Exercise training prevents skeletal muscle plasma membrane cholesterol accumulation, cortical actin filament loss, and insulin resistance in C57BL/6J mice fed a western-style high-fat diet

Abstract

Insulin action and glucose disposal are enhanced by exercise, yet the mechanisms involved remain imperfectly understood. While the causes of skeletal muscle insulin resistance also remain poorly understood, new evidence suggest excess plasma membrane (PM) cholesterol may contribute by damaging the cortical filamentous actin (F-actin) structure essential for GLUT4 glucose transporter redistribution to the PM upon insulin stimulation. Here, we investigated whether PM cholesterol toxicity was mitigated by exercise. Male C57BL/6J mice were placed on low-fat (LF, 10% kCal) or high-fat (HF, 45% kCal) diets for a total of 8 weeks. During the last 3 weeks of this LF/HF diet intervention, all mice were familiarized with a treadmill for 1 week and then either sham-exercised (0 m/min, 10% grade, 50 min) or exercised (13.5 m/min, 10% grade, 50 min) daily for 2 weeks. HF-feeding induced a significant gain in body mass by 3 weeks. Sham or chronic exercise did not affect food consumption, water intake, or body mass gain. Prior to sham and chronic exercise, "pre-intervention" glucose tolerance tests were performed on all animals and demonstrated that HF-fed mice were glucose intolerant. While sham exercise did not affect glucose tolerance in the LF or HF mice, exercised mice showed an improvement in glucose tolerance. Muscle from sham-exercised HF-fed mice showed a significant increase in PM cholesterol, loss of cortical F-actin, and decrease in insulin-stimulated glucose transport compared to sham-exercised LF-fed mice. These HF-fed skeletal muscle membrane/cytoskeletal abnormalities and insulin resistance were improved in exercised mice. These data reveal a new therapeutic aspect of exercise being regulation of skeletal muscle PM cholesterol homeostasis. Further studies on this mechanism of insulin resistance and the benefits of exercise on its prevention are needed.

Keywords: Actin; cholesterol; exercise; insulin.

Figure 1

(A) Body mass during the 5‐week acclimation period (weeks ‐5 to 0), 8‐week low‐fat (LF) or high‐fat (HF) diet (weeks 0–8), and the 2‐week sham (SH) or exercise (EX) interventions (weeks 6–8). The inset shows food intake during the 8‐week diet and 2‐week SH/EX intervention. Body mass and food intake values are means ± SEM from 17 LF + SH, 18 HF + SH, and 18 HF + EX mice. (B) Pre‐SH/EX intervention glucose tolerance was determined at 5 weeks. Blood glucose was measured before and after injection of 2 g glucose/kg body mass. Glucose values are means ± SEM from 17 LF + SH, 17 HF + SH, and 17 HF + EX mice. Two‐way ANOVA (A) and one‐way ANOVA (B, inset) post hoc analysis statistics are indicated with *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001. Note some error bars are shorter than the symbols.

Figure 2

Pre‐ and post‐SH/EX intervention glucose tolerance was determined at 5‐ or 8‐ weeks, respectively, for LF‐ (A), or HF‐ (B,C) fed mice. Blood glucose was measured before and after injection of 2 g glucose/kg body mass. All values are means ± SEM from 17 LF + SH, 17 HF + SH, and 17 HF + EX mice. AUC differences were determined using a paired two‐tailed Student's t test and statistic indicated with ^ns P > 0.05; *P < 0.05. Solid and hatched bars represent pre‐ and post‐SH/EX interventions, respectively. Note some error bars are shorter than the symbols.

Figure 3

(A) Paired extensor digitalis longus muscles were subjected to basal and insulin‐stimulated 2‐DG uptake measurements as described in Methods. Uptake values are means  ± SEM from 4 LF + SH, 4 HF + SH, and 5 HF + EX muscles. (B, C, D) Mix hindlimb skeletal muscle were subjected to fractionation for membrane cholesterol determination (B) or were labeled with antibodies against F‐actin and imaged by confocal microscopy (C) and images were digitally quantitated using MetaMorph software (D) as described in Methods. Cholesterol values are means ± SEM from 12 LF + SH, 12 HF + SH, and 12 HF + EX muscles and F‐actin values are means ±SEM from 3 to 5 images of 5 LF + SH, 5 HF + SH, and 5 HF + EX muscles. One‐way ANOVA post hoc analysis statistics are indicated with *P < 0.05 (vs. LF + SH +Insulin); #P < 0.05 (vs. HF + SH + Insulin).