Topologically heterogeneous beta cell adaptation in response to high-fat diet in mice.
ABSTRACT: AIMS:Beta cells adapt to an increased insulin demand by enhancing insulin secretion via increased beta cell function and/or increased beta cell number. While morphological and functional heterogeneity between individual islets exists, it is unknown whether regional differences in beta cell adaptation occur. Therefore we investigated beta cell adaptation throughout the pancreas in a model of high-fat diet (HFD)-induced insulin resistance in mice. METHODS:C57BL/6J mice were fed a HFD to induce insulin resistance, or control diet for 6 weeks. The pancreas was divided in a duodenal (DR), gastric (GR) and splenic (SR) region and taken for either histology or islet isolation. The capacity of untreated islets from the three regions to adapt in an extrapancreatic location was assessed by transplantation under the kidney capsule of streptozotocin-treated mice. RESULTS:SR islets showed 70% increased beta cell proliferation after HFD, whereas no significant increase was found in DR and GR islets. Furthermore, isolated SR islets showed twofold enhanced glucose-induced insulin secretion after HFD, as compared with DR and GR islets. In contrast, transplantation of islets isolated from the three regions to an extrapancreatic location in diabetic mice led to a similar decrease in hyperglycemia and no difference in beta cell proliferation. CONCLUSIONS:HFD-induced insulin resistance leads to topologically heterogeneous beta cell adaptation and is most prominent in the splenic region of the pancreas. This topological heterogeneity in beta cell adaptation appears to result from extrinsic factors present in the islet microenvironment.
Project description:AIMS/HYPOTHESIS: In this study, we used an immunodeficient mouse model to explore, in vivo, the longitudinal adaptation of human islets to an obesogenic environment. METHODS: Non-diabetic Rag2 (-/-) mice (n?=?61) were transplanted with human islets (400 islet equivalents [IEQ]) from six pancreases: four non-diabetic and two with overt metabolic dysfunction (older, high HbA(lc) or history of diabetes). Animals were fed for 12 weeks with a control or high-fat diet (HFD), and followed for weight, serum triacylglycerol, fasting blood glucose and human C-peptide. After the mice were killed, human grafts and the endogenous pancreas were analysed for endocrine volume, distribution of beta and alpha cells, and proliferation. RESULTS: Transplanted mice on an HFD gained significantly more weight (p?<?0.001) and had higher fasting glycaemia (2-12 weeks; p?=?0.0002) and consistently higher fasting human C-peptide levels (2-12 weeks; p?=?0.04) compared with those on the control diet. Histology demonstrated doubling of human islet graft volume at 12 weeks in animals on the HFD and increased beta cell volume (p?<?0.001), but no change in alpha cell volume. Human islet function (hyperbolic product HOMA2%BS) at 12 weeks was four times lower in HFD animals (p?<?0.001 vs controls) because of insufficient beta cell adaptation to decreased (70%) sensitivity (HOMA%S). Human islets obtained from donors with metabolic dysfunction failed to adapt to the HFD. CONCLUSIONS/INTERPRETATION: This longitudinal study provides direct evidence that human islets adapt both endocrine and beta cell mass, function and gene expression to obesity in vivo. The present model will facilitate the identification of mechanisms by which human islets adapt to obesity in vivo and the cell type(s) responsible, and factors predisposing human beta cells to decompensation.
Project description:Hedgehog interacting protein (Hhip) is essential for islet formation and beta-cell proliferation during pancreatic development; abnormally elevated Hhip expression has been linked to human pancreatitis. Here, we investigate the role of Hhip in modulating insulin secretion in adult Hhip mice (Hhip +/- vs. Hhip+/+) fed high fat diets (HFD). Both sexes of HFD-Hhip +/+ mice developed impaired glucose intolerance, that was only ameliorated in male HFD-Hhip +/- mice that had high levels of circulating plasma insulin, but not in female HFD-Hhip +/- mice. HFD stimulated Hhip gene expression, mainly in beta cells. Male HFD-Hhip +/+ mice had more large islets in which insulin content was reduced; islet architecture was disordered; and markers of oxidative stress (8-OHdG and Nox 2) were increased. In contrast, male HFD-Hhip +/- mice had more small islets with increased beta cell proliferation, enhanced GSIS, less oxidative stress and preserved islet integrity. In vitro, recombinant Hhip increased Nox2 and NADPH activity and decreased insulin-positive beta cells. siRNA-Hhip increased GSIS and abolished the stimulation of sodium palmitate (PA)-BSA on Nox2 gene expression. We conclude that pancreatic Hhip gene inhibits insulin secretion by altering islet integrity and promoting Nox2 gene expression in beta cells in response to HDF-mediated beta cell dysfunction, a novel finding.
Project description:Diabetes is a major complication of chronic Glucocorticoids (GCs) treatment. GCs induce insulin resistance and also inhibit insulin secretion from pancreatic beta cells. Yet, a full understanding of this negative regulation remains to be deciphered. In the present study, we investigated whether GCs could inhibit serotonin synthesis in beta cell since this neurotransmitter has been shown to be involved in the regulation of insulin secretion. To this aim, serotonin synthesis was evaluated in vitro after treatment with GCs of either islets from CD1 mice or MIN6 cells, a beta-cell line. We also explored the effect of GCs on the stimulation of serotonin synthesis by several hormones such as prolactin and GLP 1. We finally studied this regulation in islet in two in vivo models: mice treated with GCs and with liraglutide, a GLP1 analog, and mice deleted for the glucocorticoid receptor in the pancreas. We showed in isolated islets and MIN6 cells that GCs decreased expression and activity of the two key enzymes of serotonin synthesis, Tryptophan Hydroxylase 1 (Tph1) and 2 (Tph2), leading to reduced serotonin contents. GCs also blocked the induction of serotonin synthesis by prolactin or by a previously unknown serotonin activator, the GLP-1 analog exendin-4. In vivo, activation of the Glucagon-like-Peptide-1 receptor with liraglutide during 4 weeks increased islet serotonin contents and GCs treatment prevented this increase. Finally, islets from mice deleted for the GR in the pancreas displayed an increased expression of Tph1 and Tph2 and a strong increased serotonin content per islet. In conclusion, our results demonstrate an original inhibition of serotonin synthesis by GCs, both in basal condition and after stimulation by prolactin or activators of the GLP-1 receptor. This regulation may contribute to the deleterious effects of GCs on beta cells.
Project description:AIMS/HYPOTHESIS:Glucagon like peptide-1 (GLP-1) enhances glucose-dependent insulin secretion by binding to GLP-1 receptors (GLP1Rs) on pancreatic beta cells. GLP-1 mimetics are used in the clinic for the treatment of type 2 diabetes, but despite their therapeutic success, several clinical effects of GLP-1 remain unexplained at a mechanistic level, particularly in extrapancreatic tissues. The aim of this study was to generate and characterise a monoclonal antagonistic antibody for the GLP1R for use in vivo. METHODS:A naive phage display selection strategy was used to isolate single-chain variable fragments (ScFvs) that bound to GLP1R. The ScFv with the highest affinity, Glp1R0017, was converted into a human IgG1 and characterised further. In vitro antagonistic activity was assessed in a number of assays: a cAMP-based homogenous time-resolved fluorescence assay in GLP1R-overexpressing cell lines, a live cell cAMP imaging assay and an insulin secretion assay in INS-1 832/3 cells. Glp1R0017 was further tested in immunostaining of mouse pancreas, and the ability of Glp1R0017 to block GLP1R in vivo was assessed by both IPGTT and OGTT in C57/Bl6 mice. RESULTS:Antibodies to GLP1R were selected from naive antibody phage display libraries. The monoclonal antibody Glp1R0017 antagonised mouse, human, rat, cynomolgus monkey and dog GLP1R. This antagonistic activity was specific to GLP1R; no antagonistic activity was found in cells overexpressing the glucose-dependent insulinotropic peptide receptor (GIPR), glucagon like peptide-2 receptor or glucagon receptor. GLP-1-stimulated cAMP and insulin secretion was attenuated in INS-1 832/3 cells by Glp1R0017 incubation. Immunostaining of mouse pancreas tissue with Glp1R0017 showed specific staining in the islets of Langerhans, which was absent in Glp1r knockout tissue. In vivo, Glp1R0017 reversed the glucose-lowering effect of liraglutide during IPGTTs, and reduced glucose tolerance by blocking endogenous GLP-1 action in OGTTs. CONCLUSIONS/INTERPRETATION:Glp1R0017 is a monoclonal antagonistic antibody to the GLP1R that binds to GLP1R on pancreatic beta cells and blocks the actions of GLP-1 in vivo. This antibody holds the potential to be used in investigating the physiological importance of GLP1R signalling in extrapancreatic tissues where cellular targets and signalling pathways activated by GLP-1 are poorly understood.
Project description:AIMS/HYPOTHESIS:Genetic studies show coupling of genes affecting beta cell function to type 1 diabetes, but hitherto no studies on whether beta cell dysfunction could precede insulitis and clinical onset of type 1 diabetes are available. METHODS:We used 40-day-old BioBreeding (BB) DRLyp/Lyp rats (a model of spontaneous autoimmune type 1 diabetes) and diabetes-resistant DRLyp/+ and DR+/+ littermates (controls) to investigate beta cell function in vivo, and insulin and glucagon secretion in vitro. Beta cell mass was assessed by optical projection tomography (OPT) and morphometry. Additionally, measurements of intra-islet blood flow were performed using microsphere injections. We also assessed immune cell infiltration, cytokine expression in islets (by immunohistochemistry and qPCR), as well as islet Glut2 expression and ATP/ADP ratio to determine effects on glucose uptake and metabolism in beta cells. RESULTS:DRLyp/Lyp rats were normoglycaemic and without traces of immune cell infiltrates. However, IVGTTs revealed a significant decrease in the acute insulin response to glucose compared with control rats (1685.3?±?121.3 vs 633.3?±?148.7; p?<?0.0001). In agreement, insulin secretion was severely perturbed in isolated islets, and both first- and second-phase insulin release were lowered compared with control rats, while glucagon secretion was similar in both groups. Interestingly, after 5-7 days of culture of islets from DRLyp/Lyp rats in normal media, glucose-stimulated insulin secretion (GSIS) was improved; although, a significant decrease in GSIS was still evident compared with islets from control rats at this time (7393.9?±?1593.7 vs 4416.8?±?1230.5 pg islet-1 h-1; p?<?0.0001). Compared with controls, OPT of whole pancreas from DRLyp/Lyp rats revealed significant reductions in medium (4.1?×?109?±?9.5?×?107 vs 3.8?×?109?±?5.8?×?107 ?m3; p?=?0.044) and small sized islets (1.6?×?109?±?5.1?×?107 vs 1.4?×?109?±?4.5?×?107 ?m3; p?=?0.035). Finally, we found lower intra-islet blood perfusion in vivo (113.1?±?16.8 vs 76.9?±?11.8 ?l min-1 [g pancreas]-1; p?=?0.023) and alterations in the beta cell ATP/ADP ratio in DRLyp/Lyp rats vs control rats. CONCLUSIONS/INTERPRETATION:The present study identifies a deterioration of beta cell function and mass, and intra-islet blood flow that precedes insulitis and diabetes development in animals prone to autoimmune type 1 diabetes. These underlying changes in islet function may be previously unrecognised factors of importance in type 1 diabetes development.
Project description:Under conditions of high fat diet (HFD) consumption, glucose dyshomeostasis develops when β-cells are unable to adapt to peripheral insulin demands. Few studies have interrogated the molecular mechanisms of β-cell dysfunction at the level of mRNA translation under such conditions. We sought to address this issue through polyribosome profile analysis of islets from mice fed 16-weeks of 42% HFD. HFD-islet analysis revealed clear trends toward global reductions in mRNA translation with a significant reduction in the polyribosome/monoribosome ratio for Pdx1 mRNA. Transcriptional and translational analyses revealed endoplasmic reticulum stress was not the etiology of our findings. HFD-islets demonstrated evidence of oxidative stress and DNA damage, as well as activation of p53. Experiments in MIN-6 β-cells revealed that treatment with doxorubicin to directly induce DNA damage mimicked our observed effects in islets. Islets from animals treated with pioglitazone concurrently with HFD demonstrated a reversal of effects observed from HFD alone. Finally, HFD-islets demonstrated reduced expression of multiple ribosome biogenesis genes and the key translation initiation factor eIF4E. We propose a heretofore unappreciated effect of chronic HFD on β-cells, wherein continued DNA damage owing to persistent oxidative stress results in p53 activation and a resultant inhibition of mRNA translation.
Project description:A compensatory increase in β-cell mass occurs during pregnancy to counter the associated insulin resistance, and a failure in adaptation is thought to contribute to gestational diabetes. Insulin-expressing but glucose-transporter-2-low (Ins+Glut2LO) progenitor cells are present in mouse and human pancreas, being predominantly located in extra-islet β-cell clusters, and contribute to the regeneration of the endocrine pancreas following induced ablation. We therefore sought to investigate the contribution of Ins+Glut2LO cells to β-cell mass expansion during pregnancy. Female C57Bl/6 mice were time mated and pancreata were collected at gestational days (GD) 6, 9, 12, 15, and 18, and postpartum D7 (n = 4/time-point) and compared to control (non-pregnant) animals. Beta cell mass, location, proliferation (Ki67+), and proportion of Ins+Glut2LO cells were measured using immunohistochemistry and bright field or confocal microscopy. Beta cell mass tripled by GD18 and β-cell proliferation peaked at GD12 in islets (≥6 β-cells) and small β-cell clusters (1-5 β-cells). The proportion and fraction of Ins+Glut2LO cells undergoing proliferation increased significantly at GD9 in both islets and clusters, preceding the increase in β-cell mass and proliferation, and their proliferation within clusters persisted until GD15. The overall number of clusters increased significantly at GD9. Quantitative PCR showed a significant increase in Pdx1 presence at GD9 vs. GD18 or control pancreas, and Pdx1 was visualized by immunohistochemistry within both Ins+Glut2LO and Ins+Glut2HI cells within clusters. These results indicate that Ins+Glut2LO cells are likely to contribute to β-cell mass expansion during pregnancy.
Project description:AIMS/HYPOTHESIS:Loss of circadian clocks from all tissues causes defective glucose homeostasis as well as loss of feeding and activity rhythms. Little is known about peripheral tissue clocks, so we tested the hypothesis that an intrinsic circadian clock of the pancreas is important for glucose homeostasis. METHODS:We monitored real-time bioluminescence of pancreas explants from circadian reporter mice and examined clock gene expression in beta cells by immunohistochemistry and in situ hybridisation. We generated mice selectively lacking the essential clock gene Bmal1 (also known as Arntl) in the pancreas and tested mutant mice and littermate controls for glucose and insulin tolerance, insulin production and behaviour. We examined islets isolated from mutants and littermate controls for glucose-stimulated insulin secretion and total insulin content. RESULTS:Pancreas explants exhibited robust circadian rhythms. Clock genes Bmal1 and Per1 were expressed in beta cells. Despite normal activity and feeding behaviour, mutant mice lacking clock function in the pancreas had severe glucose intolerance and defective insulin production; their isolated pancreatic islets had defective glucose-stimulated insulin secretion, but normal total insulin content. CONCLUSIONS/INTERPRETATION:The mouse pancreas has an autonomous clock function and beta cells are very likely to be one of the pancreatic cell types possessing an intrinsic clock. The Bmal1 circadian clock gene is required in the pancreas, probably in beta cells, for normal insulin secretion and glucose homeostasis. Our results provide evidence for a previously unrecognised molecular regulator of pancreatic glucose-sensing and/or insulin secretion.
Project description:The progression towards type 2 diabetes depends on the allostatic response of pancreatic beta cells to synthesise and secrete enough insulin to compensate for insulin resistance. The endocrine pancreas is a plastic tissue able to expand or regress in response to the requirements imposed by physiological and pathophysiological states associated to insulin resistance such as pregnancy, obesity or ageing, but the mechanisms mediating beta cell mass expansion in these scenarios are not well defined. We have recently shown that ob/ob mice with genetic ablation of PPAR?2, a mouse model known as the POKO mouse failed to expand its beta cell mass. This phenotype contrasted with the appropriate expansion of the beta cell mass observed in their obese littermate ob/ob mice. Thus, comparison of these models islets particularly at early ages could provide some new insights on early PPAR? dependent transcriptional responses involved in the process of beta cell mass expansionHere we have investigated PPAR? dependent transcriptional responses occurring during the early stages of beta cell adaptation to insulin resistance in wild type, ob/ob, PPAR?2 KO and POKO mice. We have identified genes known to regulate both the rate of proliferation and the survival signals of beta cells. Moreover we have also identified new pathways induced in ob/ob islets that remained unchanged in POKO islets, suggesting an important role for PPAR? in maintenance/activation of mechanisms essential for the continued function of the beta cell.Our data suggest that the expansion of beta cell mass observed in ob/ob islets is associated with the activation of an immune response that fails to occur in POKO islets. We have also indentified other PPAR? dependent differentially regulated pathways including cholesterol biosynthesis, apoptosis through TGF-? signaling and decreased oxidative phosphorylation.
Project description:The tumor suppressor liver kinase B1 (LKB1), also called STK11, is a protein kinase mutated in Peutz-Jeghers syndrome. LKB1 phosphorylates AMP-activated protein kinase (AMPK) and several related protein kinases. Whereas deletion of both catalytic isoforms of AMPK from the pancreatic beta-cell and hypothalamic neurons using the rat insulin promoter (RIP2).Cre transgene (betaAMPKdKO) diminishes insulin secretion in vivo, deletion of LKB1 in the beta-cell with an inducible Pdx-1.CreER transgene enhances insulin secretion in mice. To determine whether the differences between these models reflect genuinely distinct roles for the two kinases in the beta-cell or simply differences in the timing and site(s) of deletion, we have therefore created mice deleted for LKB1 with the RIP2.Cre transgene. In marked contrast to betaAMPKdKO mice, betaLKB1KO mice showed diminished food intake and weight gain, enhanced insulin secretion, unchanged insulin sensitivity, and improved glucose tolerance. In line with the phenotype of Pdx1-CreER mice, total beta-cell mass and the size of individual islets and beta-cells were increased and islet architecture was markedly altered in betaLKB1KO islets. Signaling by mammalian target of rapamycin (mTOR) to eIF4-binding protein-1 and ribosomal S6 kinase was also enhanced. In contrast to Pdx1-CreER-mediated deletion, the expression of Glut2, glucose-induced changes in membrane potential and intracellular Ca(2+) were sharply reduced in betaLKB1KO mouse islets and the stimulation of insulin secretion was modestly inhibited. We conclude that LKB1 and AMPK play distinct roles in the control of insulin secretion and that the timing of LKB1 deletion, and/or its loss from extrapancreatic sites, influences the final impact on beta-cell function.