Role of reactive oxygen species in contraction-mediated glucose transport in mouse skeletal muscle

Abstract

Exercise increases glucose transport into skeletal muscle via a pathway that is poorly understood. We investigated the role of endogenously produced reactive oxygen species (ROS) in contraction-mediated glucose transport. Repeated contractions increased 2-deoxyglucose (2-DG) uptake roughly threefold in isolated, mouse extensor digitorum longus (fast-twitch) muscle. N-Acetylcysteine (NAC), a non-specific antioxidant, inhibited contraction-mediated 2-DG uptake by approximately 50% (P < 0.05 versus control values), but did not significantly affect basal 2-DG uptake or the uptake induced by insulin, hypoxia or 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR, which mimics AMP-mediated activation of AMP-activated protein kinase, AMPK). Ebselen, a glutathione peroxidase mimetic, also inhibited contraction-mediated 2-DG uptake (by almost 60%, P < 0.001 versus control values). Muscles from mice overexpressing Mn2+-dependent superoxide dismutase, which catalyses H2O2 production from superoxide anions, exhibited a approximately 25% higher rate of contraction-mediated 2-DG uptake versus muscles from wild-type control mice (P < 0.05). Exogenous H2O2 induced oxidative stress, as judged by an increase in the [GSSG]/[GSH + GSSG] (reduced glutathione + oxidized glutathione) ratio to 2.5 times control values, and this increase was substantially blocked by NAC. Similarly, NAC significantly attenuated contraction-mediated oxidative stress as judged by measurements of glutathione status and the intracellular ROS level with the fluorescent indicator 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein (P < 0.05). Finally, contraction increased AMPK activity and phosphorylation approximately 10-fold, and NAC blocked approximately 50% of these changes. These data indicate that endogenously produced ROS, possibly H2O2 or its derivatives, play an important role in contraction-mediated activation of glucose transport in fast-twitch muscle.

Figure 1. N-Acetylcysteine inhibits contraction-mediated glucose uptake in mouse EDL muscle

Values are means ± s.e.m. for 6–8 muscles (A) or 6 muscles (B). A, open bars, control; filled bars, NAC (20 mm). *P < 0.05 versus control values. B, NAC does not affect force generation during 10 min period of repeated contractions (○, control; •, NAC). Initial force averaged 20.8 ± 1.8 mN (mg wet weight)⁻¹ for control conditions and 23.0 ± 1.4 mN (mg wet weight)⁻¹ for NAC; n.s.).

Figure 2. N-Acetylcysteine inhibits H₂O₂-induced oxidative stress in mouse EDL muscle

Values are means ± s.e.m. for 6 muscles. TGSH is the sum of GSH and GSSG. Muscles were untreated (basal), exposed to H₂O₂ (3 mm) or to H₂O₂ + NAC (20 mm). A, GSSG; B, TGSH; and C, GSSG/TGSH (the ratio is in GSH equivalents). **P < 0.01. Muscles in the basal group were from a separate group of mice studied at the same time as the other groups; the basal values are included in the statistical analysis and serve solely as reference values.

Figure 3. N-Acetylcysteine inhibits contraction-induced oxidative stress in mouse EDL muscle

Values are means ± s.e.m. for 6 muscles. TGSH, sum of GSH and GSSG. Unfilled bars, basal; filled bars, NAC (20 mm). A, GSSG; B, TGSH; and C, GSSG/TGSH (the ratio is in GSH equivalents). *P < 0.05; **P < 0.01 versus control values.

Figure 4. N-Acetylcysteine inhibits contraction-mediated ROS formation in bundles of mouse EDL muscle fibres

Values are means ± s.e.m. Measurements of CM-H₂DCF fluorescence were performed in muscle fibres that underwent 10 min of repeated contractions in the absence (contraction, n = 6) or presence of 20 mm NAC (contraction + NAC, n = 4). Data are expressed as a percentage of the pretreatment value. *P < 0.05 versus precontraction value.

Figure 5. N-Acetylcysteine inhibits contraction-mediated activation of AMPK in mouse EDL muscle

Values are means ± s.e.m. for n = 6 (basal) or n = 5 muscles (contraction). Open bars, control; filled bars, NAC (20 mm). Muscles were stimulated for 10 min with repeated contractions. **P < 0.01 versus control values.

Figure 6. N-Acetylcysteine inhibits contraction-mediated phosphorylation of AMPK but not ACC in mouse EDL muscle

A, representative immunoblots of phosphorylated AMPK (P-AMPK) and total AMPK (AMPK). B, mean ± s.e.m. values for P-AMPK (n = 4). Open bars, control; filled bars, NAC (20 mm). The mean value of the stimulated controls is set to 100% and all the other values are expressed as a percentage of this value. Muscles were stimulated for 10 min with repeated contractions. *P < 0.05 versus control values. C, representative immunoblots of phosphorylated ACC (P-ACC) and total ACC (ACC). D, mean ± s.e.m. values for P-ACC (n = 4). See B for additional details.

Figure 7. Ebselen inhibits and overexpression of SOD2 enhances contraction-mediated glucose uptake in mouse EDL muscle

Values are means ± s.e.m. for 6–9 muscles. A, open bars, control; filled bars, ebselen (30 μm). ***P < 0.001 versus control values. B, ebselen does not affect the decline in force during repeated contractions (○, control; •, ebselen). C, open bars, wild type; filled bars, SOD2 overexpression. *P < 0.05 versus wild type. D, SOD2 overexpression does not affect force generation during repeated contractions (○, control; •, SOD2 overexpression). Initial force averaged 14.9 ± 0.8 mN (mg wet weight)⁻¹ for wild type and 16.0 ± 1.3 mN (mg wet weight)⁻¹ for SOD2 overexpression (n.s.).

Figure 8. Scheme for ROS-mediated glucose transport during muscle contraction

Following the release of Ca²⁺ from the sarcoplasmic reticulum, actomyosin interaction occurs, resulting in muscle contraction, ATP breakdown and increases in ADP and inorganic phosphate (P_i). ADP and P_i stimulate mitochondrial respiration, which can also be stimulated by increases in Ca²⁺ that activate mitochondrial dehydrogenases. Increased respiration results in superoxide anion (O₂^−·) formation through NADH dehydrogenase and semiquinone components; O₂^−· formation can also occur by extramitochondrial mechanisms (e.g. via a Ca²⁺-mediated activation of phospholipase A2). Superoxide anion is then dismutated by superoxide dismutase to H₂O₂, which results in increased LKB1-mediated phosphorylation and activation of AMPK, followed by a translocation of Glut-4 to the surface membrane.