Exercise training modifies skeletal muscle clock gene expression but not 24-hour rhythmicity in substrate metabolism of men with insulin resistance

Abstract

Twenty-four hour rhythmicity in whole-body substrate metabolism, skeletal muscle clock gene expression and mitochondrial respiration is compromised upon insulin resistance. With exercise training known to ameliorate insulin resistance, our objective was to test if exercise training can reinforce diurnal variation in whole-body and skeletal muscle metabolism in men with insulin resistance. In a single-arm longitudinal design, 10 overweight and obese men with insulin resistance performed 12 weeks of high-intensity interval training recurrently in the afternoon (between 14.00 and 18.00 h) and were tested pre- and post-exercise training, while staying in a metabolic research unit for 2 days under free-living conditions with regular meals. On the second days, indirect calorimetry was performed at 08.00, 13.00, 18.00, 23.00 and 04.00 h, muscle biopsies were taken from the vastus lateralis at 08.30, 13.30 and 23.30 h, and blood was drawn at least bi-hourly over 24 h. Participants did not lose body weight over 12 weeks, but improved body composition and exercise capacity. Exercise training resulted in reduced 24-h plasma glucose levels, but did not modify free fatty acid and triacylglycerol levels. Diurnal variation of muscle clock gene expression was modified by exercise training with period genes showing an interaction (time × exercise) effect and reduced mRNA levels at 13.00 h. Exercise training increased mitochondrial respiration without inducing diurnal variation. Twenty-four-hour substrate metabolism and energy expenditure remained unchanged. Future studies should investigate alternative exercise strategies or types of interventions (e.g. diet or drugs aiming at improving insulin sensitivity) for their capacity to reinforce diurnal variation in substrate metabolism and mitochondrial respiration. KEY POINTS: Insulin resistance is associated with blunted 24-h flexibility in whole-body substrate metabolism and skeletal muscle mitochondrial respiration, and disruptions in the skeletal muscle molecular circadian clock. We hypothesized that exercise training modifies 24-h rhythmicity in whole-body substrate metabolism and diurnal variation in skeletal muscle molecular clock and mitochondrial respiration in men with insulin resistance. We found that metabolic inflexibility over 24 h persisted after exercise training, whereas mitochondrial respiration increased independent of time of day. Gene expression of Per1-3 and Rorα in skeletal muscle changed particularly close to the time of day at which exercise training was performed. These results provide the rationale to further investigate the differential metabolic impact of differently timed exercise to treat metabolic defects of insulin resistance that manifest at a particular time of day.

Keywords: circadian rhythm; exercise; insulin resistance; mitochondria.

Figure 1. Design of pre‐ and post‐exercise laboratory testing and the exercise training protocol

A, pre‐ and post‐exercise testing. B, exercise training intervention. HIIT, high‐intensity interval training; W _max, maximal wattage. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 2. 24‐h whole‐body substrate oxidation and energy expenditure

24‐h whole‐body substrate oxidation and energy expenditure (n = 10) remain unchanged from pre‐ (blue dots and line) to post‐exercise training (red dots and line). Whole‐body resting energy expenditure (A), respiratory exchange ratio (B), carbohydrate oxidation (C), and fat oxidation (D). The dark grey area represents the sleeping period (00.00‐07.00 h). Data are presented as means ± SD. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 3. Mitochondrial oxidative capacity in skeletal muscle

Mitchondrial oxidative capacity in skeletal muscle (n = 10) increases from pre‐ (blue dots and line) to post‐exercise training (red dots and line). ADP‐stimulated respiration of permeabilized muscle fibres fuelled with the lipid substrate octanoylcarinitine (state 3 MO; A); addition of complex I substrates (state 3 MOG; B); addition of substrates for parallel electron input into complex I and II (state 3 MOGS; C); and maximal uncoupled respiration after FCCP (state U) titration (D). M, malate; O, octanoylcarnitine; G, glutamate; S, succinate. Data depict oxygen consumption per mg wet weight per second and are shown as means ± SD.*P < 0.05 based on Bonferroni post hoc tests. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 4. Core molecular clock gene expression in skeletal muscle

Changes in clock gene expression in skeletal muscle (n = 9) from pre‐ (blue dots and line) to post‐exercise training (red dots and line). mRNA expression of Bmal1 (A), Clock (B), Reverbα (C), Rorα (D), Per1 (E), Per2 (F), Per3 (G), Cry1 (H). Data are normalized to the geometric mean of two housekeeping genes. Data are presented as means ± SD. *P < 0.05 based on Bonferroni post hoc test. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 5. 24‐h plasma metabolites

Changes in 24‐h plasma metabolites (n = 9) from pre‐ (blue circles and line) to post‐exercise training (red circles and line). 24‐h plasma levels of glucose slightly decreased after exercise training (A), whereas pre‐ and postprandial insulin levels (B), 24‐h free fatty acids (C) and triacylglycerol (D) remained unchanged. The dark grey area represents the sleeping period (00.00–07.00 h). Data are presented as means ± SD. The respective individual data are shown in Supplemental Fig. 4. [Colour figure can be viewed at wileyonlinelibrary.com]