Regenerating 1 and 3b Gene Expression in the Pancreas of Type 2 Diabetic Goto-Kakizaki (GK) Rats

International audienceRegenerating (REG) proteins are associated with islet development, b-cell damage, diabetes and pancreatitis. Particularly, REG-1 and REG-3-beta are involved in cell growth/survival and/or inflammation and the Reg1 promoter contains interleukin-6 (IL-6)-responsive elements. We showed by transcriptome analysis that islets of Goto-Kakizaki (GK) rats, a model of spontaneous type 2 diabetes, overexpress Reg1, 3a, 3b and 3c, vs Wistar islets. Goto-Kakizaki rat islets also exhibit increased cytokine/chemokine expression/release, particularly IL-6. Here we analyzed Reg1 and Reg3b expression and REG-1 immuno-localization in the GK rat pancreas in relationship with inflammation. Isolated pancreatic islets and acinar tissue from male adult Wistar and diabetic GK rats were used for quantitative RT-PCR analysis. REG-1 immunohistochemistry was performed on paraffin sections with a monoclonal anti-rat REG-1 antibody. Islet cytokine/chemokine release was measured after 48 h-culture. Islet macrophage-positive area was quantified on cryostat sections using anti-CD68 and major histocompatibility complex (MHC) class II antibodies. Pancreatic exocrine-to-endocrine Reg1 and Reg3b mRNA ratios were markedly increased in Wistar vs GK rats. Conversely, both genes were upregulated in isolated GK rat islets. These findings were unexpected, because Reg genes are expressed in the pancreatic acinar tissue. However, we observed REG-1 protein labeling in acinar peri-ductal tissue close to islets and around large, often disorganized, GK rat islets, which may retain acinar cells due to their irregular shape. These large islets also showed peri-islet macrophage infiltration and increased release of various cytokines/ chemokines, particularly IL-6. Thus, IL-6 might potentially trigger acinar REG-1 expression and secretion in the vicinity of large diabetic GK rat islets. This increased acinar REG-1 expression might reflect an adaptive though unsuccessful response to deleterious microenvironment

Susceptibility to fatty acid-induced β-cell dysfunction is enhanced in prediabetic diabetes-prone biobreeding rats: A potential link between β-cell lipotoxicity and islet inflammation

β-Cell lipotoxicity is thought to play an important role in the development of type 2 diabetes. However, no study has examined its role in type 1 diabetes, which could be clinically relevant for slow-onset type 1 diabetes. Reports of enhanced cytokine toxicity in fat-laden islets are consistent with the hypothesis that lipid and cytokine toxicity maybe synergistic. Thus, β-cell lipotoxicity could be enhanced in models of autoimmune diabetes. To determine this, we examined the effects of prolonged free fatty acids elevation on β-cell secretory function in the prediabetic diabetes-prone BioBreeding (dp-BB) rat, its diabetes-resistant BioBreeding (dr-BB) control, and normal Wistar-Furth (WF) rats. Rats received a 48-h iv infusion of saline or Intralipid plus heparin (IH) (to elevate free fatty acid levels ∼2-fold) followed by hyperglycemic clamp or islet secretion studies ex vivo. IH significantly decreased β-cell function, assessed both by the disposition index (insulin secretion corrected for IH-induced insulin resistance) and in isolated islets, in dp-BB, but not in dr-BB or WF, rats, and the effect of IH was inhibited by the antioxidant N-acetylcysteine. Furthermore, IH significantly increased islet cytokine mRNA and plasma cytokine levels (monocyte chemoattractant protein-1 and IL-10) in dp-BB, but not in dr-BB or WF, rats. All dp-BB rats had mononuclear infiltration of islets, which was absent in dr-BB and WF rats. In conclusion, the presence of insulitis was permissive for IH-induced β-cell dysfunction in the BB rat, which suggests a link between β-cell lipotoxicity and islet inflammation. Copyright © 2013 by The Endocrine Society

Islet Endothelial Activation and Oxidative Stress Gene Expression Is Reduced by IL-1Ra Treatment in the Type 2 Diabetic GK Rat

Inflammation followed by fibrosis is a component of islet dysfunction in both rodent and human type 2 diabetes. Because islet inflammation may originate from endothelial cells, we assessed the expression of selected genes involved in endothelial cell activation in islets from a spontaneous model of type 2 diabetes, the Goto-Kakizaki (GK) rat. We also examined islet endotheliuml/oxidative stress (OS)/inflammation-related gene expression, islet vascularization and fibrosis after treatment with the interleukin-1 (IL-1) receptor antagonist (IL-1Ra)

Alternative signaling pathways of the glucose-dependent insulinotropic polypeptide (GIP) receptor

Glucose-dependent insulinotropic polypeptide (GIP) regulates pancreatic β -cell function by binding to its cognate Family B, G protein-coupled receptor and elevating intracellular cAMP and Ca2 + . The main rationale for the studies described in this Thesis was that multiple interacting intracellular signal transduction pathways were proposed to mediate GIP's pleiotropic actions on the pancreatic β-cell. Moreover, the ability of GIP to potentiate insulin secretion is blunted in some type 2 diabetics, implying potential defects at the receptor signaling level. Therefore, the major aim was to characterize further the intricate network of GIP receptor signaling pathways underlying β-cell processes using β-cell models. Using GIP receptor-transfected CHO-K1 cells and βTC-3 and INS-1 tumour cell lines, it was possible to correlate GIP receptor activation with the regulation of lipid signaling (arachidonic acid release), K + ATP channel-independent events, MAP kinase signaling (Raf->Mekl/2->ERKl/2->p90RSK and p38 MAPK), and CREB signaling. These events were demonstrated to be functionally relevant for insulin secretion, cell growth and survival (MAPK signaling), and insulin gene transcription (CREB signaling) respectively. Through insulin secretion studies and pharmacological approaches, GIP was shown to regulate the secretion of insulin via activation of PLA2 and K+ATP channel-independent mechanisms in βTC-3 cells. In an attempt to elucidate novel signals coupled to the GIP receptor, we examined the expression of 75 protein kinases and 25 protein phosphatases in CHO-K1, βTC-3, and INS-1 cells. This has allowed the partial mapping of intracellular signal transduction pathways for these cell models. From this, GIP receptor coupled signaling events were studied using phospho-specific antibodies, transfection techniques, pharmacological inhibitors, and gene reporter assays. Studies in CHO-K1 cells expressing the GIP receptor and in INS-1 B-cells have implicated cAMP/PKA signaling in the regulation of the mitogenic ERK 1/2 module, Raf->Mekl/2->ERKl/2->p90RSK. Results further suggest that the GIP receptor is coupled to β-cell survival via cAMP mediated inhibition of p38 MAPK and caspase-3 activity. Finally, coupling of the GIP receptor to rat insulin promoter activity was shown to occur via cAMP/PKA and a CREB family transcription factor. These events were found to be independent of phosphoregulation of S133 CREB/S117 CREM/S63 ATF-1, and suggest that the tightly regulated phosphorylation of these transcription factors by GIP may be involved in novel signals regulating β-cell function.Medicine, Faculty ofCellular and Physiological Sciences, Department ofGraduat

Macrophages, cytokines and β-cell death in Type 2 diabetes.

Abstract The pathology of islets from patients with Type 2 diabetes displays an inflammatory process characterized by the presence of immune cell infiltration, cytokines, apoptotic cells, amyloid deposits and, eventually, fibrosis. Indeed, analysis of β-cells from patients with Type 2 diabetes displays increased IL-1β (interleukin 1β) expression. Furthermore, increased islet-associated macrophages are observed in human Type 2 diabetic patients and in most animal models of diabetes. Importantly, increased numbers of macrophages are detectable very early in high-fat-fed mice islets, before the onset of diabetes. These immune cells are probably attracted by islet-derived chemokines, produced in response to metabolic stress, and under the control of IL-1β. It follows that modulation of intra-islet inflammatory mediators, particularly interleukin-1β, may prevent islet inflammation in Type 2 diabetes and therefore presents itself as a promising therapeutic approach

Interleukin-1-receptor antagonist in type 2 diabetes mellitus

BACKGROUND: The expression of interleukin-1-receptor antagonist is reduced in pancreatic islets of patients with type 2 diabetes mellitus, and high glucose concentrations induce the production of interleukin-1beta in human pancreatic beta cells, leading to impaired insulin secretion, decreased cell proliferation, and apoptosis. METHODS: In this double-blind, parallel-group trial involving 70 patients with type 2 diabetes, we randomly assigned 34 patients to receive 100 mg of anakinra (a recombinant human interleukin-1-receptor antagonist) subcutaneously once daily for 13 weeks and 36 patients to receive placebo. At baseline and at 13 weeks, all patients underwent an oral glucose-tolerance test, followed by an intravenous bolus of 0.3 g of glucose per kilogram of body weight, 0.5 mg of glucagon, and 5 g of arginine. In addition, 35 patients underwent a hyperinsulinemic-euglycemic clamp study. The primary end point was a change in the level of glycated hemoglobin, and secondary end points were changes in beta-cell function, insulin sensitivity, and inflammatory markers. RESULTS: At 13 weeks, in the anakinra group, the glycated hemoglobin level was 0.46 percentage point lower than in the placebo group (P=0.03); C-peptide secretion was enhanced (P=0.05), and there were reductions in the ratio of proinsulin to insulin (P=0.005) and in levels of interleukin-6 (P<0.001) and C-reactive protein (P=0.002). Insulin resistance, insulin-regulated gene expression in skeletal muscle, serum adipokine levels, and the body-mass index were similar in the two study groups. Symptomatic hypoglycemia was not observed, and there were no apparent drug-related serious adverse events. CONCLUSIONS: The blockade of interleukin-1 with anakinra improved glycemia and beta-cell secretory function and reduced markers of systemic inflammation. (ClinicalTrials.gov number, NCT00303394 [ClinicalTrials.gov].)

Low- and high-density lipoproteins modulate function, apoptosis, and proliferation of primary human and murine pancreatic beta-cells

A low high-density lipoprotein (HDL) plasma concentration and the abundance of small dense low-density lipoproteins (LDL) are risk factors for developing type 2 diabetes. We therefore investigated whether HDL and LDL play a role in the regulation of pancreatic islet cell apoptosis, proliferation and secretory function. Isolated mouse and human islets were exposed to plasma lipoproteins of healthy human donors. In murine and human beta-cells LDL decreased both proliferation and maximal glucose-stimulated insulin secretion. The comparative analysis of beta-cells from wild type and LDL receptor deficient mice revealed that the inhibitory effect of LDL on insulin secretion but not on proliferation requires the LDL receptor. HDL was found to modulate the survival of both human and murine islets by decreasing basal as well as IL-1beta and glucose induced apoptosis. IL-1beta induced beta-cell apoptosis was also inhibited in the presence of either the delipidated protein or the deproteinated lipid moieties of HDL, apolipoprotein A1 (the main protein component of HDL) or sphingosine-1-phosphate (a bioactive sphingolipid mostly carried by HDL). In murine beta-cells, the protective effect of HDL against IL-1beta induced apoptosis was also observed in the absence of the HDL receptor scavenger receptor class B type 1 (SRB1). Our data show that both LDL and HDL affect function or survival of beta-cells and raise the question whether dyslipidemia contributes to beta-cell failure and hence the manifestation and progression of type 2 diabetes mellitus

Islet Macrophages Shift to a Reparative State following Pancreatic Beta-Cell Death and Are a Major Source of Islet Insulin-like Growth Factor-1

Macrophages play a dynamic role in tissue repair following injury. Here we found that following streptozotocin (STZ)-induced beta-cell death, mouse islet macrophages had increased Igf1 expression, decreased proinflammatory cytokine expression, and transcriptome changes consistent with macrophages undergoing efferocytosis and having an enhanced state of metabolism. Macrophages were the major, if not sole, contributors to islet insulin-like growth factor-1 (IGF-1) production. Adoptive transfer experiments showed that macrophages can maintain insulin secretion in vivo following beta-cell death with no effects on islet cell turnover. IGF-1 neutralization during STZ treatment decreased insulin secretion without affecting islet cell apoptosis or proliferation. Interestingly, high-fat diet (HFD) combined with STZ further skewed islet macrophages to a reparative state. Finally, islet macrophages from db/db mice also expressed decreased proinflammatory cytokines and increased Igf1 mRNA. These data have important implications for islet biology and pathology and show that islet macrophages preserve their reparative state following beta-cell death even during HFD feeding and severe hyperglycemia.ISSN:2589-004