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. 2012 Jan;61(1):127-36.
doi: 10.1016/j.metabol.2011.06.001. Epub 2011 Aug 3.

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

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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

Michiko Sugita et al. Metabolism. .
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Abstract

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.

Conflict of interest statement

Disclosure Statement: no conflict of interest

Figures

Fig. 1
Fig. 1
Induction of iNOS paralleled attenuated IR- and IRS-1-mediated insulin signaling in skeletal muscle after burn injury in wild-type mice. A, Immunoblotting (IB) revealed a robust induction of iNOS expression at 3 days after burn injury. The protein expression of GAPDH was not altered following burn injury. B, C, D, Before (0) or at 1, 3 and 7 days after burn injury, insulin (5 U/kg BW) was injected and muscle was excised 90 sec thereafter. Immunoprecipitation (IP) followed by immunoblotting demonstrated that insulin-stimulated tyrosine phosphorylation (PY) of IR and IRS-1 was significantly decreased at 3 days after burn injury. The protein expression of IR was unaltered by burn injury. In contrast, IRS-1 protein expression was significantly decreased at 3 and 7 days after burn injury as compared with before burn injury. E, The ratio of insulin-stimulated IRS-1 phosphorylation normalized to IRS-1 protein expression was significantly decreased at 3 days after burn injury. *P<0.05, **P<0.01 vs before burn injury. n = 3 per group.
Fig. 2
Fig. 2
iNOS deficiency mitigated burn injury-induced impairment in IR- and IRS-1-mediated insulin signaling in skeletal muscle. At 3 days after burn injury or sham burn, insulin (5 U/kg BW) or saline was injected and 90 sec thereafter skeletal muscle was taken. A, Immunoblotting (IB) demonstrated that iNOS protein expression was induced by burn injury in wild-type, but not iNOS-deficient, mice. The protein expression of GAPDH was not affected by burn injury or iNOS deficiency. The overall interaction between genotype and burn/sham is statistically significant (P<0.001). ***P<0.001 vs sham-burned WT, §§§P<0.001 vs burned iNOS −/−. B, C, D, E, Immunoprecipitation (IP) followed by immunoblotting revealed that insulin-stimulated tyrosine phosphorylation (PY) of IR and IRS-1 was suppressed by burn injury in wild-type (WT) mice, which was ameliorated by iNOS deficiency (−/−). Neither burn injury nor iNOS deficiency altered IR protein expression. The overall interactions between genotype and other factors (burn/sham and insulin/saline) are statistically significant for phosphorylation of IR and IRS-1 (P<0.05). **P<0.01, ***P<0.001 vs sham-burned WT with insulin, §§P<0.01 vs burned iNOS −/− with insulin, †P<0.05 vs burned WT with insulin, ‡‡P<0.01 vs sham-burned WT with insulin. F, In contrast, IRS-1 protein expression was suppressed by burn injury. iNOS deficiency increased IRS-1 expression in burned mice. The overall interaction between genotype and burn/sham is statistically significant (P<0.05). **P<0.01 vs sham-burned WT, §§P<0.01 vs sham-burned iNOS −/−, †P<0.05 vs burned WT. G, The ratio of insulin-stimulated IRS-1 phosphorylation (p-IRS-1) normalized to IRS-1 protein expression was significantly decreased by burn injury in wild-type and iNOS deficient mice, as compared with sham controls. This effect of burn injury on p-IRS-1/IRS-1 ratio was not altered by iNOS deficiency. *P<0.05 vs sham-burned WT, §P<0.05 vs sham-burned iNOS −/−.
Fig. 3
Fig. 3
iNOS deficiency ameliorated attenuated IRS-1-mediated PI3K activation in skeletal muscle of burned mice. At 3 days after burn injury or sham burn, insulin (5 U/kg BW) or saline was injected and 90 sec thereafter skeletal muscle was taken. A, B, Insulin injection resulted in a robust increase in binding of p85 PI3K to IRS-1 in sham-burned mice, as judged by immunoprecipitation (IP) with anti-IRS-1 antibody followed by immunoprecipitation (IB) with anti-p85 antibody. However, insulin failed to increase p85 binding to IRS-1 in burned wild-type (WT) mice. iNOS deficiency (−/−) significantly improved insulin-stimulated binding of p85 to IRS-1 in burned mice. The protein expression of p85 was not affected by burn injury or iNOS deficiency. The overall interaction between genotype and other factors (burn/sham and insulin/saline) is statistically significant for binding of p85 PI3K to IRS-1 (P<0.05). ***P<0.001 vs sham-burned WT with insulin, §§P<0.01 vs sham-burned iNOS −/− with insulin, †P<0.05 vs burned WT with insulin. n=4 per group of animals with saline, n=8 per group of animals with insulin. C, PI3K activity was evaluated in vitro phosphorylation of phosphatidylinositol (PI). Insulin-stimulated PI3K activity was decreased in burned wild-type mice relative to sham animals. iNOS deficiency almost completely reversed decreased PI3K activity in burned mice. The overall interaction between genotype and burn/sham is statistically significant (P<0.05). **P<0.01 vs sham-burned WT, †P<0.05 vs burned WT. n=4 per group.
Fig. 4
Fig. 4
Burn injury-induced decreases in insulin-stimulated Akt phosphorylation and glucose uptake in skeletal muscle were reversed by iNOS deficiency. A, At 3 days after burn injury or sham burn, insulin (1 U/kg BW) or saline was injected and 5 min thereafter skeletal muscle was taken. Insulin-stimulated Akt phosphorylation was significantly attenuated by burn injury in wild-type (WT) mice as compared with sham animals. iNOS deficiency (−/−) almost completely reversed decreased insulin-stimulated Akt phosphorylation in burned mice. Similarly, insulin-stimulated GSK-3β phosphorylation was significantly decreased by burn injury in wild-type mice. Burn injury-induced decreased GSK-3β phosphorylation was ameliorated by iNOS deficiency. Neither burn injury nor iNOS deficiency altered Akt or GSK-3β protein expression. The overall interactions between genotype and burn/sham are statistically significant for phosphorylation of Akt (P<0.01) and GSK-3β (P<0.05). **P<0.01, ***p<0.001 vs sham-burned WT, †p<0.05, ††p<0.01 vs burned iNOS −/−, §p<0.05 vs sham-burned iNOS −/−. N.S.: not significant. n=4 per group of animals with saline, n=6 per group of animals with insulin. B, Burn injury resulted in suppression of insulin-stimulated glucose uptake by skeletal muscle ex vivo. iNOS deficiency significantly improved insulin-stimulated glucose uptake in burned mice. The interaction between genotype and burn/sham is statistically significant (P<0.05). ***P<0.001 vs sham-burned WT, ††P<0.01 vs burned iNOS−/−. N.S.: not significant. n=4–6 per group.
Fig. 5
Fig. 5
iNOS deficiency improved whole-body insulin resistance in burned mice, but did not alter decreased blood glucose levels after burn injury. A, Insulin tolerance test revealed that hypoglycemic response to insulin injection was significantly blunted in burned wild-type (WT) compared to sham animals. Gene disruption of iNOS (−/−) restored sensitivity to insulin-stimulated decrease in blood glucose levels in burned mice. The overall interactions between genotype and burn/sham at 90 and 120 min after the insulin injection are statistically significant (P<0.005). ***P<0.001 vs sham-burned WT and sham-burned and burned iNOS−/−. n=8 per group. B, Unexpectedly, burn injury resulted in decreased blood glucose levels both in wild-type and iNOS-deficient mice. iNOS deficiency did not alter blood glucose levels either in sham-burned mice or in burned mice. **P<0.01 vs sham-burned WT, ††P<0.01 vs sham-burned iNOS−/−. n=8 per group. C, Neither burn injury nor iNOS deficiency significantly affected plasma insulin concentrations.

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