ß-cell selective regulation of gene expression by nitric oxide.

Nitric oxide is produced at low micromolar levels following the induction of inducible nitric oxide synthase (iNOS) and is responsible for mediating the inhibitory actions of cytokines on glucose-stimulated insulin secretion by islets of Langerhans. It is through the inhibition of mitochondrial oxidative metabolism, specifically aconitase and complex 4 of the electron transport chain, that nitric oxide inhibits insulin secretion. Nitric oxide also attenuates protein synthesis, induces DNA damage, activates DNA repair pathways, and stimulates stress responses (unfolded protein and heat shock) in ß-cells. In this report, the time- and concentration-dependent effects of nitric oxide on the expression of 6 genes known to participate in the response of ß-cells to this free radical were examined. The genes included Gadd45α (DNA repair), Puma (apoptosis), Hmox1 (antioxidant defense), Hsp70 (heat shock), Chop (UPR), and ßPpargc1α (mitochondrial biogenesis). We show that nitric oxide stimulates ß-cell gene expression in a narrow concentration range of ~0.5-1 µM, or levels corresponding to iNOS-derived nitric oxide. At concentrations greater than 1 µM, nitric oxide fails to stimulate gene expression in ß-cells, and this is associated with the inhibition of mitochondrial oxidative metabolism. This narrow concentration range of responses is ß-cell selective, as the actions of nitric oxide in non-ß-cells (α-cells, mouse embryonic fibroblasts, and macrophages) are concentration-dependent. Our findings suggest that ß-cells respond to a narrow concentration range of nitric oxide that is consistent with the levels produced following iNOS induction, and that these concentration-dependent actions are selective for insulin-containing cells.

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.

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.

Inhibition of oxidative metabolism by nitric oxide restricts EMCV replication selectively in pancreatic beta-cells.

Environmental factors, such as viral infection, are proposed to play a role in the initiation of autoimmune diabetes. In response to encephalomyocarditis virus (EMCV) infection, resident islet macrophages release the pro-inflammatory cytokine IL-1ß, to levels that are sufficient to stimulate inducible nitric oxide synthase (iNOS) expression and production of micromolar levels of the free radical nitric oxide in neighboring ß-cells. We have recently shown that nitric oxide inhibits EMCV replication and EMCV-mediated ß-cell lysis and that this protection is associated with an inhibition of mitochondrial oxidative metabolism. Here we show that the protective actions of nitric oxide against EMCV infection are selective for ß-cells and associated with the metabolic coupling of glycolysis and mitochondrial oxidation that is necessary for insulin secretion. Inhibitors of mitochondrial respiration attenuate EMCV replication in ß-cells, and this inhibition is associated with a decrease in ATP levels. In mouse embryonic fibroblasts (MEFs), inhibition of mitochondrial metabolism does not modify EMCV replication or decrease ATP levels. Like most cell types, MEFs have the capacity to uncouple the glycolytic utilization of glucose from mitochondrial respiration, allowing for the maintenance of ATP levels under conditions of impaired mitochondrial respiration. It is only when MEFs are forced to use mitochondrial oxidative metabolism for ATP generation that mitochondrial inhibitors attenuate viral replication. In a ß-cell selective manner, these findings indicate that nitric oxide targets the same metabolic pathways necessary for glucose stimulated insulin secretion for protection from viral lysis.

Inhibition of mitochondrial oxidative metabolism attenuates EMCV replication and protects ß-cells from virally mediated lysis.

Viral infection is one environmental factor that may contribute to the initiation of pancreatic ß-cell destruction during the development of autoimmune diabetes. Picornaviruses, such as encephalomyocarditis virus (EMCV), induce a pro-inflammatory response in islets leading to local production of cytokines, such as IL-1, by resident islet leukocytes. Furthermore, IL-1 is known to stimulate ß-cell expression of iNOS and production of the free radical nitric oxide. The purpose of this study was to determine whether nitric oxide contributes to the ß-cell response to viral infection. We show that nitric oxide protects ß-cells against virally mediated lysis by limiting EMCV replication. This protection requires low micromolar, or iNOS-derived, levels of nitric oxide. At these concentrations nitric oxide inhibits the Krebs enzyme aconitase and complex IV of the electron transport chain. Like nitric oxide, pharmacological inhibition of mitochondrial oxidative metabolism attenuates EMCV-mediated ß-cell lysis by inhibiting viral replication. These findings provide novel evidence that cytokine signaling in ß-cells functions to limit viral replication and subsequent ß-cell lysis by attenuating mitochondrial oxidative metabolism in a nitric oxide-dependent manner.

The Role of Metabolic Flexibility in the Regulation of the DNA Damage Response by Nitric Oxide.

In this report, we show that nitric oxide suppresses DNA damage response (DDR) signaling in the pancreatic ß-cell line INS 832/13 and rat islets by inhibiting intermediary metabolism. Nitric oxide is known to inhibit complex IV of the electron transport chain and aconitase of the Krebs cycle. Non-ß cells compensate by increasing glycolytic metabolism to maintain ATP levels; however, ß cells lack this metabolic flexibility, resulting in a nitric oxide-dependent decrease in ATP and NAD+ Like nitric oxide, mitochondrial toxins inhibit DDR signaling in ß cells by a mechanism that is associated with a decrease in ATP. Non-ß cells compensate for the effects of mitochondrial toxins with an adaptive shift to glycolytic ATP generation that allows for DDR signaling. Forcing non-ß cells to derive ATP via mitochondrial respiration (replacing glucose with galactose in the medium) and glucose deprivation sensitizes these cells to nitric oxide-mediated inhibition of DDR signaling. These findings indicate that metabolic flexibility is necessary to maintain DDR signaling under conditions in which mitochondrial oxidative metabolism is inhibited and support the inhibition of oxidative metabolism (decreased ATP) as one protective mechanism by which nitric oxide attenuates DDR-dependent ß-cell apoptosis.

Tpcn2 knockout mice have improved insulin sensitivity and are protected against high-fat diet-induced weight gain.

Type 2 diabetes is a complex disorder affected by multiple genes and the environment. Our laboratory has shown that in response to a glucose challenge, two-pore channel 2 ( Tpcn2) knockout mice exhibit a decreased insulin response but normal glucose clearance, suggesting they have improved insulin sensitivity compared with wild-type mice. We tested the hypothesis that improved insulin sensitivity in Tpcn2 knockout mice would protect against the negative effects of a high fat diet. Male and female Tpcn2 knockout (KO), heterozygous (Het), and wild-type (WT) mice were fed a low-fat (LF) or high-fat (HF) diet for 24 wk. HF diet significantly increases body weight in WT mice relative to those on the LF diet; this HF diet-induced increase in body weight is blunted in the Het and KO mice. Despite the protection against diet-induced weight gain, however, Tpcn2 KO mice are not protected against HF-diet-induced changes in glucose or insulin area under the curve during glucose tolerance tests in female mice, while HF diet has no significant effect on glucose tolerance in the male mice, regardless of genotype. Glucose disappearance during an insulin tolerance test is augmented in male KO mice, consistent with our previous findings suggesting enhanced insulin sensitivity in these mice. Male KO mice exhibit increased fasting plasma total cholesterol and triglyceride concentrations relative to WT mice on the LF diet, but this difference disappears in HF diet-fed mice where there is increased cholesterol and triglycerides across all genotypes. These data demonstrate that knockout of Tpcn2 may increase insulin action in male, but not female, mice. In addition, both male and female KO mice are protected against diet-induced weight gain, but this protection is likely independent from glucose tolerance, insulin sensitivity, and plasma lipid levels.

Role of Protein Phosphatase 1 and Inhibitor of Protein Phosphatase 1 in Nitric Oxide-Dependent Inhibition of the DNA Damage Response in Pancreatic ß-Cells.

Nitric oxide is produced at micromolar levels by pancreatic ß-cells during exposure to proinflammatory cytokines. While classically viewed as damaging, nitric oxide also activates pathways that promote ß-cell survival. We have shown that nitric oxide, in a cell type-selective manner, inhibits the DNA damage response (DDR) and, in doing so, protects ß-cells from DNA damage-induced apoptosis. This study explores potential mechanisms by which nitric oxide inhibits DDR signaling. We show that inhibition of DDR signaling (measured by γH2AX formation and the phosphorylation of KAP1) is selective for nitric oxide, as other forms of reactive oxygen/nitrogen species do not impair DDR signaling. The kinetics and broad range of DDR substrates that are inhibited suggest that protein phosphatase activation may be one mechanism by which nitric oxide attenuates DDR signaling in ß-cells. While protein phosphatase 1 (PP1) is a primary regulator of DDR signaling and an inhibitor of PP1 (IPP1) is selectively expressed only in ß-cells, disruption of either IPP1 or PP1 does not modify the inhibitory actions of nitric oxide on DDR signaling in ß-cells. These findings support a PP1-independent mechanism by which nitric oxide selectively impairs DDR signaling and protects ß-cells from DNA damage-induced apoptosis.

Mitochondrial Voltage-Dependent Anion Channel Protein Por1 Positively Regulates the Nuclear Localization of Saccharomyces cerevisiae AMP-Activated Protein Kinase.

Snf1 protein kinase of the yeast Saccharomyces cerevisiae is a member of the highly conserved eukaryotic AMP-activated protein kinase (AMPK) family, which is involved in regulating responses to energy limitation. Under conditions of carbon/energy stress, such as during glucose depletion, Snf1 is catalytically activated and enriched in the nucleus to regulate transcription. Snf1 catalytic activation requires phosphorylation of its conserved activation loop threonine (Thr210) by upstream kinases. Catalytic activation is also a prerequisite for Snf1's subsequent nuclear enrichment, a process that is mediated by Gal83, one of three alternate ß-subunits of the Snf1 kinase complex. We previously reported that the mitochondrial voltage-dependent anion channel (VDAC) proteins Por1 and Por2 play redundant roles in promoting Snf1 catalytic activation by Thr210 phosphorylation. Here, we show that the por1Δ mutation alone, which by itself does not affect Snf1 Thr210 phosphorylation, causes defects in Snf1 and Gal83 nuclear enrichment and Snf1's ability to stimulate transcription. We present evidence that Por1 promotes Snf1 nuclear enrichment by promoting the nuclear enrichment of Gal83. Overexpression of Por2, which is not believed to have channel activity, can suppress the localization and transcription activation defects of the por1Δ mutant, suggesting that the regulatory role played by Por1 is separable from its channel function. Thus, our findings expand the positive roles of the yeast VDACs in carbon/energy stress signaling upstream of Snf1. Since AMPK/Snf1 and VDAC proteins are conserved in evolution, our findings in yeast may have implications for AMPK regulation in other eukaryotes, including humans. IMPORTANCE AMP-activated protein kinases (AMPKs) sense energy limitation and regulate transcription and metabolism in eukaryotes from yeast to humans. In mammals, AMPK responds to increased AMP-to-ATP or ADP-to-ATP ratios and is implicated in diabetes, heart disease, and cancer. Mitochondria produce ATP and are generally thought to downregulate AMPK. Indeed, some antidiabetic drugs activate AMPK by affecting mitochondrial respiration. ATP release from mitochondria is mediated by evolutionarily conserved proteins known as voltage-dependent anion channels (VDACs). One would therefore expect VDACs to serve as negative regulators of AMPK. However, our experiments in yeast reveal the existence of an opposite relationship. We previously showed that Saccharomyces cerevisiae VDACs Por1 and Por2 positively regulate AMPK/Snf1 catalytic activation. Here, we show that Por1 also plays an important role in promoting AMPK/Snf1 nuclear localization. Our counterintuitive findings could inform research in areas ranging from diabetes to cancer to fungal pathogenesis.