The Protective Effects of CD39 Overexpression in Multiple Low-Dose Streptozotocin–Induced Diabetes in Mice

Islet allograft survival limits the long-term success of islet transplantation as a potential curative therapy for type 1 diabetes. A number of factors compromise islet survival, including recurrent diabetes. We investigated whether CD39, an ectonucleotidase that promotes the generation of extracellular adenosine, would mitigate diabetes in the T cell–mediated multiple low-dose streptozotocin (MLDS) model. Mice null for CD39 (CD39KO), wild-type mice (WT), and mice overexpressing CD39 (CD39TG) were subjected to MLDS. Adoptive transfer experiments were performed to delineate the efficacy of tissue-restricted overexpression of CD39. The role of adenosine signaling was examined using mutant mice and pharmacological inhibition. The susceptibility to MLDS-induced diabetes was influenced by the level of expression of CD39. CD39KO mice developed diabetes more rapidly and with higher frequency than WT mice. In contrast, CD39TG mice were protected. CD39 overexpression conferred protection through the activation of adenosine 2A receptor and adenosine 2B receptor. Adoptive transfer experiments indicated that tissue-restricted overexpression of CD39 conferred robust protection, suggesting that this may be a useful strategy to protect islet grafts from T cell–mediated injury

Activation of the NLRP3 inflammasome complex is not required for stress-induced death of pancreatic islets.

Loss of pancreatic beta cells is a feature of type-2 diabetes. High glucose concentrations induce endoplasmic reticulum (ER) and oxidative stress-mediated apoptosis of islet cells in vitro. ER stress, oxidative stress and high glucose concentrations may also activate the NLRP3 inflammasome leading to interleukin (IL)-1β production and caspase-1 dependent pyroptosis. However, whether IL-1β or intrinsic NLRP3 inflammasome activation contributes to beta cell death is controversial. This possibility was examined in mouse islets. Exposure of islets lacking functional NLRP3 or caspase-1 to H2O2, rotenone or thapsigargin induced similar cell death as in wild-type islets. This suggests that oxidative or ER stress do not cause inflammasome-mediated cell death. Similarly, deficiency of NLRP3 inflammasome components did not provide any protection from glucose, ribose or gluco-lipotoxicity. Finally, genetic activation of NLRP3 specifically in beta cells did not increase IL-1β production or cell death, even in response to glucolipotoxicity. Overall, our results show that glucose-, ER stress- or oxidative stress-induced cell death in islet cells is not dependent on intrinsic activation of the NLRP3 inflammasome.info:eu-repo/semantics/publishe

Deficiency in type I interferon signaling prevents the early interferon–induced gene signature in pancreatic islets but not type 1 diabetes in NOD mice

Type I interferons (IFNs) have been implicated in the initiation of islet autoimmunity and development of type 1 diabetes. To directly test their involvement, we generated NOD mice deficient in type I IFN receptors (NOD.IFNAR1−/−). Expression of the type I IFN-induced genes Mx1, Isg15, Ifit1, Oas1a, and Cxcr4 was detectable in NOD islets as early as 1 week of age. Of these five genes, expression of Isg15, Ifit1, Oas1a, and Mx1 peaked at 3–4 weeks of age, corresponding with an increase in Ifnα mRNA, declined at 5–6 weeks of age, and increased again at 10–14 weeks of age. Increased IFN-induced gene expression was ablated in NOD.IFNAR1−/− islets. Loss of Toll-like receptor 2 (TLR2) resulted in reduced islet expression of Mx1 at 2 weeks of age, but TLR2 or TLR9 deficiency did not change the expression of other IFN-induced genes in islets compared with wild-type NOD islets. We observed increased β-cell major histocompatibility complex class I expression with age in NOD and NOD.IFNAR1−/− mice. NOD.IFNAR1−/− mice developed insulitis and diabetes at a similar rate to NOD controls. These results indicate type I IFN is produced within islets in young mice but is not essential for the initiation and progression of diabetes in NOD mice

Autoreactive T cells induce necrosis and not BCL-2-regulated or death receptor-mediated apoptosis or RIPK3-dependent necroptosis of transplanted islets in a mouse model of type 1 diabetes

AIMS/HYPOTHESIS: Type 1 diabetes results from T cell-mediated destruction of pancreatic beta cells. The mechanisms of beta cell destruction in vivo, however, remain unclear. We aimed to test the relative roles of the main cell death pathways: apoptosis, necrosis and necroptosis, in beta cell death in the development of CD4(+) T cell-mediated autoimmune diabetes. METHODS: We altered expression levels of critical cell death proteins in mouse islets and tested their ability to survive CD4(+) T cell-mediated attack using an in vivo graft model. RESULTS: Loss of the B cell leukaemia/lymphoma 2 (BCL-2) homology domain 3-only proteins BIM, PUMA or BID did not protect beta cells from this death. Overexpression of the anti-apoptotic protein BCL-2 or combined deficiency of the pro-apoptotic multi-BCL2 homology domain proteins BAX and BAK also failed to prevent beta cell destruction. Furthermore, loss of function of the death receptor Fas or its essential downstream signalling molecule Fas-associated death domain (FADD) in islets was also not protective. Using electron microscopy we observed that dying beta cells showed features of necrosis. However, islets deficient in receptor-interacting serine/threonine protein kinase 3 (RIPK3), a critical initiator of necroptosis, were still normally susceptible to CD4(+) T cell-mediated destruction. Remarkably, simultaneous inhibition of apoptosis and necroptosis by combining loss of RIPK3 and overexpression of BCL-2 in islets did not protect them against immune attack either. CONCLUSIONS/INTERPRETATION: Collectively, our data indicate that beta cells die by necrosis in autoimmune diabetes and that the programmed cell death pathways apoptosis and necroptosis are both dispensable for this process

Glucose toxicity of islet cells is not mediated by inflammasome activation.

<p>(A) Representative FACS profiles of DNA fragmentation in islets after treatment with 33.3 mM glucose for 6 days, 50 mM ribose for 4 days or 1 mM palmitate conjugated to 1% BSA for 6 days. The percentage of islet cells with fragmented nuclei is indicated. (B, C) DNA fragmentation was measured by flow cytometry after incubation of wild-type C57BL/6, Nlrp3<b>^−/−</b> or caspase-1<b>^−/−</b> islets with 33.3 mM glucose for 6 days (B) or 50 mM ribose for 4 days (C). Control islets were incubated in medium containing 5.5 mM glucose. Results are mean+SEM of n≥4 independent experiments. *p<0.05 glucose treated islets vs control islets of same genotype. ***p<0.001 ribose treated islets vs control islets of same genotype. (D) DNA fragmentation was measured by flow cytometry after incubation of wild-type or caspase-1<b>^−/−</b> islets with 50 mM ribose or 100 ng/mL LPS for 3 days. Results are mean+SEM of n = 3 independent experiments. **p<0.01, ***p<0.001 ribose treated islets vs controls of the same genotype without ribose. (E) DNA fragmentation was measured by flow cytometry after incubation of wild-type or caspase-1<b>^−/−</b> islets with 33.3 mM glucose or 1 mM palmitate conjugated to 1% BSA for 6 days. Control islets were incubated in a medium containing 5.5 mM glucose and 1% BSA. Results are mean+SEM of n = 3 independent experiments. *p<0.05 glucose vs glucose+palmitate-treated caspase-1<b>^−/−</b> islets, **p<0.01 glucose vs glucose+palmitate-treated wild-type islets.</p

NLRP3 activating mutation does not induce caspase-1 cleavage in islets.

<p>(<b>A</b>) Four hundred islets isolated from wild-type or Cre/+ NLRP3^A350V/A350V mice were cultured in 1 mL of medium containing 33.3 mM glucose or 50 mM ribose for 2.5 days and IL-1β concentration in the supernatant was quantified by ELISA. Control islets were incubated in a medium containing 5.5 mM glucose. Macrophages treated overnight with 1 mM palmitate were used as positive control. Results are mean+SEM of n = 3–4 independent experiments. (<b>B</b>) Four hundred islets/sample were cultured in control medium containing 1% BSA and 100 nM LPS or a medium containing 1% BSA, 100 nM LPS, 33.3 mM glucose and 1 mM palmitate for 2.5 days. Lysates were prepared in RIPA buffer and western blotting for caspase-1 (full length, FL or cleaved, p20) was performed. Genotypes 1: +/+ NLRP3^+/+, 2: Cre/+ NLRP3^+/+, 3: +/+ NLRP3^A350V/A350V and 4: Cre/+ NLRP3^A350V/A350V. As a positive control, macrophages from wild-type (A) and caspase-1^−/− (B) mice were treated with LPS+BSA+Glucose+Palmitate for 24 h or LPS+Nigericin for 1 h.</p

Neuropeptide Y1 receptor antagonism protects β-cells and improves glycemic control in type 2 diabetes

Objectives: Loss of functional β-cell mass is a key factor contributing to poor glycemic control in advanced type 2 diabetes (T2D). We have previously reported that the inhibition of the neuropeptide Y1 receptor improves the islet transplantation outcome in type 1 diabetes (T1D). The aim of this study was to identify the pathophysiological role of the neuropeptide Y (NPY) system in human T2D and further evaluate the therapeutic potential of using the Y1 receptor antagonist BIBO3304 to improve β-cell function and survival in T2D. Methods: The gene expression of the NPY system in human islets from nondiabetic subjects and subjects with T2D was determined and correlated with the stimulation index. The glucose-lowering and β-cell-protective effects of BIBO3304, a selective orally bioavailable Y1 receptor antagonist, in high-fat diet (HFD)/multiple low-dose streptozotocin (STZ)-induced and genetically obese (db/db) T2D mouse models were assessed. Results: In this study, we identified a more than 2-fold increase in NPY1R and its ligand, NPY mRNA expression in human islets from subjects with T2D, which was significantly associated with reduced insulin secretion. Consistently, the pharmacological inhibition of Y1 receptors by BIBO3304 significantly protected β cells from dysfunction and death under multiple diabetogenic conditions in islets. In a preclinical study, we demonstrated that the inhibition of Y1 receptors by BIBO3304 led to reduced adiposity and enhanced insulin action in the skeletal muscle. Importantly, the Y1 receptor antagonist BIBO3304 treatment also improved β-cell function and preserved functional β-cell mass, thereby resulting in better glycemic control in both HFD/multiple low-dose STZ-induced and db/db T2D mice. Conclusions: Our results revealed a novel causal link between increased islet NPY-Y1 receptor gene expression and β-cell dysfunction and failure in human T2D, contributing to the understanding of the pathophysiology of T2D. Furthermore, our results demonstrate that the inhibition of the Y1 receptor by BIBO3304 represents a potential β-cell-protective therapy for improving functional β-cell mass and glycemic control in T2D.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Neuropeptide Y1 receptor antagonism protects β-cells and improves glycemic control in type 2 diabetes

Objectives: Loss of functional β-cell mass is a key factor contributing to poor glycemic control in advanced type 2 diabetes (T2D). We have previously reported that the inhibition of the neuropeptide Y1 receptor improves the islet transplantation outcome in type 1 diabetes (T1D). The aim of this study was to identify the pathophysiological role of the neuropeptide Y (NPY) system in human T2D and further evaluate the therapeutic potential of using the Y1 receptor antagonist BIBO3304 to improve β-cell function and survival in T2D. Methods: The gene expression of the NPY system in human islets from nondiabetic subjects and subjects with T2D was determined and correlated with the stimulation index. The glucose-lowering and β-cell-protective effects of BIBO3304, a selective orally bioavailable Y1 receptor antagonist, in high-fat diet (HFD)/multiple low-dose streptozotocin (STZ)-induced and genetically obese (db/db) T2D mouse models were assessed. Results: In this study, we identified a more than 2-fold increase in NPY1R and its ligand, NPY mRNA expression in human islets from subjects with T2D, which was significantly associated with reduced insulin secretion. Consistently, the pharmacological inhibition of Y1 receptors by BIBO3304 significantly protected β cells from dysfunction and death under multiple diabetogenic conditions in islets. In a preclinical study, we demonstrated that the inhibition of Y1 receptors by BIBO3304 led to reduced adiposity and enhanced insulin action in the skeletal muscle. Importantly, the Y1 receptor antagonist BIBO3304 treatment also improved β-cell function and preserved functional β-cell mass, thereby resulting in better glycemic control in both HFD/multiple low-dose STZ-induced and db/db T2D mice. Conclusions: Our results revealed a novel causal link between increased islet NPY-Y1 receptor gene expression and β-cell dysfunction and failure in human T2D, contributing to the understanding of the pathophysiology of T2D. Furthermore, our results demonstrate that the inhibition of the Y1 receptor by BIBO3304 represents a potential β-cell-protective therapy for improving functional β-cell mass and glycemic control in T2D