Endothelial nitric oxide synthase is central to skeletal muscle metabolic regulation and enzymatic signaling during exercise in vivo

Abstract

Endothelial nitric oxide synthase (eNOS) is associated with a number of physiological functions involved in the regulation of metabolism; however, the functional role of eNOS is poorly understood. We tested the hypothesis that eNOS is critical to muscle cell signaling and fuel usage during exercise in vivo, using 16-wk-old catheterized (carotid artery and jugular vein) C57BL/6J mice with wild-type (WT), partial (+/-), or no expression (-/-) of eNOS. Quantitative reductions in eNOS expression ( approximately 40%) elicited many of the phenotypic effects observed in enos(-/-) mice under fasted, sedentary conditions, with expression of oxidative phosphorylation complexes I to V and ATP levels being decreased, and total NOS activity and Ca(2+)/CaM kinase II Thr(286) phosphorylation being increased in skeletal muscle. Despite these alterations, exercise tolerance was markedly impaired in enos(-/-) mice during an acute 30-min bout of exercise. An eNOS-dependent effect was observed with regard to AMP-activated protein kinase signaling and muscle perfusion. Muscle glucose and long-chain fatty acid uptake, and hepatic and skeletal muscle glycogenolysis during the exercise bout was markedly accelerated in enos(-/-) mice compared with enos(+/-) and WT mice. Correspondingly, enos(-/-) mice exhibited hypoglycemia during exercise. Thus, the ablation of eNOS alters a number of physiological processes that result in impaired exercise capacity in vivo. The finding that a partial reduction in eNOS expression is sufficient to induce many of the changes associated with ablation of eNOS has implications for chronic metabolic diseases, such as obesity and insulin resistance, which are associated with reduced eNOS expression.

Fig. 1.

Baseline characteristics of 16-wk-old chow-fed C57BL/6J mice with wild type (WT), partial (+/−) or no expression (−/−) of endothelial nitric oxide synthase (eNOS). Protein expression of eNOS (A), oxidative phosphorylation (OXPHOS) complexes I to V of the electron transport chain (B and C), NOS activity (D), AMP-activated protein kinase (AMPK) α2 activity (E), and Ca²⁺/calmodulin protein kinase (CaMK) II Thr²⁸⁶ phosphorylation (F) was determined in gastrocnemius muscle of sedentary mice that had been fasted for 5 h. Data are expressed as means ± SE for n = 5–7 per group. *P < 0.05 vs. WT; †P < 0.05 vs. WT and enos^+/−; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; +ve, positive control (rat brain mitochondria).

Fig. 2.

AMP-activated protein kinase (AMPK) α Thr¹⁷² phosphorylation (A), linear regression analysis for the exercise-induced increase in AMPKα Thr¹⁷² phosphorylation (B), acetyl-CoA carboxylase (ACC) β Ser²²¹ phosphorylation (C), linear regression analysis for the exercise-induced increase in ACCβ Ser²²¹ phosphorylation (D), AMPKα2 activity (E), nitric oxide synthase (NOS) activity (F), and Ca²⁺/calmodulin protein kinase (CaMK) II Thr²⁸⁶ phosphorylation (G) in gastrocnemius muscle of 16-wk-old chow fed C57BL/6J mice with wild-type (WT), partial (+/−), or no expression (−/−) of eNOS. Mice either remained sedentary (Sed) or performed a maximum of 30 min of treadmill exercise (Ex) following a 5-h fast. Data are expressed as means ± SE for n = 5–7 per group. ‡ P < 0.05 vs. corresponding rest value; *P < 0.05 vs. WT; †P < 0.05 vs. WT and enos^+/−.

Fig. 3.

Arterial blood glucose (A), plasma nonesterified fatty acids (NEFAs; B), plasma insulin (C), and plasma lactate (D) at rest and during exercise in 16-wk-old chow fed C57BL/6J mice with wild-type (WT), partial (+/−), or no expression (−/−) of eNOS. Following a 5-h fast, chronically catheterized mice performed a maximum of 30 min of running on a motorized treadmill, and arterial blood was sampled at times shown. Data are expressed as means ± SE for n = 7 (WT), n = 15 (enos^+/−), and n = 10 (enos^−/−). ‡P < 0.05 vs. corresponding rest value: *P < 0.001 vs. WT; †P < 0.001 vs. WT and enos^+/−; fMain effect for genotype, P < 0.05; §Main effect for time, P < 0.05.

Fig. 4.

Exercise-induced increase in percent cardiac output to gastrocnemius muscle (%Q_G) in 16-wk-old chow fed C57BL/6J mice with wild-type (WT), partial (+/−), or no expression (−/−) of eNOS. Following a 5-h fast, mice either remained sedentary or performed a maximum of 30 min of running on a motorized treadmill. At the end of exercise, 50 μl of yellow DYE-TRAK Microspheres were injected into the carotid artery to determine %Q_G (see materials and methods). A: %Q_G in sedentary mice and exercised mice. B: exercise-induced increase in %Q_G. eNOS protein expression in gastrocnemius muscle is also shown. Data are expressed as means ± SE; n = 5 per group. *P < 0.01 vs. WT; ‡P < 0.05 vs. corresponding rest value.

Fig. 5.

Tissue-specific indexes of glucose clearance (K_g) and glucose uptake (R_g) in gastrocnemius (Gastroc; A, B), superficial vastus lateralis (SVL; C, D), and cardiac muscle (E, F) at rest and during exercise in 16-wk-old chow fed C57BL/6J mice with wild-type (WT), partial (+/−) or no expression (−/−) of eNOS. Mice were chronically catheterized and allowed to recover for 7 days. Following a 5-h fast, mice either remained sedentary (Sed) or performed a maximum of 30 min of treadmill exercise (Ex). At 5 min, mice were given a bolus injection containing 13 μCi of 2[¹⁴C]deoxyglucose. K_g and R_g were calculated as described previously (see materials and methods). In Sed, data are expressed as means ± SE for n = 6 (WT), n = 7 (enos^+/−) and n = 4 (enos^−/−). For Ex, n = 6, n = 12, and n = 7, respectively. †P < 0.05 vs. WT and enos^+/−; ‡P < 0.05 vs. corresponding sedentary value; *P < 0.05 vs. WT.

Fig. 6.

Tissue-specific indexes of long-chain fatty acid (LCFA) clearance (K_f) and LCFA uptake (R_f) in gastrocnemius (Gastroc; A, B), superficial vastus lateralis (SVL; C, D), and cardiac muscle (E, F) at rest and during exercise in 16-wk-old chow fed C57BL/6J mice with wild-type (WT), partial (+/−) or no expression (−/−) of eNOS. Mice were chronically catheterized and allowed to recover for 7 days. Following a 5-h fast, mice either remained sedentary (Sed) or performed a maximum of 30 min of treadmill exercise (Ex). At 5 min, mice were given a bolus injection containing 26 μCi of [9,10-³H]-(R)-2-bromopalmitate. K_f and R_f were calculated as described previously (see materials and methods). In Sed, data are expressed as means ± SE for n = 7 (WT), n = 9 (enos^+/−) and n = 4 (enos^−/−). For Ex, n = 7, n = 12, and n = 7, respectively. †P < 0.05 vs. WT and enos^+/−; ‡P < 0.05 vs. corresponding sedentary value.