Insulin-secreting Beta Cells Research Articles

Maternal exercise attenuates the lower skeletal muscle glucose uptake and insulin secretion caused by paternal obesity in female adult rat offspring.

Key points Paternal obesity negatively influences metabolic outcomes in adult rat offspring.Maternal voluntary physical activity has previously been reported to improve glucose metabolism in adult rat offspring sired by healthy fathers.Here, we investigated whether a structured programme of maternal exercise training before and during gestation can attenuate the negative impacts that paternal obesity has on insulin sensitivity and secretion in female adult offspring.Exercise before and during pregnancy normalised the lower insulin sensitivity in skeletal muscle and the lower insulin secretion observed in female offspring sired by obese fathers.This paper presents a feasible, low‐cost and translatable intervention strategy that can be applied perinatally to support multifactor interventions to break the cycle of metabolic dysfunction caused by paternal obesity. We investigated whether maternal exercise before and during gestation could attenuate the negative metabolic effects of paternal high‐fat diet‐induced obesity in female adult rat offspring. Fathers consumed a normal chow or high‐fat diet before mating. Mothers exercised on a treadmill before and during gestation or remained sedentary. In adulthood, female offspring were assessed using intraperitoneal insulin and glucose tolerance tests (IPITT and IPGTT, respectively), pancreatic morphology, ex vivo skeletal muscle insulin‐stimulated glucose uptake and mitochondrial respiratory function. Paternal obesity impaired whole‐body and skeletal muscle insulin sensitivity and insulin secretion in adult offspring. Maternal exercise attenuated the lower insulin‐stimulated glucose uptake in offspring sired by obese fathers but distal insulin signalling components (p‐AKT Thr308 and Ser473, p‐TBC1D4 Thr642 and GLUT4) remained unchanged (P > 0.05). Maternal exercise increased citrate synthase activity only in offspring sired by obese fathers. Maternal exercise also reversed the lower insulin secretion in vivo observed in offspring of obese fathers, probably due to an attenuation of the decrease in pancreatic beta cell mass. In summary, maternal exercise before and during pregnancy in rats attenuated skeletal muscle insulin resistance and attenuated the decrease in pancreatic beta cell mass and insulin secretion observed in the female offspring of obese fathers.

Insulin clearance as the major player in the hyperinsulinaemia of black African men without diabetes.

To investigate relationships between insulin clearance, insulin secretion, hepatic fat accumulation and insulin sensitivity in black African (BA) and white European (WE) men. Twenty-three BA and twenty-three WE men with normal glucose tolerance, matched for age and body mass index, underwent a hyperglycaemic clamp to measure insulin secretion and clearance, hyperinsulinaemic-euglycaemic clamp with stable glucose isotope infusion to measure whole-body and hepatic-specific insulin sensitivity, and magnetic resonance imaging to quantify intrahepatic lipid (IHL). BA men had higher glucose-stimulated peripheral insulin levels (48.1 [35.5, 65.2] × 103 vs. 29.9 [23.3, 38.4] × 103 pmol L-1 × min, P = .017) and lower endogeneous insulin clearance (771.6 [227.8] vs. 1381 [534.3] mL m-2 body surface area min -1 , P < .001) compared with WE men. There were no ethnic differences in beta-cell insulin secretion or beta-cell responsivity to glucose, even after adjustment for prevailing insulin sensitivity. In WE men, endogenous insulin clearance was correlated with whole-body insulin sensitivity (r = 0.691, P = .001) and inversely correlated with IHL (r = -0.674, P = .001). These associations were not found in BA men. While normally glucose-tolerant BA men have similar insulin secretory responses to their WE counterparts, they have markedly lower insulin clearance, which does not appear to be explained by either insulin resistance or hepatic fat accumulation. Low insulin clearance may be the primary mechanism of hyperinsulinaemia in populations of African origin.

Combination of ferric ammonium citrate with cytokines involved in apoptosis and insulin secretion of human pancreatic beta cells related to diabetes in thalassemia.

BackgroundDiabetes mellitus (DM) is a common complication found in β-thalassemia patients. The mechanism of DM in β-thalassemia patients is still unclear, but it could be from an iron overload and increase of some cytokines, such as interleukin1-β (IL-1β) and tumor necrosis factor-α (TNF-α). The objective of this study was to study the effect of interaction between ferric ammonium citrate (FAC) and cytokines, IL-1β and TNF-α, on 1.1B4 human pancreatic β-cell line.MethodsThe effect of the combination of FAC and cytokines on cell viability was studied by MTT assay. Insulin secretion was assessed by the enzyme-linked immunosorbent assay (ELISA). The reactive oxygen species (ROS) and cell apoptosis in normal and high glucose condition were determined by flow cytometer. In addition, gene expression of apoptosis, antioxidant; glutathione peroxidase 1 (GPX1) and superoxide dismutase 2 (SOD2), and insulin secretory function were studied by real-time polymerase chain reaction (Real-time PCR).ResultsThe findings revealed that FAC exposure resulted in the decrease of cell viability and insulin-release, and the induction of ROS and apoptosis in pancreatic cells. Interestingly, a combination of FAC and cytokines had an additive effect on SOD2 antioxidants’ genes expression and endoplasmic reticulum (ER) stress. In addition, it reduced the insulin secretion genes expression; insulin (INS), glucose kinase (GCK), protein convertase 1 (PSCK1), and protein convertase 2 (PSCK2). Moreover, the highest ROS and the lowest insulin secretion were found in FAC combined with IL-1β and TNF-α in the high-glucose condition of human pancreatic beta cell, which could be involved in the mechanism of DM development in β-thalassemia patients.

2072-P: Deletion of the Mitofusins 1 and 2 (Mfn1 and Mfn2) in the Pancreatic Beta Cell Disrupts Mitochondrial Structure and Function In Vitro and Strongly Impairs Glucose-Stimulated Insulin Secretion In Vivo

Background and Aims: Mitochondrial metabolism of glucose is vital for the initiation of insulin secretion in pancreatic beta cells. Whether mitochondrial ultra-structure, and the proteins controlling their fission and fusion, are essential for glucose recognition is less clear. In the present study, beta cell-selective adult mouse restricted deletion of Mfn1 and Mfn2 was used to investigate the role of mitochondrial fusion in insulin secretion and glucose homeostasis in vivo and in vitro. Methods: C57BL6 mice bearing Mfn1 and Mfn2 alleles with FloxP sites, were crossed to animals carrying an inducible Cre recombinase at the Pdx1 locus (PdxCreERT). After induction of recombination with tamoxifen, live beta cell fluorescence imaging of mitochondrial [Ca2+] and membrane potential were performed on isolated islets. Mitochondrial ultra-structure was measured using super resolution fluorescence and transmission electron microscopy. Intraperitoneal glucose tolerance, and insulin secretion in vivo and in vitro, were measured using standard techniques. Results: Mitochondrial length was strongly (to 77±0.9% of controls, p&amp;lt;0.0001) and selectively diminished in beta cells from Mfn1/2 KO mice. KO animals displayed higher fasting glycaemia than control animals at 11-12 weeks (8.6 vs. 6.4 mmol/L, p&amp;gt;0.05). An increase in circulating glucose levels was also observed (p&amp;lt;0.05 at 30 min and p&amp;lt;0.01 at 60 min) and was linked to a considerable (&amp;gt;five-fold) decrease in plasma insulin (5-15 min, p&amp;lt;0.0001) post-intraperitoneal glucose injection. Mitochondrial Ca2+ accumulation and membrane potential were significantly reduced (p&amp;lt;0.01) in response to high glucose in KO animals. Conclusions: Mitochondrial fusion and fission cycles are indispensable in the beta cell in order to preserve normal mitochondrial bioenergetics and glucose sensing, and may be disrupted in some forms of diabetes. Disclosure E. Georgiadou: None. P.L. Chabosseau: None. A. Tomas: None. I. Leclerc: Consultant; Spouse/Partner; Sun Pharmaceutical Industries Ltd. Research Support; Spouse/Partner; Servier. G.A. Rutter: Consultant; Self; Sun Pharmaceutical Industries Ltd. Research Support; Self; Servier, Sun Pharmaceutical Industries Ltd. Funding UK Wellcome Trust (WT098424AIA, 212625/Z/18/Z); UK Medical Researh Council (MR/R022259/1, MR/J0003042/1, MR/L020149/1, MR/L02036X/1, MR/N00275X/1); Diabetes UK (BDA/11/0004210, BDA/15/0005275, BDA16/0005485)

2078-P: APPL2 Deficiency Diminishes Glucose-Stimulated Insulin Secretion in Pancreatic Beta Cells

In response to elevated postprandial blood glucose, remodeling of F-actin enables exocytosis of insulin granules from pancreatic β cells. Dysregulation of F-actin remodeling contributes to defective glucose-stimulated insulin secretion (GSIS) in β cells and subsequent type 2 diabetes. The adaptor protein APPL1 potentiates GSIS and prevents β cell loss in diabetes, but the role of its close homolog APPL2 in β cell function remains unknown. Here we demonstrated that APPL2 controls GSIS by Rac1-mediated F-actin depolymerization via interaction with Rac GTPase activating protein 1 (RacGAP1). β-cell specific deletion of APPL2 induced defective GSIS and glucose intolerance in mice. Ex vivo study revealed a dramatic reduction in both first- and second-phase GSIS in islets, accompanied by a dysregulation of F-actin remodeling. Consistently, knockdown of APPL2 caused impaired glucose-induced F-actin remodeling and insulin secretion in INS-1E β cells. The defects were mainly due to the inactivation of Rac1- the key player controlling insulin granule secretion via F-actin remodeling. Ectopic expression of constitutively active Rac1 mutant rescued the defective GSIS and F-actin remodeling in APPL2 deficient β cells. Furthermore, we identified RacGAP1 as an interacting partner of APPL2. RacGAP1 interacted with and regulate activity of Rac1, and overexpression of RacGAP1 significantly suppressed glucose-induced F-actin depolymerization, whereas co-expression with APPL2 antagonized this suppressive effect. In addition, the defects in APPL2 knockdown β cells were largely reversed by simultaneous downregulation of RacGAP1 or co-expression of constitutively active Rac1 mutant. In summary, our study suggest that APPL2 potentiates GSIS by antagonizing the inhibitory effect of RacGAP1 on Rac1 activation and F-actin remodeling and insulin secretion in β cells, highlighting the importance of APPL2 in the regulation of GSIS in pancreatic β cells and glucose homeostasis. Disclosure H. Lin: None. Funding National Natural Science Foundation of China (81970675)

86-OR: High-Fat Diet Prevents Autoimmune Diabetes in NOD Mice

Type 1 diabetes (T1D) has a strong genetic predisposition and requires an environmental trigger to initiate autoimmune destruction of beta cells. The rate of childhood obesity has risen in parallel to the rate of T1D, suggesting high fat diet (HFD) may be an environmental trigger in the development of autoimmune diabetes. Supporting this hypothesis, HFD can induce insulin resistance, beta cell stress, dysfunctional insulin production, and beta cell death. To explore the role of HFD in the development of T1D, 4 week-old non-obese diabetic (NOD) female mice were placed on HFD or chow diet (CD). At 10 weeks of age NOD mice fed with HFD demonstrated a significant increase in body weight and non-fasting blood glucose along with mild glucose intolerance compared with NOD CD fed mice. Histological analysis at 10 weeks of age revealed significantly less pancreatic immune cell infiltration and increased beta cell mass in HFD fed mice compared to CD fed mice. By 15 weeks of age, HFD fed NOD animals demonstrated impaired glucose tolerance similar to CD fed mice, increased glucose-stimulated insulin secretion and total insulin content, and insulin resistance. Surprisingly, at 25 weeks of age the HFD fed NOD mice were completely protected from the development of diabetes, while as expected 70% of NOD CD fed mice had developed diabetes. In summary, this study demonstrates that in the NOD model of T1D HFD reduced islet immune cell infiltration, increased beta cell mass, insulin content and insulin secretion protecting the mice from development of diabetes. The mechanism underlying this protection seems to be two-fold: expansion of beta cell mass and prevention of autoimmune-mediated beta cell death. Our data correlate with other recent studies indicating that early stimulation of beta cell replication can induce immune tolerance in NOD animals preventing T1D. Identification of the mechanisms by which HFD leads to prevention of diabetes in NOD animals may lead to the development of novel therapeutic interventions to prevent T1D in humans. Disclosure A.L. Clark: None. Z. Yan: None. S.X. Chen: None. M. Fuess: None. G. McGinn: None. M.S. Remedi: None. Funding National Institutes of Health (P30DK020579, K12HD076224-04)

249-LB: Effects of Oxidative Phosphorylation Complex Defects on Beta-Cell Biology

Background: The insulin-secreting pancreatic beta cell is a highly metabolic cell type relying on mitochondrial metabolism for stimulus-secretion coupling. Specifically, the ATP-producing oxidative phosphorylation (OXPHOS) pathway is central to insulin secretion. Although disruptions in ATP production occur in beta cell dysfunction, the exact mechanisms remain unknown. We have generated beta cell specific OXPHOS complex inducible knock out mouse models of Complex I, III, and IV to determine the direct effects OXPHOS complexes on beta cell function. We hypothesize that individual complexes contribute distinctly to beta cell function. Methods: Eight-week-old, male OXPHOSf/f - MIPcreERT mice were administered tamoxifen (150mg/kg) or vehicle by I.P. injection for 5 consecutive days. Non-fasting glycemia and body weight were measured one week prior and once per week up to 5 weeks after induction. Glucose tolerance was assessed by glucose tolerance tests (IP-GTT) 1 week before and 3 weeks after induction for all groups. Mice were anesthetized with ketamine/xylazine and perfused with sterile PBS followed by 4% PFA. Pancreata were dissected, embedded in OCT, and cut at 40µM sections for immunofluorescent staining of insulin, glucagon, and somatostatin. Results: No changes in body weight were observed throughout the experimental period for any group. Complex III knockout animals developed severe hyperglycemia and significant glucose intolerance at 3 weeks after induction. Although the other models display a minor trend towards hyperglycemia starting at 5 weeks after induction, they retain their ability to regulate glucose at 3 weeks as seen by IP-GTT. Interestingly, no changes in islet cytoarchitecture are observed in any of the groups at the 3-week timepoint. Conclusions: Mice with Complex III deficient beta cells show a robust diabetic phenotype compared to Complex I and IV models. These results support the hypothesis that specific OXPHOS complexes contribute differentially to beta cell function. Disclosure A.L. Lang: None. A. Caicedo: None. Funding National Institute of Environmental Health Sciences (R21ES025673-01)

2082-P: A Negative Regulatory Role of Insulinoma Beta Cell–Derived Exosomes in Glucose-Stimulated Insulin Secretion of Beta-Cells by Upregulating Exosomal MicroRNAs-Associated Signals

Background: In conditions of diabetes, exosomes have been suggested to play the roles in the pathological processes in both type 1 and type 2 diabetes. Exosomes released from both high-concentration glucose- and cytokine-treated β-cells induced β-cell apoptosis. Pancreatic cancer-derived exosomes have also been found to cause paraneoplastic β-cell dysfunction. Here, we further investigated the effects and mechanisms of insulinoma β-cell-derived exosomes on glucose-stimulated insulin secretion. Methods: Exosomes from insulinoma β-cell lines RIN-m5f or NIT-1 were prepared by ExoQuick-TC™, and identified its characteristics by Western blotting and nanoparticle tracking analysis. For in vitro study, exosomes at the concentrations of 1-30 μg/mL were used. For in vivo study, ICR mice were treated with PBS or exosomes (30 μg/100 μL) for 4 weeks by tail vein intravenous injection twice a week. The miRNA profile was determined by Next Generation Sequencing (NGS) analysis. Results: Exosomes from insulinoma β-cells did not affect the cell viability, but significantly inhibited the glucose-stimulated insulin secretion of naïve β-cells. Exogenously administration of exosomes derived from insulinoma β-cells to mice also showed the dysregulation of glucose tolerance and serum insulin levels. Moreover, the results of NGS showed that exosomes may transfer miR-1224, -122-5p, -133a-3p, -10b-5p, -423-5p, -409a-3p to affect insulin secretion-related genes/proteins in β-cells. Exosomes also significantly reduced the protein expressions of phosphorylated Akt, Ca2+/calmodulin-dependent protein kinase-II, and glucose transporter-2 in β-cells, which could be reversed by inhibition of exosome uptake. Conclusion: Exosomes released from insulinoma β-cells are capable of upregulating exosomal miRNAs-associated signals to dysregulate glucose-stimulated insulin secretion in naïve β-cells. Disclosure S. Liu: None. C. Wu: None. Funding Ministry of Science and Technology of Taiwan (107-2314-B-002-031-MY3)

A BAD portion of glucose can be good for inflamed beta cells.

Exposure to high glucose under inflammatory conditions is detrimental to insulin-secreting beta cells in the pancreas. Fu and colleagues describe a metabolic axis that decreases production of the ‘danger molecule’ nitric oxide and improves the survival of beta cells exposed to an inflammatory milieu, thus paving the way to new interventions for diabetes.

E2f8 and Dlg2 genes have independent effects on impaired insulin secretion associated with hyperglycaemia.

Reduced insulin secretion results in hyperglycaemia and diabetes involving a complex aetiology that is yet to be fully elucidated. Genetic susceptibility is a key factor in beta cell dysfunction and hyperglycaemia but the responsible genes have not been defined. The Collaborative Cross (CC) is a recombinant inbred mouse panel with diverse genetic backgrounds allowing the identification of complex trait genes that are relevant to human diseases. The aim of this study was to identify and characterise genes associated with hyperglycaemia. Using an unbiased genome-wide association study, we examined random blood glucose and insulin sensitivity in 53 genetically unique mouse strains from the CC population. The influences of hyperglycaemia susceptibility quantitative trait loci (QTLs) were investigated by examining glucose tolerance, insulin secretion, pancreatic histology and gene expression in the susceptible mice. Expression of candidate genes and their association with insulin secretion were examined in human islets. Mechanisms underlying reduced insulin secretion were studied in MIN6 cells using RNA interference. Wide variations in blood glucose levels and the related metabolic traits (insulin sensitivity and body weight) were observed in the CC population. We showed that elevated blood glucose in the CC strains was not due to insulin resistance nor obesity but resulted from reduced insulin secretion. This insulin secretory defect was demonstrated to be independent of abnormalities in islet morphology, beta cell mass and pancreatic insulin content. Gene mapping identified the E2f8 (p = 2.19 × 10-15) and Dlg2 loci (p = 3.83 × 10-8) on chromosome 7 to be significantly associated with hyperglycaemia susceptibility. Fine mapping the implicated regions using congenic mice demonstrated that these two loci have independent effects on insulin secretion in vivo. Significantly, our results revealed that increased E2F8 and DLG2 gene expression are correlated with enhanced insulin secretory function in human islets. Furthermore, loss-of-function studies in MIN6 cells demonstrated that E2f8 is involved in insulin secretion through an ATP-sensitive K+ channel-dependent pathway, which leads to a 30% reduction in Abcc8 expression. Similarly, knockdown of Dlg2 gene expression resulted in impaired insulin secretion in response to glucose and non-glucose stimuli. Collectively, these findings suggest that E2F transcription factor 8 (E2F8) and discs large homologue 2 (DLG2) regulate insulin secretion. The CC resource enables the identification of E2f8 and Dlg2 as novel genes associated with hyperglycaemia due to reduced insulin secretion in pancreatic beta cells. Taken together, our results provide better understanding of the molecular control of insulin secretion and further support the use of the CC resource to identify novel genes relevant to human diseases.

MiD51 Is Important for Maintaining Mitochondrial Health in Pancreatic Islet and MIN6 Cells.

Background: Mitochondrial dynamics are important for glucose-stimulated insulin secretion in pancreatic beta cells. The mitochondrial elongation factor MiD51 has been proposed to act as an anchor that recruits Drp1 from the cytosol to the outer mitochondrial membrane. Whether MiD51 promotes mitochondrial fusion by inactivation of Drp1 is a controversial issue. Since both the underlying mechanism and the effects on mitochondrial function remain unknown, this study was conducted to investigate the role of MiD51 in beta cells.Methods: Overexpression and downregulation of MiD51 in mouse insulinoma 6 (MIN6) and mouse islet cells was achieved using the pcDNA expression vector and specific siRNA, respectively. Expression of genes regulating mitochondrial dynamics and autophagy was analyzed by quantitative Real-Time PCR, glucose-stimulated insulin secretion by ELISA, and cellular oxygen consumption rate by optode sensor technology. Mitochondrial membrane potential and morphology were visualized after TMRE and MitoTracker Green staining, respectively. Immunofluorescence analyses were examined by confocal microscopy.Results: MiD51 is expressed in insulin-positive mouse and human pancreatic islet and MIN6 cells. Overexpression of MiD51 resulted in mitochondrial fragmentation and cluster formation in MIN6 cells. Mitochondrial membrane potential, glucose-induced oxygen consumption rate and glucose-stimulated insulin secretion were reduced in MIN6 cells with high MiD51 expression. LC3 expression remained unchanged. Downregulation of MiD51 resulted in inhomogeneity of the mitochondrial network in MIN6 cells with hyperelongated and fragmented mitochondria. Mitochondrial membrane potential, maximal and glucose-induced oxygen consumption rate and insulin secretion were diminished in MIN6 cells with low MiD51 expression. Furthermore, reduced Mfn2 and Parkin expression was observed. Based on MiD51 overexpression and downregulation, changes in the mitochondrial network structure similar to those in MIN6 cells were also observed in mouse islet cells.Conclusion: We have demonstrated that MiD51 plays a pivotal role in regulating mitochondrial function and hence insulin secretion in MIN6 cells. We propose that this anchor protein of Drp1 is important to maintain a homogeneous mitochondrial network and to avoid morphologies such as hyperelongation and clustering which are inaccessible for degradation by autophagy. Assuming that insulin granule degradation frequently suppresses autophagy in beta cells, MiD51 could be a key element maintaining mitochondrial health.

Pharmacological Targeting of the Actin Cytoskeleton to Drive Endocrinogenesis

In vitro generation of insulin-secreting beta cells from human pluripotent stem cells (hPSCs) opens new avenues for treating and modeling diabetes. Hogrebe and colleaguesestablished a new 2D differentiation protocol where they targeted the cytoskeleton pharmacologically for controlled endocrine induction and generation of hPSC-derived beta cells with improved function.

Exogenous Ghrelin Increases Plasma Insulin Level in Diabetic Rats.

Ghrelin, a 28-amino acid peptide, is a strong growth hormone secretagogue and a regulator of food intake. In addition, ghrelin is thought to play a role in insulin secretion and in glucose homeostasis. A lot of contradictory data have been reported in the literature regarding the co-localization of ghrelin with other hormones in the islet of Langerhans, its role in insulin secretion and attenuation of type 2 diabetes mellitus. In this study, we investigate the effect of chronic ghrelin treatment on glucose, body weight and insulin level in normal and streptozotocin-induced diabetic male Wistar rats. We have also examined the distribution pattern and co-localization of ghrelin with insulin in pancreatic islet cells using immunohistochemistry and immune-electron microscopy and the ability of ghrelin to stimulate insulin release from the CRL11065 beta cell line. Control, non-diabetic groups received intraperitoneal injection of normal saline, while treated groups received intraperitoneal injection of 5 µg/kg body weight of ghrelin (amino acid chain 24–51) on a daily basis for a duration of four weeks. Our results show that the administration of ghrelin increases the number of insulin-secreting beta cells and serum insulin level in both normal and diabetic rats. We also demonstrated that ghrelin co-localizes with insulin in pancreatic islet cells and that the pattern of ghrelin distribution is altered after the onset of diabetes. Moreover, ghrelin at a dose of 10−6 M and 10−12 M increased insulin release from the CRL11065 beta cell line. In summary, ghrelin co-localizes with insulin in the secretory granules of pancreatic beta cells and enhances insulin production.

A genetic screen identifies Crat as a regulator of pancreatic beta-cell insulin secretion

ObjectivesGlucose-stimulated insulin secretion is a critical function in the regulation of glucose homeostasis, and its deregulation is associated with the development of type 2 diabetes. Here, we performed a genetic screen using islets isolated from the BXD panel of advanced recombinant inbred (RI) lines of mice to search for novel regulators of insulin production and secretion. MethodsPancreatic islets were isolated from 36 RI BXD lines and insulin secretion was measured following exposure to 2.8 or 16.7 mM glucose with or without exendin-4. Islets from the same RI lines were used for RNA extraction and transcript profiling. Quantitative trait loci (QTL) mapping was performed for each secretion condition and combined with transcriptome data to prioritize candidate regulatory genes within the identified QTL regions. Functional studies were performed by mRNA silencing or overexpression in MIN6B1 cells and by studying mice and islets with beta-cell-specific gene inactivation. ResultsInsulin secretion under the 16.7 mM glucose plus exendin-4 condition was mapped significantly to a chromosome 2 QTL. Within this QTL, RNA-Seq data prioritized Crat (carnitine O-acetyl transferase) as a strong candidate regulator of the insulin secretion trait. Silencing Crat expression in MIN6B1 cells reduced insulin content and insulin secretion by ∼30%. Conversely, Crat overexpression enhanced insulin content and secretion by ∼30%. When islets from mice with beta-cell-specific Crat inactivation were exposed to high glucose, they displayed a 30% reduction of insulin content as compared to control islets. We further showed that decreased Crat expression in both MIN6B1 cells and pancreatic islets reduced the oxygen consumption rate in a glucose concentration-dependent manner. ConclusionsWe identified Crat as a regulator of insulin secretion whose action is mediated by an effect on total cellular insulin content; this effect also depends on the genetic background of the RI mouse lines. These data also show that in the presence of the stimulatory conditions used the insulin secretion rate is directly related to the insulin content.

PKR inhibitors suppress endoplasmic reticulum stress and subdue glucolipotoxicity-mediated impairment of insulin secretion in pancreatic beta cells.

Type 2 diabetes mellitus is characterized by insulin resistance and hypersecretion of insulin from the pancreas to compensate for decreased insulin sensitivity in the peripheral tissues. In later stages of the disease insulin-secreting beta cell degeneration commences and patients require insulin replacement therapy in order to accomplish proper regulation of their blood glucose. Endoplasmic reticulum (ER) stress in the beta cells is one of the factors contributing to this detrimental effect. Protein kinase R (PKR) is a cellular stress kinase activated by ER stress and contributing to degeneration of pancreatic islets. In order to determine whether inhibition of PKR activation by specific small molecule inhibitors of PKR ameliorates pancreatic insulin secretion capacity, we treated beta cells with two imidazole/oxindole-derived inhibitors of PKR kinase, imoxin (C16) and 2-aminopurine (2-AP), in the presence of ER stress. Our results demonstrate that PKR inhibition suppresses tunicamycin-mediated ER stress without altering the insulin production capacity of the cells. Palmitic acid-mediated suppression of insulin secretion, however, was subdued significantly by PKR inhibitor treatment through an ER stress-related mechanism. We suggest that PKR inhibitor treatment may be used to increase the insulin secretion capacity of the pancreas in later stages of diabetes.

ER stress increases store-operated Ca2+ entry (SOCE) and augments basal insulin secretion in pancreatic beta cells

Type 2 diabetes mellitus (T2DM) is characterized by impaired glucose-stimulated insulin secretion and increased peripheral insulin resistance. Unremitting endoplasmic reticulum (ER) stress can lead to beta-cell apoptosis and has been linked to type 2 diabetes. Although many studies have attempted to link ER stress and T2DM, the specific effects of ER stress on beta-cell function remain incompletely understood. To determine the interrelationship between ER stress and beta-cell function, here we treated insulin-secreting INS-1(832/13) cells or isolated mouse islets with the ER stress-inducer tunicamycin (TM). TM induced ER stress as expected, as evidenced by activation of the unfolded protein response. Beta cells treated with TM also exhibited concomitant alterations in their electrical activity and cytosolic free Ca2+ oscillations. As ER stress is known to reduce ER Ca2+ levels, we tested the hypothesis that the observed increase in Ca2+ oscillations occurred because of reduced ER Ca2+ levels and, in turn, increased store-operated Ca2+ entry. TM-induced cytosolic Ca2+ and membrane electrical oscillations were acutely inhibited by YM58483, which blocks store-operated Ca2+ channels. Significantly, TM-treated cells secreted increased insulin under conditions normally associated with only minimal release, e.g. 5 mm glucose, and YM58483 blocked this secretion. Taken together, these results support a critical role for ER Ca2+ depletion-activated Ca2+ current in mediating Ca2+-induced insulin secretion in response to ER stress.

Arf6 Heterozygosity Results in Impaired Glucose Tolerance Due to Abberant Hepatic Gluconeogenesis

Adenosine diphosphate (ADP) ribosylation factor 6 (Arf6) is a small GTPase that plays a critical role in numerous cellular processes including proliferation and membrane trafficking. Arf6 has been found to be dysregulated in diseases such as diabetic retinopathy and tumor metastasis. Furthermore, genetic insufficiency or pharmacological inhibition of Arf6 is protective in the aforementioned diseases, suggesting that Arf6 may be a potential therapeutic target. Here, we assessed the metabolic consequences of a reduction in Arf6 expression by performing glucose (GTT: 2 g/kg, ip), insulin (ITT: 1 U/kg, ip) and pyruvate (PTT: 2 g/kg, ip) tolerance tests in 3–4 mo old whole body Arf6 heterozygote (Arf6+/−) and wildtype (Arf6+/+) littermate control mice. Both protein and gene expression of Arf6 in the liver were 40–50% lower (p&lt;0.05) in Arf6+/− mice compared to Arf6+/+. Body mass was higher in normal chow fed Arf6+/− compared to Arf6+/+ mice (30.3±0.9 vs 36.4±2.0 g, p&lt;0.05). Blood glucose at 15, 30, and 60 min after injection during the GTT were 20–30% higher in Arf6+/− mice compared to Arf6+/+ (each p&lt;0.05). Likewise, the area under the GTT time response curve was ~15% higher in Arf6+/− compared to Arf6+/+ (p&lt;0.05), indicating impaired glucose metabolism. To examine the underlying cause of this glucose intolerance, we performed an ITT and assessed insulin‐stimulated suppression of plasma free fatty acids 15, and 30 min after insulin injection. We found no groups differences between the ITT time response curves (p&gt;0.05) and no difference in the percent decrease in plasma FFAs from fasted between Arf6+/− and Arf6+/+ mice (~50% vs ~50% p&gt;0.05). Likewise, fasted and glucose‐stimulated (2g/kg, ip) serum insulin were not different between the groups (2.44±0.58 vs 1.73±0.28, fasted; 4.54±1.74 vs 4.03±1.68 insulin stimulated; both p&gt;0.05), suggesting that reductions in Arf6 expression do not impact pancreatic beta cell insulin secretion. Collectively, these measures suggest that neither peripheral insulin resistance nor impaired pancreatic beta cell function underlie glucose intolerance in Arf6+/− mice. To determine the contribution of hepatic gluconeogenesis to the observed glucose intolerance in the Arf6+/− mice, we performed a PTT and found that Arf6+/− mice exhibited a 15–25% increase in blood glucose over time compared to Arf6+/+ mice (p&lt;0.05), suggesting a role for elevated hepatic gluconeogenesis. Taken together, this study demonstrates that Arf6 heterozygosity impairs glucose metabolism that is associated with aberrant hepatic gluconeogenesis but is independent of peripheral insulin resistance or pancreatic beta cell dysfunction.Support or Funding InformationNIA R01 AG048366, R01 AG050238 and K02 AG045339, R01 AG060395 and US Department of Veterans Affairs I01 BX002151, I01 BX004492.

Lactational metformin exposure programs offspring white adipose tissue glucose homeostasis and resilience to metabolic stress in a sex-dependent manner.

We previously demonstrated that exposing mouse dams to metformin during gestation results in increased beta-cell mass at birth and increased beta-cell insulin secretion in adult male offspring. Given these favorable changes after a gestational maternal metformin exposure, we wanted to understand the long-term metabolic impact on offspring after exposing dams to metformin during the postnatal window. The newborn period provides a feasible clinical window for intervention and is important for beta-cell proliferation and metabolic tissue development. Using a C57BL/6 model, we administered metformin to dams from the day of birth to postnatal day 21. We monitored maternal health and offspring growth during the lactation window, as well as adult glucose homeostasis through in vivo testing. At necropsy we assessed pancreas and adipocyte morphology using histological and immunofluorescent staining techniques. We found that metformin exposure programmed male and female offspring to be leaner with a higher proportion of small adipocytes in the gonadal white adipose tissue (GWAT). Male, but not female, offspring had an improvement in glucose tolerance as young adults concordant with a mild increase in insulin secretion in response to glucose in vivo. These data demonstrate long-term metabolic programming of offspring associated with maternal exposure to metformin during lactation.

AE804, AE806, AE999, AF017, AF041 and AF131 antibodies label mouse insulin-secreting beta cells by immunohistochemistry

The recombinant antibodies AE804, AE806, AE999, AF017, AF041 and AF131 detect by immunohistochemistry the insulin-secreting beta cells in mouse pancreatic islets. The most efficient staining was obtained with AE999.

Islet Complexins are Complex

While Ca2+ activity is required for islet hormone secretion, proper glucose stimulated glucagon, insulin and somatostatin secretion requires a synergistic combination of electrical, paracrine, juxtacrine and cell intrinsic signaling pathways. This complexity can be unraveled by reconciling islet cell electrical activity, second messenger response and secretory vesicle mobilization in the presence and absence of paracrine regulatory factors. One molecule of interest is Complexin 2, a Protein Kinase A (PKA) dependent SNARE binding protein known to be expressed in alpha, beta and delta cells. Previous alpha cell studies showed that paracrine signaling by insulin and somatostatin combine to reduce glucagon secretion by lowering cAMP and PKA activity independent of Ca2+ activity. In contrast, beta cell insulin secretion is increased by cAMP/PKA and Ca2+ activity. Here, we test the hypothesis that Complexin 2 regulates insulin and glucagon secretion at the secretory vesicle level via phosphorylation by Protein Kinase A (PKA) which is in turn regulated by somatostatin paracrine signaling. Using islets from mice that are null for Complexin 2 gene (Cplxn2 KO), we measured insulin, glucagon and somatostatin secretion in response to glucose. We found increases in both glucagon and insulin output despite a concomitant increase in somatostatin secretion. In order to image Ca2+ signaling in Cplxn2 KO mice we used 2D confocal and 3D dual inverted selective plane illumination microscopy (diSPIM), which performs at high speed using light sheet illumination and camera detection. Live islet imaging experiments with GCaMP6 expressed in Cplxn 2 KO alpha cells showed that somatostatin, which normally inhibits Ca2+ oscillations, was unable to attenuate Ca2+ oscillations in Cplxn2 KO alpha cells during direct alpha cell cAMP/PKA activation.