Inducible Nitric Oxide Synthase Plays a Role in LPS-induced Hyperglycemia and Insulin Resistance

American Journal of Physiology. Endocrinology and Metabolism. Feb, 2002  |  Pubmed ID: 11788371

The molecular mechanisms underlying endotoxin-induced insulin resistance remain unclear. Endotoxin or lipopolysaccharide (LPS) injection is a potent stimulator of inducible nitric oxide synthase (iNOS). This study in rats, using the specific iNOS inhibitor aminoguanidine, investigated the role of iNOS in endotoxin-induced hyperglycemia and insulin resistance. LPS injection led to hyperglycemia, insulin resistance, and increased iNOS protein expression and activity. Aminoguanidine prevented LPS-induced hyperglycemia without affecting insulin levels or iNOS expression. Aminoguanidine attenuated the LPS-induced insulin resistance, reflected by the requirement for a higher glucose infusion rate to maintain euglycemia during a hyperinsulinemic clamp study. Aminoguanidine completely blocked the LPS-elevated hepatic glucose output and also inhibited LPS-induced increases in hepatic glycogen phosphorylase activities and phosphoenolpyruvate carboxykinase (PEPCK) mRNA expression, key enzymes for glycogenolysis and gluconeogenesis, respectively. Thus, these data demonstrate an important role for iNOS in LPS-induced insulin resistance, evidenced by the attenuation of LPS-induced hyperglycemia and reversal of increased hepatic glucose output by aminoguanidine. The protective effect of aminoguanidine on insulin resistance is probably by attenuation of hepatic glucose output via its inhibition of key enzymes for glycogenolysis and gluconeogenesis, including glycogen phosphorylase and PEPCK.

Comparison of Comet Assay, Electron Microscopy, and Flow Cytometry for Detection of Apoptosis

The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society. Jul, 2003  |  Pubmed ID: 12810838

Differentiating apoptosis from necrosis is a challenge in single cells and in parenchymal tissues. The techniques available, including in situ TUNEL (Terminal deoxyribonucleotide transferase-mediated dUTP-X Nick End-Labeling) staining, DNA ladder assay, and flow cytometry, suffer from low sensitivity or from a high false-positive rate. This study, using a Jurkat cell model, initially evaluated the specificity of the neutral comet assay and flow cytometry compared to the gold standard, electron microscopy, for detection of apoptosis and necrosis. Neutral comet assay distinguished apoptosis from necrosis in Jurkat cells, as evidenced by the increased comet score in apoptotic cells and the almost zero comet score in necrotic cells. These findings were consistent with those of electron microscopy and flow cytometry. Furthermore, using rats with burn or ischemia/reperfusion injury, well-established models of skeletal and cardiac muscle tissue apoptosis, respectively, we applied the comet assay to detect apoptosis in these muscles. Neutral comet assay was able to detect apoptotic changes in both models. In the muscle samples from rats with burn or ischemia-reperfusion injury, the comet score was higher than that of muscle samples from their respective controls. These studies confirm the consistency of the comet assay for detection of apoptosis in single cells and provide evidence for its applicability as an additional method to detect apoptosis in parenchymal cells.

Up-regulation of Acetylcholine Receptors During Subchronic Infusion of Pancuronium is Caused by a Posttranscriptional Mechanism Related to Disuse

Critical Care Medicine. Feb, 2004  |  Pubmed ID: 14758171

Contrasting with the classic theory that competitive block of the acetylcholine receptor induces up-regulation of the receptor, recent studies show that irreversible block of acetylcholine receptors with alpha-bungarotoxin decreases acetylcholine receptor number within hours. This study investigated the early effects of competitive acetylcholine receptor block with the reversible, competitive muscle relaxant, pancuronium.

Steroidal Nondepolarizing Muscle Relaxants Do Not Simulate the Effects of Glucocorticoids on Glucocorticoid Receptor-mediated Transcription in Cultured Skeletal Muscle Cells

Anesthesiology. Jun, 2004  |  Pubmed ID: 15166588

Burn Injury Impairs Insulin-stimulated Akt/PKB Activation in Skeletal Muscle

American Journal of Physiology. Endocrinology and Metabolism. Mar, 2005  |  Pubmed ID: 15536206

The molecular bases underlying burn- or critical illness-induced insulin resistance still remain unclarified. Muscle protein catabolism is a ubiquitous feature of critical illness. Akt/PKB plays a central role in the metabolic actions of insulin and is a pivotal regulator of hypertrophy and atrophy of skeletal muscle. We therefore examined the effects of burn injury on insulin-stimulated Akt/PKB activation in skeletal muscle. Insulin-stimulated phosphorylation of Akt/PKB was significantly attenuated in burned compared with sham-burned rats. Insulin-stimulated Akt/PKB kinase activity, as judged by immune complex kinase assay and phosphorylation status of the endogenous substrate of Akt/PKB, glycogen synthase kinase-3beta (GSK-3beta), was significantly impaired in burned rats. Furthermore, insulin consistently failed to increase the phosphorylation of p70 S6 kinase, another downstream effector of Akt/PKB, in rats with burn injury, whereas phosphorylation of p70 S6 kinase was increased by insulin in controls. The protein expression of Akt/PKB, GSK-3beta, and p70 S6 kinase was unaltered by burn injury. However, insulin-stimulated activation of ERK, a signaling pathway parallel to Akt/PKB, was not affected by burn injury. These results demonstrate that burn injury impairs insulin-stimulated Akt/PKB activation in skeletal muscle and suggest that attenuated Akt/PKB activation may be involved in deranged metabolism and muscle wasting observed after burn injury.

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

The Journal of Biological Chemistry. Apr, 2005  |  Pubmed ID: 15805118

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.

Proton NMR Spectroscopy Shows Lipids Accumulate in Skeletal Muscle in Response to Burn Trauma-induced Apoptosis

FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology. Sep, 2005  |  Pubmed ID: 16126910

Burn trauma triggers hypermetabolism and muscle wasting via increased cellular protein degradation and apoptosis. Proton nuclear magnetic resonance (1H NMR) spectroscopy can detect mobile lipids in vivo. To examine the local effects of burn in skeletal muscle, we performed in vivo 1H NMR on mice 3 days after burn trauma; and ex vivo, high-resolution, magic angle spinning (1)H NMR on intact excised mouse muscle samples before and 1 and 3 days after burn. These samples were then analyzed for apoptotic nuclei using a terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. To confirm our NMR and cell biology results, we used transcriptome analysis to demonstrate that burn trauma alters the expression of genes involved in lipid metabolism and apoptosis. Our results demonstrate that burn injury results in a localized intramyocellular lipid accumulation, which in turn is accompanied by burn-induced apoptosis and mitochondrial dysfunction, as seen by the up-regulation of apoptotic genes and down-regulation of genes that encode lipid oxidation and the peroxisomal proliferator activator receptor gamma coactivator PGC-1beta. Moreover, the increased levels of bisallylic methylene fatty acyl protons (2.8 ppm) and vinyl protons (5.4 ppm), in conjunction with the TUNEL assay results, further suggest that burn trauma results in apoptosis. Together, our results provide new insight into the local physiological changes that occur in skeletal muscle after severe burn trauma.

Adipocyte Apoptosis After Burn Injury is Associated with Altered Fat Metabolism

Journal of Burn Care & Research : Official Publication of the American Burn Association. May-Jun, 2006  |  Pubmed ID: 16679908

Burn injury often is associated with the abnormal lipid metabolism, including hyperlipidemia, desensitization to lipolytic responses to catecholamines, and reduction in the size of the white adipose tissue. Understanding the biological mechanisms for the decrease in fat mass despite desensitization to catecholamines is important both for the study of lipid metabolism and for the study of its relationship to concomitant insulin resistance. Using epididymal adipose tissue from adult male Sprague-Dawley rats after burn injury (n = 102) or sham-burn injury (n = 102), we tested the hypothesis that a whole-body burn injury causes apoptosis in that tissue. At 1, 3, and 7 days after 40% to 50% body burn injury to the rat, epidydimal adipose tissue was harvested and studied for apoptotic changes and lipolytic properties. For apoptosis, paraformaldehyde-fixed tissue sections were analyzed by in situ TdT-mediated dUTP-X nick-end labeling (TUNEL) staining, and tissue homogenates were also analyzed for DNA fragmentation by enzyme-linked immunoassay and ligation-mediated polymerase chain reaction ladder assay. Isolated adipocytes were stimulated with isoprotenerol, and glycerol production was measured as a reflector of effectiveness of lipolysis. Epididymal adipose tissue showed increased apoptosis manifested by the positive TUNEL staining and increased DNA fragmentation by enzyme-linked immunoassay at day 3 and 7 after burn injury. The DNA fragmentation was confirmed further by the ligation-mediated polymerase chain reaction ladder assay. This elevated DNA fragmentation persisted in the burned animals from day 3 until day 7 after burn injury, the end of observation period. Increase in apoptosis was correlated with decrease in DNA content and tissue weight in the epidydimis. At the functional level, a significant decrease in isoproterenol-induced lipolytic activity (glycerol production) was observed to almost 50% of control level at day 3 and 7 but was not decreased at day 1. Apoptosis of adipocytes may play a role in the altered lipid metabolism, including hyperlipidemia observed in burned subjects.

Mitochondria, Endoplasmic Reticulum, and Alternative Pathways of Cell Death in Critical Illness

Critical Care Medicine. Sep, 2007  |  Pubmed ID: 17713398

Dying cells are distinguished by their biochemical and morphologic traits and categorized into three subtypes: apoptosis, oncosis (necrosis), and cell death with autophagy. Each of these types of cell death plays critical roles in tissue morphogenesis during normal development and in the pathogenesis of human diseases. Given that tissue homeostasis is controlled by the intricate balance between degeneration and regeneration, it is essential to understand the mechanisms of different forms of cell death to establish and improve therapeutic interventions for prevention and rescue of these cell death-related disorders. Critical illness, including sepsis, trauma, and burn injury, is often complicated by multiple organ dysfunction syndrome and is accompanied by increased cell death in parenchymal and nonparenchymal tissues. Accumulating evidence suggests that augmented cell death plays an important role in the organ failure in critical illness. We discuss possible therapeutic approaches for prevention of cell death, particularly apoptotic cell death.

Primary Role of Functional Ischemia, Quantitative Evidence for the Two-hit Mechanism, and Phosphodiesterase-5 Inhibitor Therapy in Mouse Muscular Dystrophy

PloS One. 2007  |  Pubmed ID: 17726536

Duchenne Muscular Dystrophy (DMD) is characterized by increased muscle damage and an abnormal blood flow after muscle contraction: the state of functional ischemia. Until now, however, the cause-effect relationship between the pathogenesis of DMD and functional ischemia was unclear. We examined (i) whether functional ischemia is necessary to cause contraction-induced myofiber damage and (ii) whether functional ischemia alone is sufficient to induce the damage.

Obesity-induced Insulin Resistance and Hyperglycemia: Etiologic Factors and Molecular Mechanisms

Anesthesiology. Jul, 2008  |  Pubmed ID: 18580184

Obesity is a major cause of type 2 diabetes, clinically evidenced as hyperglycemia. The altered glucose homeostasis is caused by faulty signal transduction via the insulin signaling proteins, which results in decreased glucose uptake by the muscle, altered lipogenesis, and increased glucose output by the liver. The etiology of this derangement in insulin signaling is related to a chronic inflammatory state, leading to the induction of inducible nitric oxide synthase and release of high levels of nitric oxide and reactive nitrogen species, which together cause posttranslational modifications in the signaling proteins. There are substantial differences in the molecular mechanisms of insulin resistance in muscle versus liver. Hormones and cytokines from adipocytes can enhance or inhibit both glycemic sensing and insulin signaling. The role of the central nervous system in glucose homeostasis also has been established. Multipronged therapies aimed at rectifying obesity-induced anomalies in both central nervous system and peripheral tissues may prove to be beneficial.

Combined Off-resonance Imaging and T2 Relaxation in the Rotating Frame for Positive Contrast MR Imaging of Infection in a Murine Burn Model

Journal of Magnetic Resonance Imaging : JMRI. Nov, 2010  |  Pubmed ID: 21031524

To develop novel magnetic resonance (MR) imaging methods to monitor accumulation of macrophages in inflammation and infection. Positive-contrast MR imaging provides an alternative to negative-contrast MRI, exploiting the chemical shift induced by ultra-small superparamagnetic iron-oxide (USPIO) nanoparticles to nearby water molecules. We introduce a novel combination of off-resonance (ORI) positive-contrast MRI and T(2ρ) relaxation in the rotating frame (ORI-T(2ρ)) for positive-contrast MR imaging of USPIO.

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

Metabolism: Clinical and Experimental. Jan, 2012  |  Pubmed ID: 21816442

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.