of INS-1 cells exposed to DEHP (0-1600 μM) were decreased in a concentration-dependent manner. DEHP caused significant increases of DNA migration and oxidative damage in INS-1 cells. Lysosomal membrane permeability was increased and mitochondrial membrane potential was reduced after INS-1 cells treated with DEHP. DEHP was also shown to induce ROS production and cause GSH depletion in INS-1 cells. DEHP brought a significant decrease in super oxide dismutase (SOD) and led to accumulation of malondialdehyde (MDA) in the INS-1 cells. DEHP increased significantly the expression of P53 and ATM gene of INS-1 cell at high dose levels. Simultaneously, Pyrroloquinoline Quinone (PQQ), an antioxidant, and alcohol were used in the study to determine their effects on DEHP-induced INS-1 cells damage. PQQ could protect the INS-1 cells from the damage induced by DEHP to some extent, while alcohol aggravated the toxic effects of DEHP. These results indicate that DEHP-mediated INS-1 cell dysfunction through a lysosomal-mitochondrial pathway, involving oxidative stress and p53 and ATM activation., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Accumulating evidence supports the "glucagonocentric hypothesis", in which antecedent α-cell failure and inhibition of glucagon secretion are responsible for diabetes progression. Protein kinase C (PKC) is involved in glucagon secretion from α-cells, although which PKC isozyme is involved and the mechanism underlying this PKC-regulated glucagon secretion remains unknown. Here, the involvement of PKCδ in the onset and progression of diabetes was elucidated. Immunofluorescence studies revealed that PKCδ was expressed and activated in α-cells of STZ-induced diabetic model mice. Phorbol 12-myristate 13-acetate (PMA) stimulation significantly augmented glucagon secretion from isolated islets. Pre-treatment with quercetin and rottlerin, PKCδ signaling inhibitors, significantly suppressed the PMA-induced elevation of glucagon secretion. While Go6976, a Ca 2+ -dependent PKC selective inhibitor did not suppress glucagon secretion. Quercetin suppressed PMA-induced phosphorylation of Tyr 311 of PKCδ in isolated islets. However, quercetin itself had no effect on either glucagon secretion or glucagon mRNA expression. Our data suggest that PKCδ signaling inhibitors suppressed glucagon secretion. Elucidation of detailed signaling pathways causing PKCδ activation in the onset and progression of diabetes followed by the augmentation of glucagon secretion could lead to the identification of novel therapeutic target molecules and the development of novel therapeutic drugs for diabetes. J. Med. Invest. 64: 122-128, February, 2017.

Nicotine is the more abundant and most significant components of cigarette smoke. Epidemiological evidence strongly suggests an association between cigarette smoking and pancreatic injury. Although effects of smoking on endocrine pancreas are still controversial Here, we examined the impact and underlying mechanisms of action of folic acid and vitamin B12 on nicotine induced damage in pancreatic islets of rats. Male Wistar rats were treated with nicotine (3mg/kg body weight/day, intraperitonealy) with or without folic acid (36μg/kg body weight/day, orally) and vitamin B 12 (0.63μg/kg body weight/day, orally) for 21days. Supplementation with folic acid and vitamin B 12 suppressed the nicotine induced changes in HbA1c, insulin, TNF-α, IL-6, generation of reactive oxygen species, and attenuated the changes in markers of oxidative stress. Moreover, folic acid and vitamin B 12 also counteracted the increased expression of protein and mRNA contents of TNF-α and iNOS produced by nicotine. Further, folic acid and vitamin B 12 in combination limits the nicotine induced changes in cell cycle and excessive apoptosis of the pancreatic β-cells and also successfully blunted the nicotine induced alteration in loss of mitochondrial membrane potential. In conclusion, data demonstrate that folic acid and vitamin B 12 may be possible nutritional intervention against cellular oxidative stress, which is a critical step in nicotine-mediated islet injury, and improves islet cell functional status by scavenging free radicals and by inhibiting the generation of pro-inflammatory mediators., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Context Oxidative stress induces apoptosis within Islets of Langerhans in diabetes mellitus (DM). Enicostemma littorale blume, herb of the Gentianaceae family is used as an anti-diabetic agent across rural India. Objective This report demonstrates potent anti-apoptotic and cyto-protective activity of Enicostemma littorale MeOH extract (EL MeOH ext.) against 50 μM H2O2 in isolated rat Islets. Materials and methods In this study, the whole plant methanolic extract of EL with doses 0.25-4 mg/mL each for the preincubation duration of 0.5-4 h against 50 μM H2O2 were tested for optimum protective dose and time by Trypan blue dye exclusion assay. Islet intracellular reactive oxygen species (ROS) was quantified by DCFDA staining and cell death using PS/PI & FDA/PI staining. Further, comet assay, biochemical assessment of caspase-3 and antioxidant enzyme activities along with immunoblotting of PARP-1, caspase-3, TNF-α activation and p-P38 MapK (stress kinase) induction was performed. Results The optimized dose of EL MeOH ext. 2 mg/mL for 2 h was used throughout the study, which significantly decreased total Intracellular ROS and cell death. Further, caspase-3 activity, PARP-1 cleavage, p-P38 MapK (stress kinase) activation and TNF-α levels, which had been significantly elevated, were normalized. Antioxidant enzymes like catalase, superoxide dismutase, reduced glutathione and glutathione peroxidase, along with Comet assay, demonstrated that pretreatment with EL MeOH ext. can augment antioxidant enzyme activities and protect from DNA damage. Discussion and conclusions Significant anti-apoptotic and cyto-protective effects were mediated by EL with Islets of Langerhans subjected to oxidative stress-induced cell death.

Inositol hexakisphosphate kinase 3 (IP6K3) generates inositol pyrophosphates, which regulate diverse cellular functions. However, little is known about its own physiological role. Here, we show the roles of IP6K3 in metabolic regulation. We detected high levels of both mouse and human IP6K3 mRNA in myotubes and muscle tissues. In human myotubes, IP6K3 was upregulated by dexamethasone treatment, which is known to inhibit glucose metabolism. Furthermore, Ip6k3 expression was elevated under diabetic, fasting, and disuse conditions in mouse skeletal muscles. Ip6k3(-/-) mice demonstrated lower blood glucose, reduced circulating insulin, deceased fat mass, lower body weight, increased plasma lactate, enhanced glucose tolerance, lower glucose during an insulin tolerance test, and reduced muscle Pdk4 expression under normal diet conditions. Notably, Ip6k3 deletion extended animal lifespan with concomitant reduced phosphorylation of S6 ribosomal protein in the heart. In contrast, Ip6k3(-/-) mice showed unchanged skeletal muscle mass and no resistance to the effects of high fat diet. The current observations suggest novel roles of IP6K3 in cellular regulation, which impact metabolic control and lifespan.

Sphingosine-1-phosphate (S1P) is a sphingolipid metabolite that regulates basic cell functions through metabolic and signaling pathways. Intracellular metabolism of S1P is controlled, in part, by two homologous S1P phosphatases (SPPases), 1 and 2, which are encoded by the Sgpp1 and Sgpp2 genes, respectively. SPPase activity is needed for efficient recycling of sphingosine into the sphingolipid synthesis pathway. SPPase 1 is important for skin homeostasis, but little is known about the functional role of SPPase 2. To identify the functions of SPPase 2 in vivo, we studied mice with the Sgpp2 gene deleted. In contrast to Sgpp1(-/-) mice, Sgpp2(-/-) mice had normal skin and were viable into adulthood. Unexpectedly, WT mice expressed Sgpp2 mRNA at high levels in pancreatic islets when compared with other tissues. Sgpp2(-/-) mice had normal pancreatic islet size; however, they exhibited defective adaptive β-cell proliferation that was demonstrated after treatment with either a high-fat diet or the β-cell-specific toxin, streptozotocin. Importantly, β-cells from untreated Sgpp2(-/-) mice showed significantly increased expression of proteins characteristic of the endoplasmic reticulum stress response compared with β-cells from WT mice, indicating a basal islet defect. Our results show that Sgpp2 deletion causes β-cell endoplasmic reticulum stress, which is a known cause of β-cell dysfunction, and reveal a juncture in the sphingolipid recycling pathway that could impact the development of diabetes., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Oxamate (OXA) is a pyruvate analogue that directly inhibits the lactate dehydrogenase (LDH)-catalyzed conversion process of pyruvate into lactate. Earlier and recent studies have shown elevated blood lactate levels among insulin-resistant and type 2 diabetes subjects and that blood lactate levels independently predicted the development of incident diabetes. To explore the potential of OXA in the treatment of diabetes, db/db mice were treated with OXA in vivo. Treatment of OXA (350-750 mg/kg of body weight) for 12 weeks was shown to decrease body weight gain and blood glucose and HbA1c levels and improve insulin secretion, the morphology of pancreatic islets, and insulin sensitivity in db/db mice. Meanwhile, OXA reduced the lactate production of adipose tissue and skeletal muscle and serum lactate levels and decreased serum levels of TG, FFA, CRP, IL-6, and TNF-α in db/db mice. The PCR array showed that OXA downregulated the expression of Tnf, Il6, leptin, Cxcr3, Map2k1, and Ikbkb, and upregulated the expression of Irs2, Nfkbia, and Pde3b in the skeletal muscle of db/db mice. Interestingly, LDH-A expression increased in the islet cells of db/db mice, and both treatment of OXA and pioglitazone decreased LDH-A expression, which might be related to the improvement of insulin secretion. Taken together, increased lactate production of adipose tissue and skeletal muscle may be at least partially responsible for insulin resistance and diabetes in db/db mice. OXA improved glycemic control and insulin sensitivity in db/db mice primarily via inhibition of tissue lactate production. Oxamic acid derivatives may be a potential drug for the treatment of type 2 diabetes.

Glucagon-like peptide-1 (GLP-1) is a glucoincretin hormone that can act through its receptor (GLP-1R) on pancreatic β-cells and increase insulin secretion and production. GLP-1R agonists are used clinically to treat type 2 diabetes. GLP-1 may also regulate the exocrine pancreas at multiple levels, including inhibition through the central nervous system, stimulation indirectly through insulin, and stimulation directly on acinar cells. However, it has been unclear whether GLP-1R is present in pancreatic acini and what physiological functions these receptors regulate. In the current study we utilized GLP-1R knockout (KO) mice to study the role of GLP-1R in acinar cells. RNA expression of GLP-1R was detected in acutely isolated pancreatic acini. Acinar cell morphology and expression of digestive enzymes were not affected by loss of GLP-1R. GLP-1 induced amylase secretion in wild-type (WT) acini. In GLP-1R KO mice, this effect was abolished, whereas vasoactive intestinal peptide-induced amylase release in KO acini showed a pattern similar to that in WT acini. GLP-1 stimulated cAMP production and increased protein kinase A-mediated protein phosphorylation in WT acini, and these effects were absent in KO acini. These data show that GLP-1R is present in pancreatic acinar cells and that GLP-1 can regulate secretion through its receptor and cAMP signaling pathway., (Copyright © 2016 the American Physiological Society.)

We have reported a high expression of IGF-I in pancreatic islet β-cells of transgenic mice under the metallothionein promoter. cDNA microarray analysis of the islets revealed that the expression of 82 genes was significantly altered compared to wild-type mice. Of these, 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1), which is responsible for the conversion of inert cortisone (11-dehydrocorticosterone, DHC in rodents) to active cortisol (corticosterone) in the liver and adipose tissues, has not been identified previously as an IGF-I target in pancreatic islets. We characterized the changes in its protein level, enzyme activity and glucose-stimulated insulin secretion. In freshly isolated islets, the level of 11β-HSD1 protein was significantly lower in MT-IGF mice. Using dual-labeled immunofluorescence, 11β-HSD1 was observed exclusively in glucagon-producing, islet α-cells but at a lower level in transgenic vs. wild-type animals. MT-IGF islets also exhibited reduced enzymatic activities. Dexamethasone (DEX) and DHC inhibited glucose-stimulated insulin secretion from freshly isolated islets of wild-type mice. In the islets of MT-IGF mice, 48-h pre-incubation of DEX caused a significant decrease in insulin release, while the effect of DHC was largely blunted consistent with diminished 11β-HSD1 activity. In order to establish the function of intracrine glucocorticoids, we overexpressed 11β-HSD1 cDNA in MIN6 insulinoma cells, which together with DHC caused apoptosis and a significant decrease in proliferation. Both effects were abolished with the treatment of an 11β-HSD1 inhibitor. Our results demonstrate an inhibitory effect of IGF-I on 11β-HSD1 expression and activity within the pancreatic islets, which may mediate part of the IGF-I effects on cell proliferation, survival and insulin secretion.

Objective: To demonstrate the role of islet glucokinase, glucose metabolism, and intracellular insulin mediators in the enhancing effect of islet neogenesis-associated protein pentadecapeptide (INGAP-PP) on glucose-induced insulin secretion., Methods: Islets from normal rats were cultured for 4 days in the absence or presence of 10 μg/mL INGAP-PP, with/without Wortmannin or LY294002. Islets were incubated with different glucose concentrations to measure insulin secretion and content, hexokinase and glucokinase activity, glucose oxidation and utilization, glucokinase, insulin receptor, insulin receptor substrate (IRS)-1/2, and PI3K concentration and phosphorylation., Results: The INGAP-PP significantly increased insulin release at high but not at low glucose concentration, glucokinase activity, glucose metabolism, glucokinase, insulin receptor, IRS-2 and PI3K protein concentration, insulin receptor and IRS-1/2 tyrosine phosphorylation, and the association of p85 with IRS-1. Wortmannin and LY294002 blocked INGAP-PP effect on insulin secretion and glucokinase protein levels in a dose-dependent manner., Conclusions: The enhancing effect of INGAP-PP on glucose-induced insulin release could be partly ascribed to its effect on glucokinase activity and glucose metabolism and is mainly mediated by the PI3K/AKT pathway. These results, together with the low hypoglycemia risk associated with the use of INGAP-PP, offer a new alternative for diabetes prevention and treatment.

SET7/9 is an enzyme that methylates histone 3 at lysine 4 (H3K4) to maintain euchromatin architecture. Although SET7/9 is enriched in islets and contributes to the transactivation of β cell-specific genes, including Ins1 and Slc2a, SET7/9 has also been reported to bind the p65 subunit of nuclear factor κB in non-β cells and modify its transcriptional activity. Given that inflammation is a central component of β cell dysfunction in Type 1 and Type 2 diabetes, the aim of this study was to elucidate the role of SET7/9 in proinflammatory cytokine signaling in β cells. To induce inflammation, βTC3 insulinoma cells were treated with IL-1β, TNF-α, and IFN-γ. Cytokine treatment led to increased expression of inducible nitric-oxide synthase, which was attenuated by the diminution of SET7/9 using RNA interference. Consistent with previous reports, SET7/9 was co-immunoprecipitated with p65 and underwent cytosolic to nuclear translocation in response to cytokines. ChIP analysis demonstrated augmented H3K4 mono- and dimethylation of the proximal Nos2 promoter with cytokine exposure. SET7/9 was found to occupy this same region, whereas SET7/9 knockdown attenuated cytokine-induced histone methylation of the Nos2 gene. To test this relationship further, islets were isolated from SET7/9-deficient and wild-type mice and treated with IL-1β, TNF-α, and IFN-γ. Cytokine-induced Nos2 expression was reduced in the islets from SET7/9 knock-out mice. Together, our findings suggest that SET7/9 contributes to Nos2 transcription and proinflammatory cytokine signaling in the pancreatic β cell through activating histone modifications., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Although the insulin secretion deficit in hypothyroid male rats has been documented, the underling mechanisms of the effect of hypothyroidism on insulin secretion are not clear. Isolated islets of the PTU-induced hypothyroid and control rats were exposed to glibenclamide, acetylcholine, and nifedipine in the presence of glucose concentrations of 2.8 or 8.3 and 16.7 mmol/L. Glucokinase and hexokinase specific activity, glucokinase content, and glucose transporter 2 protein expression were also determined in the isolated islets. Isolated islets from the hypothyroid rats showed a defect in insulin secretion in response to high glucose. In the presence of glibenclamide or acetylcholine, the isolated islets from the hypothyroid and control rats stimulated by glucose concentration of 16.7 mmol/L secreted similar amounts of insulin. In the presence of glucose concentrations of 8.3 mmol/L and 16.7 mmol/L, nifedipine was able to diminish insulin secretion from isolated islets of both groups, indicating that probably the defect may not arise from L type calcium channels or the steps beyond depolarization or the elements involved in the acetylcoline signaling pathway. Glucokinase content and hexokinase specific activity were also the same in the control and hypothyroid groups. On the other hand, glucokinase specific activity and glucose transporter 2 protein expression were significantly (p<0.001 and p<0.01 respectively) lower in the islets isolated from the hypothyroid rats (6.50 ± 0.46 mU/min/mg protein and 0.55 ± 0.09 arbitrary unit) compared to the controls (10.93 ± 0.83 mU/min/mg protein and 0.98 ± 0.07 arbitrary unit) respectively. In conclusion, the results of this study indicated that hypothyroidism reduced insulin secretion from isolated pancreatic islets, which confirms the finding of the previous studies; in addition, the insulin secretion deficit observed in hypothyroid rats may arise from the abnormalities in some parts of the glucose sensor apparatus of the pancreatic islets including glucokinase activity and glucose transporter 2 protein expression.

Background: We have previously shown that an immunomodulatory enzyme, indoleamine 2,3-dioxygenase (IDO) in dermal fibroblasts generates a tryptophan-deficient environment that selectively inhibits proliferation and induces apoptosis of bystander CD4+ and CD8+ T cells, but not pancreatic islets. Because these immune cells are involved in islet allograft rejection, we hypothesized that transplantation of islets embedded in a novel 3-dimensional composite scaffold within which stable IDO-expressing fibroblasts serve as source of local immunosuppression would lead to normoglycemia in a streptozotocin-induced diabetic mouse model., Methods: Islet grafts were prepared by embedding stable IDO-expressing fibroblasts and allogeneic islets into a protease-resistant composite scaffold. Islets function and survival were evaluated in vitro using immunohistochemistry. Allografts were transplanted under the kidney capsule of streptozotocin-induced diabetic mice; viability, function, and criteria for graft take were evaluated. Flow cytometry was performed to determine specific intragraft, draining lymph nodes and spleen T-cell population, and splenocytes alloantigen responsiveness of graft recipients., Results: The results of a series of in vitro experiments revealed that IDO-expressing fibroblasts do not compromise islet function or survival. The expression of IDO suppressed the proliferation of alloantigen-stimulated splenocytes. The in vivo experiments revealed that local IDO expression delivered by lentiviral vector prolonged islet allograft survival (51.0 ± 2.9 days) by increasing the population of FOXP3+ regulatory T cells at the graft site and graft-draining lymph nodes and preventing T-cell infiltration., Conclusions: This study shows that incorporation of islets within our novel matrix that is equipped with stable IDO-expressing fibroblasts prolongs allograft survival.

Background: Islet isolation currently requires collagenase, neutral protease and other components. Thermolysin (TL) from Bacillus thermoproteolyticus is the gold standard neutral protease. However, we speculated that neutral protease derived from Clostridium histolyticum (Ch; ChNP) would be biologically superior for islet isolation. Tryptic-like activity has also been reported to be important. Therefore, we focused on clostripain (CP), since it is one of the main proteases in Clostridium histolyticum which possesses tryptic-like activity. We then examined the synergistic effects of highly purified ChNP and CP on rat islet isolation., Methods: The same amount of collagenase was used in all four groups (TL, ChNP, TL+CP and ChNP+CP; n = 12/group). The efficiency was evaluated by the islet yield and function. An immunohistochemical analysis, in vitro digestion assay for each enzyme component and evaluation of the activation of endogenous exocrine proteases during islet isolation were also performed., Results: The islet yield of the TL group was significantly higher than that of the ChNP group (P < 0.01). The islet yield was dose dependently increased in the ChNP+CP group, but was decreased in the TL + CP group. The islet yield in the ChNP + CP group was significantly higher than that in the TL group, but their islet function was similar. Different specificities for laminin, especially laminin-511, were observed in the TL, ChNP, and CP groups., Conclusions: Clostripain had a strong synergistic effect with ChNP, but not with TL. Therefore, ChNP and CP, in combination with collagenase derived from the same bacteria, may effectively increase the isolation efficiency without affecting the quality of islets.

Deposition of β cell toxic islet amyloid is a cardinal finding in type 2 diabetes. In addition to the main amyloid component islet amyloid polypeptide (IAPP), heparan sulfate proteoglycan is constantly present in the amyloid deposit. Heparan sulfate (HS) side chains bind to IAPP, inducing conformational changes of the IAPP structure and an acceleration of fibril formation. We generated a double-transgenic mouse strain (hpa-hIAPP) that overexpresses human heparanase and human IAPP but is deficient of endogenous mouse IAPP. Culture of hpa-hIAPP islets in 20 mm glucose resulted in less amyloid formation compared with the amyloid load developed in cultured islets isolated from littermates expressing human IAPP only. A similar reduction of amyloid was achieved when human islets were cultured in the presence of heparin fragments. Furthermore, we used CHO cells and the mutant CHO pgsD-677 cell line (deficient in HS synthesis) to explore the effect of cellular HS on IAPP-induced cytotoxicity. Seeding of IAPP aggregation on CHO cells resulted in caspase-3 activation and apoptosis that could be prevented by inhibition of caspase-8. No IAPP-induced apoptosis was seen in HS-deficient CHO pgsD-677 cells. These results suggest that β cell death caused by extracellular IAPP requires membrane-bound HS. The interaction between HS and IAPP or the subsequent effects represent a possible therapeutic target whose blockage can lead to a prolonged survival of β cells., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Purpose of Review: Proprotein convertase subtilisin kexin type 9 (PCSK9) acts as an endogenous natural inhibitor of the LDL receptor pathway, by targeting the receptor to lysosomes for degradation. Beside the liver, PCSK9 is also expressed at significant levels in other tissues, where its function remains unclear. The current review focuses on the extrahepatic actions of PCSK9., Recent Findings: The generation of liver-specific PCSK9 knockout mice has clearly indicated that PCSK9 affects cholesterol homeostasis via its action on extrahepatic organs. PCSK9 is highly expressed in the intestine, where it controls the production of triglyceride-rich lipoproteins and the transintestinal cholesterol excretion. The role of PCSK9 in the endocrine pancreas and glucose homeostasis remains unclear because conflicting data exist concerning the metabolic phenotype of PCSK9-deficient mice. Sparse data suggest that PCSK9 might also play a role in kidneys, vascular smooth muscle cells, and neurons., Summary: Based on the virtuous combination of genetic and pharmacological approaches, the major function of PCSK9 as a key regulator of hepatic LDL receptor metabolism had quickly emerged. Accumulating evidence indicates that intestinal PCSK9 is also involved in the modulation of lipid homeostasis. Additional studies are warranted to decipher the physiological function of PCSK9 in other extrahepatic tissues and thus to better assess the safety of PCSK9 inhibitors.

Benzene (C6H6) is an organic compound used in petrochemicals and numerous other industries. It is abundantly released to our environment as a chemical pollutant causing widespread human exposure. This study mainly focused on benzene induced toxicity on rat pancreatic islets with respect to oxidative damage, insulin secretion and glucokinase (GK) activity. Benzene was dissolved in corn oil and administered orally at doses 200, 400 and 800mg/kg/day, for 4 weeks. In rats, benzene significantly raised the concentration of plasma insulin. Also the effect of benzene on the release of glucose-induced insulin was pronounced in isolated islets. Benzene caused oxidative DNA damage and lipid peroxidation, and also reduced the cell viability and total thiols groups, in the islets of exposed rats. In conclusion, the current study revealed that pancreatic glucose metabolism is susceptible to benzene toxicity and the resultant oxidative stress could lead to functional abnormalities in the pancreas., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The aim of this study is to explore the effect of zerumbone, a natural sesquiterpene isolated from Zingiber zerumbet Smith, on high glucose-induced cytotoxicity in pancreatic β cells. INS-1 rat pancreatic β cells were treated with 33 mM glucose with or without different concentrations of zerumbone and cell viability and apoptosis were assessed. The involvement of reactive oxygen species (ROS) and mitogen-activated protein kinase (MAPK) signaling in the action of zerumbone was examined. Notably, zerumbone significantly (P < 0.05) prevented the reduction of cell viability induced by high glucose. Such protection was in a concentration-dependent fashion up to 60 μM of zerumbone. Annexin-V/propidium iodide staining analysis showed that zerumbone impaired the apoptotic response of high glucose-treated INS-1 cells, which was coupled with a significant decline in cleaved caspase-3 and caspase-9. Pretreatment with the ROS inhibitor N-acetylcysteine abrogated the phosphorylation of p38 and JNK induced by high glucose. Zerumbone significantly (P < 0.05) decreased the generation of ROS and the phosphorylation of p38 and JNK MAPKs in high glucose-treated INS-1 cells. Pharmacological activation of p38 and JNK with anisomycin reversed the anti-apoptotic effect of zerumbone. Additionally, simultaneous inhibition of p38 and JNK significantly (P < 0.05) reduced the apoptotic response in high glucose-treated INS-1 cells. In conclusion, zerumbone confers protection against high glucose-induced apoptosis of INS-1 pancreatic β cells, largely through interfering with ROS production and p38 and JNK activation. Zerumbone may have potential therapeutic effects against hyperglycemia-induced β cell damage in diabetes., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Fibroblast activation protein (FAP, seprase, EC 3.4.21.B28) and dipeptidyl peptidase-IV (DPP-IV, CD26, EC 3.4.14.5) are homologous serine proteases implicated in the modulation of the bioavailability and thus the function of a number of biologically active peptides. In spite of their generally nonoverlapping expression patterns, DPP-IV and FAP are co-expressed and probably co-regulated in certain cell types suggesting that for some biological processes their functional synergy is essential. By an in situ enzymatic activity assay, we show an abundant DPP-IV-like enzymatic activity sensitive to a highly specific DPP-IV inhibitor sitagliptin and corresponding DPP-IV immunoreactivity in the adult human islets of Langerhans. Moreover, the homologous protease FAP was present in the human endocrine pancreas and was co-expressed with DPP-IV. DPP-IV and FAP were found in the pancreatic alpha cells as determined by the co-localization with glucagon immunoreactivity. In summary, we show abundant enzymatic activity of the canonical DPP-IV (CD26) in Langerhans islets in the natural tissue context and demonstrate for the first time the co-expression of FAP and DPP-IV in pancreatic alpha cells in adult humans. Given their ability to proteolytically modify several biologically active peptides, both proteases have the potential to modulate the paracrine signaling in the human Langerhans islets.

Protein restriction in the early stages of life can result in several changes in pancreatic function. These alterations include documented reductions in insulin secretion and in cytoplasmic calcium concentration [Ca(2+)]i. However, the mechanisms underlying these changes have not been completely elucidated and may result, in part, from alterations in signaling pathways that potentiate insulin secretion in the presence of glucose. Our findings suggest that protein restriction disrupts the insulin secretory synergism between Cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) and Ca(2+)-dependent protein kinase C (PKC) in isolated islets. Western blot analysis demonstrated reduced levels of both phospho-cAMP response element-binding protein (phospho-CREB) at Ser-133 and substrates phosphorylated by PKCs (Phospho-(Ser) PKC substrate), suggesting that PKA and PKC activity was impaired in islets from rats fed a low-protein diet (LP). cAMP levels and global Ca(2+) entry were also reduced in LP islets. In summary, our findings showed that protein restriction altered the crosstalk between PKA and PKC signaling pathways, resulting in the alteration of secretory synergism in isolated islets., (Copyright © 2015 Elsevier Inc. All rights reserved.)

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.

Transplantation of islets of Langerhans that have been isolated from whole pancreas is an attractive alternative for the reversal of Type 1 diabetes. However, in vitro culture of isolated pancreatic islets has been reported to cause a decrease in glucose response over time. Hence, the improvement in islet culture conditions is an important goal in islet transplantation. Heme Oxygenase-1 (HO-1) is a stress protein that has been described as an inducible protein with the capacity of preventing apoptosis and cytoprotection via radical scavenging. Therefore, this study was aimed to assess the influence of endogenous HO-1 gene transfer on insulin secretion of caprine islets. The full-length cDNA sequence of Capra hircus HO-1 was determined using specific designed primers and rapid amplification of cDNA ends of pancreatic tissue. The HO-1 cDNA was then cloned into the prokaryotic expression vectors and transfected into caprine islets using lipid carriers. Efficiency of lipid carriers to transfect caprine islets was determined by flow cytometry. Insulin secretion assay was carried out by ovine insulin ELISA. The finding demonstrated that endogenous HO-1 gene transfer could improve caprine islet function in in vitro culture. Consequently, strategies using HO-1 gene transfer to islets might lead to better outcome in islet transplantation.

Incretins, such as glucagon-like peptide-1 (GLP)-1, have been shown to elevate plasma insulin concentration. The purpose of this study is to investigate the cellular and molecular basis of the beneficial effects of GLP-1. Normal and diabetic male Wistar rats were treated with GLP-1 (50 ng/kg body weight) for 10 weeks. At the end of the experiment, pancreatic tissues were taken for immunohistochemistry, immunoelectron microscopy and real-time polymerase chain reaction studies. Samples of blood were retrieved from the animals for the measurement of enzymes and insulin. The results show that treatment of diabetic rats with GLP-1 caused significant (P < 0.05) reduction in body weight gain and blood glucose level. GLP-1 (10(-12)-10(-6) M) induced significant (P < 0.01) dose-dependent increases in insulin release from the pancreas of normal and diabetic rats compared to basal. Diabetes-induced abnormal liver (aspartate aminotransferase and alanine aminotransferase) and kidney (blood urea nitrogen and uric acid) parameters were corrected in GLP-1-treated rats compared to controls. GLP-1 treatment induced significant (P < 0.05) elevation in the expression of pancreatic duodenal homeobox-1, heat shock protein-70, glutathione peroxidase, insulin receptor and GLP-1-receptor genes in diabetic animals compared to controls. GLP-1 is present in pancreatic beta cells and significantly (P < 0.05) increased the number of insulin-, glutathione reductase- and catalase-immunoreactive islet cells. The results of this study show that GLP-1 is co-localized with insulin and seems to exert its beneficial effects by increasing cellular concentrations of endogenous antioxidant genes and other genes involved in the maintenance of pancreatic beta cell structure and function.

Objectives: A functional vacuolar adenosine triphosphatase (v-ATPase) complex regulates canonical Wnt/β-catenin signaling. The goal of this study was to identify the distribution of the v-ATPase in human and murine models of pancreatic intraepithelial neoplasms (PanINs) and assess its role in Wnt/β-catenin signaling., Methods: We evaluated the immunolabeling pattern of the v-ATPase in human PanIN specimens and murine PanIN-1 and PanIN-2 lesions obtained from Ptf1a(Cre/+); LSL-Kras(G12D) mice. Wnt/β-catenin signaling was interrogated in primary PanIN cells by examining the phosphorylated levels of its surface coreceptor, low-density lipoprotein receptor-related protein-6 (LRP6), and its intracellular effector, nonphosphorylated β-catenin. The response of primary PanIN cells to epidermal growth factor (EGF) was assessed in the absence and presence of the v-ATPase inhibitor, concanamycin., Results: In advanced (PanIN-2), but not early (PanIN-1), lesions, the v-ATPase assumed a polarized phenotype. Blocking the v-ATPase disrupted Wnt/β-catenin signaling in primary PanIN cells despite significantly higher levels of the total and activated Wnt cell surface coreceptor, LRP6. Vacuolar adenosine triphosphatase blockade significantly decreased the total and activated levels of EGF receptor, a determinant of PanIN progression. The activation of EGF receptor and its intracellular mediator, p44/42 mitogen-activated protein kinase, was also reduced by v-ATPase blockade. This led to diminished proliferation in response to EGF ligand., Conclusions: The v-ATPase regulates Wnt/β-catenin and EGF receptor signaling in PanINs.

Dipeptidyl peptidase 4 (DPP-4) was recently found to be expressed in human and mouse islets with different expression patterns. However, whether species-dependent expression pattern is a generalized phenomenon and whether islet DPP-4 activity is regulated are not known. This study was conducted to investigate DPP-4 localization in several different species, and to examine the impact of glucose, incretin hormones, and insulin on islet DPP-4 activity. It was shown by immuofluorescent staining that there were two distinct species-specific expression patterns of islet DPP-4. The enzyme was expressed exclusively in α-cells in human and pig islets, but primarily in β-cells in mouse and rat islets. INS-1 832/13 cells also expressed DPP-4, and inhibition of DPP-4 enhanced insulin secretion in the presence of glucagon-like peptide-1 (GLP-1) in the cells. DPP-4 activity was remarkably robust when cultured with high glucose, incretin hormones, and insulin in mouse and human islets as well as INS-1 832/13 cells and islet DPP-4 activity and expression pattern was not altered in double incretin receptor knockout mice, compared to wild type mice. We conclude that islet DPP-4 is species-specifically expressed in α-cell and β-cell dominant patterns in several species and both patterns remained robust in enzyme activity during short-term metabolic challenge., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Group X secretory phospholipase A2 (GX sPLA2) potently hydrolyzes membrane phospholipids to release arachidonic acid (AA). While AA is an activator of glucose-stimulated insulin secretion (GSIS), its metabolite prostaglandin E2 (PGE2) is a known inhibitor. In this study, we determined that GX sPLA2 is expressed in insulin-producing cells of mouse pancreatic islets and investigated its role in beta cell function. GSIS was measured in vivo in wild-type (WT) and GX sPLA2-deficient (GX KO) mice and ex vivo using pancreatic islets isolated from WT and GX KO mice. GSIS was also assessed in vitro using mouse MIN6 pancreatic beta cells with or without GX sPLA2 overexpression or exogenous addition. GSIS was significantly higher in islets isolated from GX KO mice compared with islets from WT mice. Conversely, GSIS was lower in MIN6 cells overexpressing GX sPLA2 (MIN6-GX) compared with control (MIN6-C) cells. PGE2 production was significantly higher in MIN6-GX cells compared with MIN6-C cells and this was associated with significantly reduced cellular cAMP. The effect of GX sPLA2 on GSIS was abolished when cells were treated with NS398 (a COX-2 inhibitor) or L-798,106 (a PGE2-EP3 receptor antagonist). Consistent with enhanced beta cell function, GX KO mice showed significantly increased plasma insulin levels following glucose challenge and were protected from age-related reductions in GSIS and glucose tolerance compared with WT mice. We conclude that GX sPLA2 plays a previously unrecognized role in negatively regulating pancreatic insulin secretion by augmenting COX-2-dependent PGE2 production., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)