Jessica R. Weaver

Integration of pro-inflammatory cytokines, 12-lipoxygenase and NOX-1 in pancreatic islet beta cell dysfunction.

Elevated cellular reactive species, which can be produced by diabetic serum conditions such as elevated inflammatory cytokines, lipotoxicity or glucotoxicity contribute to islet beta cell dysfunction and cell death. Cellular pathways that result in beta cell oxidative stress are poorly resolved. In this study, stimulation of human donor islets, primary mouse islets or homogeneous beta cell lines with a cocktail of inflammatory cytokines (TNFα, IL-1β, and INFγ) significantly induced NADPH oxidase-1 (NOX-1) gene expression (p<0.05). This pro-inflammatory cytokine cocktail concomitantly induced loss of islet glucose stimulated insulin response (p<0.05), elevated expression of MCP-1 (p<0.01), increased cellular reactive oxygen species (ROS) and induced cell death. Inhibitors of NADPH oxidase, apocynin and diphenyleneiodonium, and a dual selective NOX1/4 inhibitor, blocked ROS generation (p<0.01) and induction of MCP-1 (p<0.05) by pro-inflammatory cytokines in beta cells. It has previously been reported that pro-inflammatory cytokine stimulation induces 12-lipoxygenase (12-LO) expression in human islets. 12-Hydroxyeicosatetraenoic acid (12-HETE), a product of 12-LO activity, stimulated NOX-1 expression in human islets (p<0.05). A novel selective inhibitor of 12-LO blocked induction of NOX-1, production of ROS and pro-caspase 3 cleavage by pro-inflammatory cytokines in INS-1 beta cells (p<0.01). Inhibition was not seen with a structurally related but inactive analog. Importantly, islets from human type 2 diabetic donors have an elevated expression of NOX-1 (p<0.05). This study describes an integrated pathway in beta cells that links beta cell dysfunction induced by pro-inflammatory cytokines with 12-lipoxygenase and NADPH oxidase (NOX-1) activation. Inhibitors of this pathway may provide a new therapeutic strategy to preserve beta cell mass in diabetes.

Production and function of IL-12 in islets and beta cells.

Aims/hypothesisIL-12 is an important cytokine in early inflammatory responses and is implicated in the immune-mediated pathogenesis of pancreatic islets in diabetes. However, little is known about the direct effects of IL-12 on islets and beta cells.MethodsIn this study, beta cell function, gene expression and protein production were assessed in primary human donor islets and murine beta cell lines in response to stimulation with IL-12 or a pro-inflammatory cytokine cocktail (TNF-α, IL-1β and IFN-γ).ResultsThe pro-inflammatory cytokine cocktail induced islet dysfunction and potently increased the expression and production of IL-12 ligand and IL-12 receptor in human islets. In human islets, the receptor for IL-12 co-localised to the cell surface of insulin-producing cells. Both IL-12 ligand and IL-12 receptor are expressed in the homogeneous beta cell line INS-1. IL-12 induced changes in gene expression, including a dose-dependent upregulation of IFNγ (also known as IFNG), in INS-1 cells. A neutralising antibody to IL-12 directly inhibited IFNγ gene expression in human donor islets induced by either IL-12 or pro-inflammatory cytokine stimulation. Functionally, IL-12 impaired glucose-stimulated insulin secretion (GSIS) in INS-1 cells and human donor islets. A neutralising antibody to IL-12 reversed the beta cell dysfunction (uncoupling of GSIS or induction of caspase-3 activity) induced by pro-inflammatory cytokines.Conclusions/interpretationThese data identify beta cells as a local source of IL-12 ligand and suggest a direct role of IL-12 in mediating beta cell pathology.

Regulation of NOX-1 expression in beta cells: A positive feedback loop involving the Src-kinase signaling pathway

NADPH oxidase-1 (NOX-1) is upregulated in beta cells in response to pro-inflammatory cytokines. Inhibition of NADPH oxidase activity blocked stimulated NOX-1 expression (p<0.05). Regulation of NOX-1 expression in beta cells followed modulation of cellular reactive oxygen species (ROS); pro-oxidants increased NOX-1 (p<0.001) and anti-oxidants decreased NOX-1 (p<0.05). Activation of Src-kinase followed ROS elevation. Inhibition of Src-kinase decreased NOX-1 expression (p<0.01). Beta cell dysfunction, measured by elevated MCP-1 expression, loss of glucose-sensitive insulin secretion or cell death, was induced by pro-inflammatory cytokine stimulation. Importantly, inhibition of Src-kinase or NOX-1 preserved beta cell function and survival. Collectively, these data indicate that expression of NOX-1 in beta cells is regulated in a feed-forward loop mediated by ROS and Src-kinase. Uncoupling of this feed-forward activation could provide new approaches to preserve and protect beta cells in diabetes.

Selective inhibition of 12-lipoxygenase protects islets and beta cells from inflammatory cytokine-mediated beta cell dysfunction

Aims/hypothesisIslet inflammation leads to loss of functional pancreatic beta cell mass. Increasing evidence suggests that activation of 12-lipoxygenase leads to inflammatory beta cell loss. This study evaluates new specific small-molecule inhibitors of 12-lipoxygenase for protecting rodent and human beta cells from inflammatory damage.MethodsMouse beta cell lines and mouse and human islets were treated with inflammatory cytokines IL-1β, TNFα and IFNγ in the absence or presence of novel selective 12-lipoxygenase inhibitors. Glucose-stimulated insulin secretion (GSIS), gene expression, cell survival and 12-S-hydroxyeicosatetraenoic acid (12-S-HETE) levels were evaluated using established methods. Pharmacokinetic analysis was performed with the lead inhibitor in CD1 mice.ResultsInflammatory cytokines led to the loss of human beta cell function, elevated cell death, increased inflammatory gene expression and upregulation of 12-lipoxygenase expression and activity (measured by 12-S-HETE generation). Two 12-lipoxygenase inhibitors, Compounds 5 and 9, produced a concentration-dependent reduction of stimulated 12-S-HETE levels. GSIS was preserved in the presence of the 12-lipoxygenase inhibitors. 12-Lipoxygenase inhibition preserved survival of primary mouse and human islets. When administered orally, Compound 5 reduced plasma 12-S-HETE in CD1 mice. Compounds 5 and 9 preserved the function and survival of human donor islets exposed to inflammatory cytokines.Conclusions/interpretationSelective inhibition of 12-lipoxygenase activity confers protection to beta cells during exposure to inflammatory cytokines. These concept validation studies identify 12-lipoxygenase as a promising target in the prevention of loss of functional beta cells in diabetes.

Targeted expression of islet neogenesis associated protein to beta cells enhances glucose tolerance and confers resistance to streptozotocin-induced hyperglycemia

Islet neogenesis associated protein (INGAP) stimulates experimental pancreatic islet growth, as evidenced by elevated markers of beta cell mass, in rodents, dogs and primates. Previous analyses of mice that have a transgenic expression of INGAP targeted to the exocrine pancreas have indicated additional biological activity attributed to INGAP. In this study we report on mice with a targeted expression of INGAP to the islet beta cell. The beta cell transgenic mice (IP-INGAP) showed enhanced normalization of blood glucose during IPGTT. Further, IP-INGAP mice had a significant delay in development of hyperglycemia following a diabetogenic dose of streptozotocin. INGAP conferred beta cell protection and enhanced islet function. Analysis of oxidative stress genes in IP-INGAP mice revealed a decrease in islet expression of the NADPH oxidase, NOX1, in both basal state and in response to pro-inflammatory cytokine stimulation. These data are consistent with a pleiotropic role for INGAP and reveal new pathways to target in the discovery of improved diabetic therapies.

Interleukin-12 (IL-12)/STAT4 Axis Is an Important Element for β-Cell Dysfunction Induced by Inflammatory Cytokines

Pathology driving β-cell loss in diabetes is poorly defined. Chronic subclinical inflammation is associated with β-cell dysfunction. Acute in vitro exposure of islets and β-cells to an inflammatory cytokine cocktail (IL-1β/TNF-α/IFN-γ) results in loss of cell function and viability. The contribution of each cytokine alone or in combination has been evaluated in homogeneous mouse β-cell lines and primary mouse islets. Cytokine cooperation is required for β-cell apoptosis with the most potent combinations including IL-1β. Single cytokine exposure did not induce β-cell apoptosis. Expression of endogenous interleukin-12 in β-cells correlated with inflammatory cytokine combinations that induced β-cell apoptosis. Uncoupling of the IL-12 axis by a block of IL-12 production, inhibition of IL-12 receptor/ligand interaction or disruption of IL-12 receptor signaling conferred protection to β-cells from apoptosis induced by inflammatory cytokine stimulation. Signaling through STAT4 is indicated since disruption of IL-12 concomitantly reduced inflammatory cytokine stimulation of endogenous IFN-γ expression. Primary mouse islets isolated from mice deficient in STAT4 show resistance to inflammatory-cytokine-induced cell death when compared to islets isolated from wild type mice. Collectively, the data identify IL-12 as an important mediator of inflammation induced β-cell apoptosis. Modulation of IL-12/STAT4 signaling may be a valuable therapeutic strategy to preserve islet/β-cell viability in established diabetes.

12-Lipoxygenase Inhibitor Improves Functions of Cytokine-Treated Human Islets and Type 2 Diabetic Islets

Context The 12-lipoxygenase (12-LO) pathway produces proinflammatory metabolites, and its activation is implicated in islet inflammation associated with type 1 and type 2 diabetes (T2D). Objectives We aimed to test the efficacy of ML355, a highly selective, small molecule inhibitor of 12-LO, for the preservation of islet function. Design Human islets from nondiabetic donors were incubated with a mixture of tumor necrosis factor α , interluekin-1β, and interferon-γ to model islet inflammation. Cytokine-treated islets and human islets from T2D donors were incubated in the presence and absence of ML355. Setting In vitro study. Participants Human islets from organ donors aged >20 years of both sexes and any race were used. T2D status was defined from either medical history or most recent hemoglobin A1c value >6.5%. Intervention Glucose stimulation. Main Outcome Measures Static and dynamic insulin secretion and oxygen consumption rate (OCR). Results ML355 prevented the reduction of insulin secretion and OCR in cytokine-treated human islets and improved both parameters in human islets from T2D donors. Conclusions ML355 was efficacious in improving human islet function after cytokine treatment and in T2D islets in vitro. The study suggests that the blockade of the 12-LO pathway may serve as a target for both form of diabetes and provides the basis for further study of this small molecule inhibitor in vivo.

Relationship of NADPH Oxidase-1 expression to beta cell dysfunction induced by inflammatory cytokines

Redox stress related loss of beta cell function is a feature of diabetes. Exposure of beta cells and islets to inflammatory mediators elevates reactive oxygen species (ROS) and beta cell dysfunction. Direct molecular manipulation of NADPH oxidase-1 (NOX-1) has identified a key role for NOX-1 in cytokine-induced beta cell dysfunction. Plasmid driven elevation of NOX-1 resulted in elevated ROS, loss of glucose-stimulated-insulin-secretion and increased apoptosis. These outcomes on beta cell function are analogous to cytokine treatment. In contrast, reduction of NOX-1 expression, by shRNA, conferred protection to beta cells and islets from the damaging effects of inflammatory cytokines. Collectively, these data support the therapeutic potential for NOX-1 inhibition in diabetes.

EXPRESSION OF HIF-1α IN A PANCREATIC DUCTAL ADENOCARCINOMA IN A PATIENT WITH NEWLY DIAGNOSED TYPE 2 DIABETES

Objective: Newly diagnosed type 2 diabetes mellitus (T2DM) increases the risk of pancreatic cancer by an unknown mechanism. We report a patient diagnosed with T2DM <1 year who developed an invasive pancreatic ductal adenocarcinoma expressing hypoxia-inducible factor 1-alpha (HIF-1α). Methods: HIF-1α staining was performed using the immunohistochemistry protocol from Novus (NB100-105), with minor modifications. Results: A 47-year-old African American male with a history of T2DM for 6 months presented with abdominal pain, weight loss, elevated liver and pancreatic enzymes, and a significant raise in carcinoembryonic antigen and cancer antigen 19-9 levels. A 2.5-cm mass was found on the head of the pancreas/uncinate process. A pylorus-sparing pancreaticoduodenectomy was performed that included removal of the gallbladder and a portion of the omentum. Pathology report showed an invasive ductal adenocarcinoma of the head of the pancreas (stage IIB: pT3N1M0). Immunohistochemistry of sections of the adenocarcinom...