ß-cell-selective inhibition of DNA damage response signaling by nitric oxide is associated with an attenuation in glucose uptake.

Nitric oxide (NO) plays a dual role in regulating DNA damage response (DDR) signaling in pancreatic ß-cells. As a genotoxic agent, NO activates two types of DDR signaling; however, when produced at micromolar levels by the inducible isoform of NO synthase, NO inhibits DDR signaling and DDR-induced apoptosis in a ß-cell-selective manner. DDR signaling inhibition by NO correlates with mitochondrial oxidative metabolism inhibition and decreases in ATP and NAD+. Unlike most cell types, ß-cells do not compensate for impaired mitochondrial oxidation by increasing glycolytic flux, and this metabolic inflexibility leads to a decrease in ATP and NAD+. Here, we used multiple analytical approaches to determine changes in intermediary metabolites in ß-cells and non-ß-cells treated with NO or complex I inhibitor rotenone. In addition to ATP and NAD+, glycolytic and tricarboxylic acid cycle intermediates as well as NADPH are significantly decreased in ß-cells treated with NO or rotenone. Consistent with glucose-6-phosphate residing at the metabolic branchpoint for glycolysis and the pentose phosphate pathway (NADPH), we show that mitochondrial oxidation inhibitors limit glucose uptake in a ß-cell-selective manner. Our findings indicate that the ß-cell-selective inhibition of DDR signaling by NO is associated with a decrease in ATP to levels that fall significantly below the KM for ATP of glucokinase (glucose uptake) and suggest that this action places the ß-cell in a state of suspended animation where it is metabolically inert until NO is removed, and metabolic function can be restored.

Deletion of Thioredoxin Reductase Disrupts Redox Homeostasis and Impairs ß-Cell Function.

Reactive oxygen species (ROS) have been implicated as mediators of pancreatic ß-cell damage. While ß-cells are thought to be vulnerable to oxidative damage, we have shown, using inhibitors and acute depletion, that thioredoxin reductase, thioredoxin, and peroxiredoxins are the primary mediators of antioxidant defense in ß-cells. However, the role of this antioxidant cycle in maintaining redox homeostasis and ß-cell survival in vivo remains unclear. Here, we generated mice with a ß-cell specific knockout of thioredoxin reductase 1 (Txnrd1fl/fl; Ins1Cre/+ , ßKO). Despite blunted glucose-stimulated insulin secretion, knockout mice maintain normal whole-body glucose homeostasis. Unlike pancreatic islets with acute Txnrd1 inhibition, ßKO islets do not demonstrate increased sensitivity to ROS. RNA-sequencing analysis revealed that Txnrd1-deficient ß-cells have increased expression of nuclear factor erythroid 2-related factor 2 (Nrf2)-regulated genes, and altered expression of genes involved in heme and glutathione metabolism, suggesting an adaptive response. Txnrd1-deficient ß-cells also have decreased expression of factors controlling ß-cell function and identity which may explain the mild functional impairment. Together, these results suggest that Txnrd1-knockout ß-cells compensate for loss of this essential antioxidant pathway by increasing expression of Nrf2-regulated antioxidant genes, allowing for protection from excess ROS at the expense of normal ß-cell function and identity.

Regulation of ATR-dependent DNA damage response by nitric oxide.

We have shown that nitric oxide limits ataxia-telangiectasia mutated signaling by inhibiting mitochondrial oxidative metabolism in a ß-cell selective manner. In this study, we examined the actions of nitric oxide on a second DNA damage response transducer kinase, ataxia-telangiectasia and Rad3-related protein (ATR). In ß-cells and non-ß-cells, nitric oxide activates ATR signaling by inhibiting ribonucleotide reductase; however, when produced at inducible nitric oxide synthase-derived (low micromolar) levels, nitric oxide impairs ATR signaling in a ß-cell selective manner. The inhibitory actions of nitric oxide are associated with impaired mitochondrial oxidative metabolism and lack of glycolytic compensation that result in a decrease in ß-cell ATP. Like nitric oxide, inhibitors of mitochondrial respiration reduce ATP levels and limit ATR signaling in a ß-cell selective manner. When non-ß-cells are forced to utilize mitochondrial oxidative metabolism for ATP generation, their response is more like ß-cells, as nitric oxide and inhibitors of mitochondrial respiration attenuate ATR signaling. These studies support a dual role for nitric oxide in regulating ATR signaling. Nitric oxide activates ATR in all cell types examined by inhibiting ribonucleotide reductase, and in a ß-cell selective manner, inducible nitric oxide synthase-derived levels of nitric oxide limit ATR signaling by attenuating mitochondrial oxidative metabolism and depleting ATP.

SurfaceGenie: a web-based application for prioritizing cell-type-specific marker candidates.

MOTIVATION: Cell-type-specific surface proteins can be exploited as valuable markers for a range of applications including immunophenotyping live cells, targeted drug delivery and in vivo imaging. Despite their utility and relevance, the unique combination of molecules present at the cell surface are not yet described for most cell types. A significant challenge in analyzing 'omic' discovery datasets is the selection of candidate markers that are most applicable for downstream applications. RESULTS: Here, we developed GenieScore, a prioritization metric that integrates a consensus-based prediction of cell surface localization with user-input data to rank-order candidate cell-type-specific surface markers. In this report, we demonstrate the utility of GenieScore for analyzing human and rodent data from proteomic and transcriptomic experiments in the areas of cancer, stem cell and islet biology. We also demonstrate that permutations of GenieScore, termed IsoGenieScore and OmniGenieScore, can efficiently prioritize co-expressed and intracellular cell-type-specific markers, respectively. AVAILABILITY AND IMPLEMENTATION: Calculation of GenieScores and lookup of SPC scores is made freely accessible via the SurfaceGenie web application: www.cellsurfer.net/surfacegenie. CONTACT: Rebekah.gundry@unmc.edu. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Cation-Independent Mannose 6-Phosphate Receptor Deficiency Enhances ß-Cell Susceptibility to Palmitate.

Palmitate attenuates insulin secretion and reduces the viability of insulin-producing cells. Previous studies identified the aberrant palmitoylation or mispalmitoylation of proteins as one mechanism by which palmitate causes ß-cell damage. In this report, we identify a role for lysosomal protein degradation as a mechanism by which ß cells defend themselves against excess palmitate. The cation-independent mannose 6-phosphate receptor (CI-MPR) is responsible for the trafficking of mannose 6-phosphate-tagged proteins to lysosomes via Golgi sorting and from extracellular locations through endocytosis. RINm5F cells, which are highly sensitive to palmitate, lack CI-MPR. The reconstitution of CI-MPR expression attenuates the induction of endoplasmic reticulum (ER) stress and the toxic effects of palmitate on RINm5F cell viability. INS832/13 cells express CI-MPR and are resistant to the palmitate-mediated loss of cell viability. The reduction of CI-MPR expression increases the sensitivity of INS832/13 cells to the toxic effects of palmitate treatment. The inhibition of lysosomal acid hydrolase activity by weak base treatment of islets under glucolipotoxic conditions causes islet degeneration that is prevented by the inhibition of protein palmitoylation. These findings indicate that defects in lysosomal function lead to the enhanced sensitivity of insulin-producing cells to palmitate and support a role for normal lysosomal function in the protection of ß cells from excess palmitate.

The role of reactive oxygen species and proinflammatory cytokines in type 1 diabetes pathogenesis.

Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease characterized by the destruction of insulin-secreting pancreatic ß cells. In humans with T1D and in nonobese diabetic (NOD) mice (a murine model for human T1D), autoreactive T cells cause ß-cell destruction, as transfer or deletion of these cells induces or prevents disease, respectively. CD4(+) and CD8(+) T cells use distinct effector mechanisms and act at different stages throughout T1D to fuel pancreatic ß-cell destruction and disease pathogenesis. While these adaptive immune cells employ distinct mechanisms for ß-cell destruction, one central means for enhancing their autoreactivity is by the secretion of proinflammatory cytokines, such as IFN-γ, TNF-α, and IL-1. In addition to their production by diabetogenic T cells, proinflammatory cytokines are induced by reactive oxygen species (ROS) via redox-dependent signaling pathways. Highly reactive molecules, proinflammatory cytokines are produced upon lymphocyte infiltration into pancreatic islets and induce disease pathogenicity by directly killing ß cells, which characteristically possess low levels of antioxidant defense enzymes. In addition to ß-cell destruction, proinflammatory cytokines are necessary for efficient adaptive immune maturation, and in the context of T1D they exacerbate autoimmunity by intensifying adaptive immune responses. The first half of this review discusses the mechanisms by which autoreactive T cells induce T1D pathogenesis and the importance of ROS for efficient adaptive immune activation, which, in the context of T1D, exacerbates autoimmunity. The second half provides a comprehensive and detailed analysis of (1) the mechanisms by which cytokines such as IL-1 and IFN-γ influence islet insulin secretion and apoptosis and (2) the key free radicals and transcription factors that control these processes.

Cytokine-mediated ß-cell damage in PARP-1-deficient islets.

Poly(ADP)-ribose polymerase (PARP) is an abundant nuclear protein that is activated by DNA damage; once active, it modifies nuclear proteins through attachment of poly(ADP)-ribose units derived from ß-nicotinamide adenine dinucleotide (NAD(+)). In mice, the deletion of PARP-1 attenuates tissue injury in a number of animal models of human disease, including streptozotocin-induced diabetes. Also, inflammatory cell signaling and inflammatory gene expression are attenuated in macrophages isolated from endotoxin-treated PARP-1-deficient mice. In this study, the effects of PARP-1 deletion on cytokine-mediated ß-cell damage and macrophage activation were evaluated. There are no defects in inflammatory mediator signaling or inflammatory gene expression in macrophages and islets isolated from PARP-1-deficient mice. While PARP-1 deficiency protects islets against cytokine-induced islet cell death as measured by biochemical assays of membrane polarization, the genetic absence of PARP-1 does not effect cytokine-induced inhibition of insulin secretion or cytokine-induced DNA damage in islets. While PARP-1 deficiency appears to provide protection from cell death, it fails to provide protection against the inhibitory actions of cytokines on insulin secretion or the damaging actions on islet DNA integrity.

IRE1-dependent activation of AMPK in response to nitric oxide.

While there can be detrimental consequences of nitric oxide production at pathological concentrations, eukaryotic cells have evolved protective mechanisms to defend themselves against this damage. The unfolded-protein response (UPR), activated by misfolded proteins and oxidative stress, is one adaptive mechanism that is employed to protect cells from stress. Nitric oxide is a potent activator of AMP-activated protein kinase (AMPK), and AMPK participates in the cellular defense against nitric oxide-mediated damage in pancreatic ß-cells. In this study, the mechanism of AMPK activation by nitric oxide was explored. The known AMPK kinases LKB1, CaMKK, and TAK1 are not required for the activation of AMPK by nitric oxide. Instead, this activation is dependent on the endoplasmic reticulum (ER) stress-activated protein IRE1. Nitric oxide-induced AMPK phosphorylation and subsequent signaling to AMPK substrates, including Raptor, acetyl coenzyme A carboxylase, and PGC-1α, is attenuated in IRE1α-deficient cells. The endoribonuclease activity of IRE1 appears to be required for AMPK activation in response to nitric oxide. In addition to nitric oxide, stimulation of IRE1 endoribonuclease activity with the flavonol quercetin leads to IRE1-dependent AMPK activation. These findings indicate that the RNase activity of IRE1 participates in AMPK activation and subsequent signaling through multiple AMPK-dependent pathways in response to nitrosative stress.

FoxO1 and SIRT1 regulate beta-cell responses to nitric oxide.

For many cell types, including pancreatic ß-cells, nitric oxide is a mediator of cell death; paradoxically, nitric oxide can also activate pathways that promote the repair of cellular damage. In this report, a role for FoxO1-dependent transcriptional activation and its regulation by SIRT1 in determining the cellular response to nitric oxide is provided. In response to nitric oxide, FoxO1 translocates from the cytoplasm to the nucleus and stimulates the expression of the DNA repair gene GADD45α, resulting in FoxO1-dependent DNA repair. FoxO1-dependent gene expression appears to be regulated by the NAD(+)-dependent deacetylase SIRT1. In response to SIRT1 inhibitors, the FoxO1-dependent protective actions of nitric oxide (GADD45α expression and DNA repair) are attenuated, and FoxO1 activates a proapoptotic program that includes PUMA (p53-up-regulated mediator of apoptosis) mRNA accumulation and caspase-3 cleavage. These findings support primary roles for FoxO1 and SIRT1 in regulating the cellular responses of ß-cells to nitric oxide.

Selective mtDNA mutation accumulation results in beta-cell apoptosis and diabetes development.

To test the hypothesis that somatic mitochondrial (mt)DNA mutation accumulation predisposes mice to beta-cell loss and diabetes development, transgenic mice expressing a proofreading-deficient mtDNA polymerase-gamma under the control of the rat insulin-1 promoter were generated. At 6 wk of age, mtDNA mutations reached 0.01% (1.05 mutations/10,000 bp) in islets isolated from transgenic mice. This mutational burden is associated with impaired glucose tolerance and a diabetes prevalence of 52% in male transgenic mice. Female transgenic mice maintain slightly elevated fasting glucose levels, mild glucose intolerance, and a diabetes prevalence of 14%. Diabetes in transgenic animals is associated with insulin insufficiency that results from a significant reduction in beta-cell mass. Importantly, apoptosis of beta-cells is increased 7-fold in female and 11-fold in male transgenic mice compared with littermate controls. These results are consistent with a causative role of somatic mtDNA mutation accumulation in the loss of beta-cell mass and diabetes development.

Chemically chaperoning the actions of insulin.

The role of inflammation as a mediator of insulin resistance in type 2 diabetes and obesity has been a major focus of studies over the past ten years. In mouse models of obesity and type 2 diabetes, the development of insulin resistance correlates with elevated levels of endoplasmic reticulum stress and induction of the unfolded protein response. Activation of N-terminal C-Jun kinase is known to be associated with unfolded protein response activation, and has been shown to participate in the inhibition of insulin action by stimulating serine phosphorylation of the insulin receptor substrate 1, an event that attenuates insulin signaling. 'Chemical chaperones' are small molecules that have been shown to attenuate unfolded protein response activation. The exciting new findings of Ozcan et al. indicate that chemical chaperones improve glucose tolerance and insulin action in a mouse model of type 2 diabetes. These findings offer a potential new target for therapeutic strategies designed to improve insulin action and glucose tolerance in diabetic individuals.

Distinct mechanisms of glucose lowering by specific agonists for peroxisomal proliferator activated receptor gamma and retinoic acid X receptors.

Agonists for the nuclear receptor peroxisomal proliferator-activated receptor-gamma (PPARgamma) and its heterodimeric partner, retinoid X receptor (RXR), are effective agents for the treatment of type 2 diabetes. To gain insight into the antidiabetic action of these compounds, we treated female Zucker diabetic rats (ZFF) with AGN194204, which we show to be a homodimer-specific RXR agonist, or the PPARgamma agonist, troglitazone. Hyperinsulinemic-euglycemic clamps in ZFF showed that troglitazone and AGN194204 reduced basal endogenous glucose production (EGP) approximately 30% and doubled the insulin suppression of EGP. AGN194204 had no effect on peripheral glucose utilization, whereas troglitazone increased insulin-stimulated glucose utilization by 50%, glucose uptake into skeletal muscle by 85%, and de novo skeletal muscle glycogen synthesis by 300%. Troglitazone increased skeletal muscle Irs-1 and phospho-Akt levels following in vivo insulin treatment, whereas AGN194204 increased hepatic Irs-2 and insulin stimulated phospho-Akt in liver. Gene profiles of AGN194204-treated mouse liver analyzed by Ingenuity Pathway Analysis identified increases in fatty acid synthetic genes, including Srebp-1 and fatty acid synthase, a pathway previously shown to be induced by RXR agonists. A network of down-regulated genes containing Foxa2, Foxa3, and G-protein subunits was identified, and decreases in these mRNA levels were confirmed by quantitative reverse transcription-PCR. Treatment of HepG2 cells with AGN194204 resulted in inhibition of glucagon-stimulated cAMP accumulation suggesting the G-protein down-regulation may provide an additional mechanism for hepatic insulin sensitization by RXR. These studies demonstrate distinct molecular events lead to insulin sensitization by high affinity RXR and PPARgamma agonists.

Incretin hormones and insulin sensitivity.

Incretin hormones such as glucagon-like peptide-1 (GLP-1) and the longer lasting analog exendin-4 show clinical promise for the treatment of diabetes because of glucoregulatory activities that enhance beta-cell function and growth, and actions in the central nervous system that induce satiety and decrease caloric intake. The actions of these peptides on insulin-responsive tissues is less clear, but recent advances indicate that chronic treatment with exendin-4 increases insulin sensitivity via two distinct mechanisms: one is attributable to changes in food intake and the subsequent improvements in glycemia; the second is largely independent of reductions in blood glucose. In addition, exendin-4 might also have direct effects on beta-cell neogenesis that are independent of insulin demand.

A peroxovanadium compound stimulates muscle glucose transport as powerfully as insulin and contractions combined.

Stimulation of glucose transport by insulin involves tyrosine phosphorylation of the insulin receptor (IR) and IR substrates (IRSs). Peroxovanadates inhibit tyrosine phosphatases, also resulting in tyrosine phosphorylation of the IRSs. Muscle contractions stimulate glucose transport by a mechanism independent of the insulin-signaling pathway. We found that the peroxovanadate compound bis-peroxovanadium,1,10-phenanthrolene [bpV(phen)] stimulates glucose transport to the same extent as the additive effects of maximal insulin and contraction stimuli. Translocation of GLUT4 to the cell surface mediates stimulation of glucose transport. There is evidence suggesting there are separate insulin- and contraction-stimulated pools of GLUT4-containing vesicles. We tested the hypothesis that bpV(phen) stimulates both the insulin- and the contraction-activated pathways. Stimulation of glucose transport and GLUT4 translocation by bpV(phen) was completely blocked by the phosphatidylinositol 3-kinase (PI 3-K) inhibitors wortmannin and LY294002. The combined effect of bpV(phen) and contractions was no greater than that of bpV(phen) alone. Activation of the IRS-PI 3-K signaling pathway was much greater with bpV(phen) than with insulin. Our results suggest that the GLUT4 vesicles that are normally translocated in response to contractions but not insulin can respond to the signal generated via the IRS-PI 3-K pathway if it is sufficiently powerful.