Nobuyuki Shimizu

Inducible Nitric-oxide Synthase and NO Donor Induce Insulin Receptor Substrate-1 Degradation in Skeletal Muscle Cells

Chronic inflammation plays an important role in insulin resistance. Inducible nitric-oxide synthase (iNOS), a mediator of inflammation, has been implicated in many human diseases including insulin resistance. However, the molecular mechanisms by which iNOS mediates insulin resistance remain largely unknown. Here we demonstrate that exposure to NO donor or iNOS transfection reduced insulin receptor substrate (IRS)-1 protein expression without altering the mRNA level in cultured skeletal muscle cells. NO donor increased IRS-1 ubiquitination, and proteasome inhibitors blocked NO donor-induced reduction in IRS-1 expression in cultured skeletal muscle cells. The effect of NO donor on IRS-1 expression was cGMP-independent and accentuated by concomitant oxidative stress, suggesting an involvement of nitrosative stress. Inhibitors for phosphatidylinositol-3 kinase, mammalian target of rapamycin, and c-Jun amino-terminal kinase failed to block NO donor-induced IRS-1 reduction, whereas these inhibitors prevented insulin-stimulated IRS-1 decrease. Moreover iNOS expression was increased in skeletal muscle of diabetic (ob/ob) mice compared with lean wild-type mice. iNOS gene disruption or treatment with iNOS inhibitor ameliorated depressed IRS-1 expression in skeletal muscle of diabetic (ob/ob) mice. These findings indicate that iNOS reduces IRS-1 expression in skeletal muscle via proteasome-mediated degradation and thereby may contribute to obesity-related insulin resistance.

A short duration of high-fat diet induces insulin resistance and predisposes to adverse left ventricular remodeling after pressure overload

Insulin resistance is an increasingly prevalent condition in humans that frequently clusters with disorders characterized by left ventricular (LV) pressure overload, such as systemic hypertension. To investigate the impact of insulin resistance on LV remodeling and functional response to pressure overload, C57BL6 male mice were fed a high-fat (HFD) or a standard diet (SD) for 9 days and then underwent transverse aortic constriction (TAC). LV size and function were assessed in SD- and HFD-fed mice using serial echocardiography before and 7, 21, and 28 days after TAC. Serial echocardiography was also performed on nonoperated SD- and HFD-fed mice over a period of 6 wk. LV perfusion was assessed before and 7 and 28 days after TAC. Nine days of HFD induced systemic and myocardial insulin resistance (assessed by myocardial 18F-fluorodeoxyglucose uptake), and myocardial perfusion response to acetylcholine was impaired. High-fat feeding for 28 days did not change LV size and function in nonbanded mice; however, TAC induced greater hypertrophy, more marked LV systolic and diastolic dysfunction, and decreased survival in HFD-fed compared with SD-fed mice. Compared with SD-fed mice, myocardial perfusion reserve was decreased 7 days after TAC, and capillary density was decreased 28 days after TAC in HFD-fed mice. A short duration of HFD induces insulin resistance in mice. These metabolic changes are accompanied by increased LV remodeling and dysfunction after TAC, highlighting the impact of insulin resistance in the development of pressure-overload-induced heart failure.

Liver-specific Inducible Nitric-oxide Synthase Expression Is Sufficient to Cause Hepatic Insulin Resistance and Mild Hyperglycemia in Mice

Inducible nitric-oxide synthase (iNOS), a major mediator of inflammation, plays an important role in obesity-induced insulin resistance. Inhibition of iNOS by gene disruption or pharmacological inhibitors reverses or ameliorates obesity-induced insulin resistance in skeletal muscle and liver in mice. It is unknown, however, whether increased expression of iNOS is sufficient to cause insulin resistance in vivo. To address this issue, we generated liver-specific iNOS transgenic (L-iNOS-Tg) mice, where expression of the transgene, iNOS, is regulated under mouse albumin promoter. L-iNOS-Tg mice exhibited mild hyperglycemia, hyperinsulinemia, insulin resistance, and impaired insulin-induced suppression of hepatic glucose output, as compared with wild type (WT) littermates. Insulin-stimulated phosphorylation of insulin receptor substrate-1 (IRS-1) and -2, and Akt was significantly attenuated in liver, but not in skeletal muscle, of L-iNOS-Tg mice relative to WT mice without changes in insulin receptor phosphorylation. Moreover, liver-specific iNOS expression abrogated insulin-stimulated phosphorylation of glycogen synthase kinase-3β, forkhead box O1, and mTOR (mammalian target of rapamycin), endogenous substrates of Akt, along with increased S-nitrosylation of Akt relative to WT mice. However, the expression of insulin receptor, IRS-1, IRS-2, Akt, glycogen synthase kinase-3β, forkhead box O1, protein-tyrosine phosphatase-1B, PTEN (phosphatase and tensin homolog), and p85 phosphatidylinositol 3-kinase was not altered by iNOS transgene. Hyperglycemia was associated with elevated glycogen phosphorylase activity and decreased glycogen synthase activity in the liver of L-iNOS-Tg mice, whereas phosphoenolpyruvate carboxykinase, glucose-6-phosphatase, and proliferator-activated receptor γ coactivator-1α expression were not altered. These results clearly indicate that selective expression of iNOS in liver causes hepatic insulin resistance along with deranged insulin signaling, leading to hyperglycemia and hyperinsulinemia. Our data highlight a critical role for iNOS in the development of hepatic insulin resistance and hyperglycemia.

Inflammatory stimuli induce inhibitory S-nitrosylation of the deacetylase SIRT1 to increase acetylation and activation of p53 and p65

S-nitrosylation of the deacetylase SIRT1 functions as a proinflammatory switch in aging and inflammatory disorders. Flipping the SIRT1 Switch During Inflammation In aging-related diseases, chronic inflammation is associated with increased production of nitric oxide. Nitric oxide causes a posttranslational modification of proteins known as S-nitrosylation. SIRT1 is a protein deacetylase that inhibits the transcription factors p53 and NF-κB, which are involved in mediating cell death by apoptosis and promoting inflammatory responses. S-nitrosylation inhibits SIRT1 activity. Shinozaki et al. found that in cultured mammalian cells, S-nitrosylation of SIRT1 prevented it from deacetylating and inhibiting p53 and NF-κB. In mouse models of systemic inflammation, neurodegeneration, or muscle aging, S-nitrosylation of SIRT1 and the associated activation of p53 and NF-κB required the activity of nitric oxide synthases. Thus, S-nitrosylation of SIRT1 may be a critical factor in promoting apoptotic and inflammatory responses in aging-related diseases. Inflammation increases the abundance of inducible nitric oxide synthase (iNOS), leading to enhanced production of nitric oxide (NO), which can modify proteins by S-nitrosylation. Enhanced NO production increases the activities of the transcription factors p53 and nuclear factor κB (NF-κB) in several models of disease-associated inflammation. S-nitrosylation inhibits the activity of the protein deacetylase SIRT1. SIRT1 limits apoptosis and inflammation by deacetylating p53 and p65 (also known as RelA), a subunit of NF-κB. We showed in multiple cultured mammalian cell lines that NO donors or inflammatory stimuli induced S-nitrosylation of SIRT1 within CXXC motifs, which inhibited SIRT1 by disrupting its ability to bind zinc. Inhibition of SIRT1 reduced deacetylation and promoted activation of p53 and p65, leading to apoptosis and increased expression of proinflammatory genes. In rodent models of systemic inflammation, Parkinson’s disease, or aging-related muscular atrophy, S-nitrosylation of SIRT1 correlated with increased acetylation of p53 and p65 and activation of p53 and NF-κB target genes, suggesting that S-nitrosylation of SIRT1 may represent a proinflammatory switch common to many diseases and aging.

Reduction of cardiomyocyte S-nitrosylation by S-nitrosoglutathione reductase protects against sepsis-induced myocardial depression

Myocardial depression is an important contributor to morbidity and mortality in septic patients. Nitric oxide (NO) plays an important role in the development of septic cardiomyopathy, but also has protective effects. Recent evidence has indicated that NO exerts many of its downstream effects on the cardiovascular system via protein S-nitrosylation, which is negatively regulated by S-nitrosoglutathione reductase (GSNOR), an enzyme promoting denitrosylation. We tested the hypothesis that reducing cardiomyocyte S-nitrosylation by increasing GSNOR activity can improve myocardial dysfunction during sepsis. Therefore, we generated mice with a cardiomyocyte-specific overexpression of GSNOR (GSNOR-CMTg mice) and subjected them to endotoxic shock. Measurements of cardiac function in vivo and ex vivo showed that GSNOR-CMTg mice had a significantly improved cardiac function after lipopolysaccharide challenge (LPS, 50 mg/kg) compared with wild-type (WT) mice. Cardiomyocytes isolated from septic GSNOR-CMTg mice showed a corresponding improvement in contractility compared with WT cells. However, systolic Ca(2+) release was similarly depressed in both genotypes after LPS, indicating that GSNOR-CMTg cardiomyocytes have increased Ca(2+) sensitivity during sepsis. Parameters of inflammation were equally increased in LPS-treated hearts of both genotypes, and no compensatory changes in NO synthase expression levels were found in GSNOR-overexpressing hearts before or after LPS challenge. GSNOR overexpression however significantly reduced total cardiac protein S-nitrosylation during sepsis. Taken together, our results indicate that increasing the denitrosylation capacity of cardiomyocytes protects against sepsis-induced myocardial depression. Our findings suggest that specifically reducing protein S-nitrosylation during sepsis improves cardiac function by increasing cardiac myofilament sensitivity to Ca(2+).

Inducible nitric-oxide synthase and nitric oxide donor decrease insulin receptor substrate-2 protein expression by promoting proteasome-dependent degradation in pancreatic beta-cells: involvement of glycogen synthase kinase-3beta.

Insulin receptor substrate-2 (IRS-2) plays a critical role in the survival and function of pancreatic β-cells. Gene disruption of IRS-2 results in failure of the β-cell compensatory mechanism and diabetes. Nonetheless, the regulation of IRS-2 protein expression in β-cells remains largely unknown. Inducible nitric-oxide synthase (iNOS), a major mediator of inflammation, has been implicated in β-cell damage in type 1 and type 2 diabetes. The effects of iNOS on IRS-2 expression have not yet been investigated in β-cells. Here, we show that iNOS and NO donor decreased IRS-2 protein expression in INS-1/832 insulinoma cells and mouse islets, whereas IRS-2 mRNA levels were not altered. Interleukin-1β (IL-1β), alone or in combination with interferon-γ (IFN-γ), reduced IRS-2 protein expression in an iNOS-dependent manner without altering IRS-2 mRNA levels. Proteasome inhibitors, MG132 and lactacystin, blocked the NO donor-induced reduction in IRS-2 protein expression. Treatment with NO donor led to activation of glycogen synthase kinase-3β (GSK-3β) and c-Jun N-terminal kinase (JNK/SAPK) in β-cells. Inhibition of GSK-3β by pharmacological inhibitors or siRNA-mediated knockdown significantly prevented NO donor-induced reduction in IRS-2 expression in β-cells. In contrast, a JNK inhibitor, SP600125, did not effectively block reduced IRS-2 expression in NO donor-treated β-cells. These data indicate that iNOS-derived NO reduces IRS-2 expression by promoting protein degradation, at least in part, through a GSK-3β-dependent mechanism. Our findings suggest that iNOS-mediated decreased IRS-2 expresssion may contribute to the progression and/or exacerbation of β-cell failure in diabetes.

Inducible nitric oxide synthase deficiency ameliorates skeletal muscle insulin resistance but does not alter unexpected lower blood glucose levels after burn injury in C57BL/6 mice.

Burn injury is associated with inflammatory responses and metabolic alterations including insulin resistance. Impaired insulin receptor substrate-1 (IRS-1)-mediated insulin signal transduction is a major component of insulin resistance in skeletal muscle following burn injury. To further investigate molecular mechanisms that underlie burn injury-induced insulin resistance, we study a role of inducible nitric oxide synthase (iNOS), a major mediator of inflammation, on burn-induced muscle insulin resistance in iNOS-deficient mice. Full-thickness third-degree burn injury comprising 12% of total body surface area was produced in wild-type and iNOS-deficient C57BL/6 mice. Insulin-stimulated activation (phosphorylation) of IR, IRS-1, and Akt was assessed by immunoblotting and immunoprecipitation. Insulin-stimulated glucose uptake by skeletal muscle was evaluated ex vivo. Burn injury caused induction of iNOS in skeletal muscle of wild-type mice. The increase of iNOS expression paralleled the increase of insulin resistance, as evidenced by decreased tyrosine phosphorylation of IR and IRS-1, IRS-1 expression, insulin-stimulated activation of phosphatidylinositol 3-kinase and Akt/PKB, and insulin-stimulated glucose uptake in mouse skeletal muscle. The absence of iNOS in genetically engineered mice significantly lessened burn injury-induced insulin resistance in skeletal muscle. In wild-type mice, insulin tolerance test revealed whole-body insulin resistance in burned mice compared with sham-burned controls. This effect was reversed by iNOS deficiency. Unexpectedly, however, blood glucose levels were depressed in both wild-type and iNOS-deficient mice after burn injury. Gene disruption of iNOS ameliorated the effect of burn on IRS-1-mediated insulin signaling in skeletal muscle of mice. These findings indicate that iNOS plays a significant role in burn injury-induced skeletal muscle insulin resistance.

NO donor induces Nec-1-inhibitable, but RIP1-independent, necrotic cell death in pancreatic β-cells

Nitric oxide (NO) has been implicated in pancreatic β‐cell death in the development of diabetes. The mechanisms underlying NO‐induced β‐cell death have not been clearly defined. Recently, receptor‐interacting protein‐1 (RIP1)‐dependent necrosis, which is inhibited by necrostatin‐1, an inhibitor of RIP1, has emerged as a form of regulated necrosis. Here, we show that NO donor‐induced β‐cell death was inhibited by necrostatin‐1. Unexpectedly, however, RIP1 knockdown neither inhibited cell death nor altered the protective effects of necrostatin‐1 in NO donor‐treated β‐cells. These results indicate that NO donor induces necrostatin‐1‐inhibitable necrotic β‐cell death independent of RIP1. Our findings raise the possibility that NO‐mediated β‐cell necrosis may be a novel form of signal‐regulated necrosis, which play a role in the progression of diabetes.

iNOS inhibitor, L-NIL, reverses burn-induced glycogen synthase kinase-3β activation in skeletal muscle of rats.

OBJECTIVES Recent studies suggest that activation of glycogen synthase kinase (GSK)-3β may be involved in burn injury-induced metabolic derangements and protein breakdown in skeletal muscle. However, the mechanism for GSK-3β activation after burn injury is unknown. To investigate the role of inducible nitric oxide synthase (iNOS) in this scenario, a major mediator of inflammation, we examined the effects of a specific inhibitor for iNOS, L-NIL, on GSK-3β activity in skeletal muscle of burned rats. MATERIALS/METHODS Full-thickness third degree burn injury comprising 40% of total body surface area was produced under anesthesia in male Sprague-Dawley rats (160-190g) by immersing the back of the trunk for 15s and the abdomen for 8s in 80°C water. Burned and sham-burned rats were treated with L-NIL (60mg/kg BW, b.i.d., IP) or phosphate-buffered saline for three days. GSK-3β activity in skeletal muscle was evaluated by immune complex kinase assay, and by phosphorylation status of GSK-3β and its endogenous substrate, glycogen synthase. RESULTS GSK-3β activity was increased in a time-dependent manner in skeletal muscle after burn injury, concomitant with the induction of iNOS expression. iNOS inhibitor, L-NIL, reverted the elevated GSK-3β activity in skeletal muscle of burned rats, although L-NIL did not alter GSK-3β activity in sham-burned rats. CONCLUSIONS Our results clearly indicate that iNOS plays an important role in burn injury-induced GSK-3β activation in skeletal muscle. These findings suggest that iNOS may contribute to burn injury-induced metabolic derangements, in part, by activating GSK-3β.

Could insulin sensitization be used as an alternative to intensive insulin therapy to improve the survival of intensive care unit patients with stress-induced hyperglycemia?

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