Exercise and Weight Loss Improve Muscle Mitochondrial Respiration, Lipid Partitioning, and Insulin Sensitivity After Gastric Bypass Surgery

Paul M. Coen; Elizabeth V. Menshikova; Giovanna Distefano; Donghai Zheng; Charles J. Tanner; Robert A. Standley; Nicole L. Helbling; Gabriel S. Dubis; Vladimir B. Ritov; Hui Xie; Marisa E. Desimone; Steven R. Smith; Maja Stefanovic-Racic; Frederico G.S. Toledo; Joseph A. Houmard; Bret H. Goodpaster

Disclosures

Diabetes. 2015;64(11):3737-3750. 

In This Article

Results

Study Participants

The characteristics of the study groups before and after the interventions are shown in Table 1. There were no baseline differences in age, sex, race, mass, fat mass, or BMI between groups. The average time from the date of RYGB surgery to randomization into study groups was 77 ± 24 days. There was no difference between groups in average time from surgery to randomization. All muscle biopsy procedures and IVGTTs were conducted 1 day before randomization. No serious adverse events occurred in either study group, and no differences were found in reported adverse events between groups. By design, all participants completed the interventions, and participants in the EX group performed an average ± SEM of 154 ± 17 min/week of structured exercise. Supplementary Table 1 contains further exercise compliance data. Both groups reported similar medication use at baseline, and no difference was found in medication use after intervention (data not shown).

Weight, Body Composition, and Cardiorespiratory Fitness

Both groups experienced similar reductions in mass, fat mass, BMI, and waist circumference after the interventions (Table 1). Changes in blood pressure (systolic and diastolic) and blood lipids (total, LDL, and HDL cholesterol and triglycerides) were reduced to a similar degree in the EX and CON groups similar to that reported in the parent trial.[4] EX but not CON had a significantly improved VO2peak (Table 1), an index of cardiorespiratory fitness and an effective predictor of future morbidity and mortality.

Intravenous Glucose Tolerance Test

The baseline insulin sensitivity index (SI) for both groups was similar (Table 1). Baseline fasting insulin, glucose, and HOMA for insulin resistance were slightly higher in the CON than in the EX group. After the interventions, SI was improved in both groups. The EX group, however, had a significantly greater improvement in SI than the CON group. The intervention effects on weight, body composition, fitness, and SI for these groups were the same or similar as those reported in the parent trial.[4]

Muscle CL and OXPHOS Content

CL and OXPHOS protein expression were measured as surrogates of mitochondrial content. Total CL did not change in either intervention group, indicating no change in inner mitochondrial membrane content (Fig. 1A). However, exercise altered individual molecular CL species (based on fatty acyl carbon chain length and degree of saturation). Exercise increased the CL-(C18:2)4 species and decreased the CL-(C18:2)3(C18:1)1, CL-(C18:2)2(C18:1)2, and CL-(C18:2)3(C18:0)1 species. CL-(C18:2)3(C18:3)1 was not altered by exercise (Fig. 1B). These data indicate that the exercise intervention remodeled the CL profile in mitochondria independent of changes in total content. We also found that neither the total OXPHOS content nor each of the five ETS subunit proteins were significantly changed by either the CON or the EX group (Fig. 1CE). Relative change of each ETS subunit did not change for either intervention (Fig. 1F).

Mitochondrial Respiration

Measuring mitochondrial respiration is a widely used approach to assess mitochondrial ETS function. In the first assay protocol, which used carbohydrate-derived substrates, we found that exercise improved CI P and CI&II P respiration. ETS capacity was also improved with exercise. In contrast, the CON group had no change in steady-state respiration (Fig. 2A). The second assay protocol used fatty acid (palmitoylcarnitine) and carbohydrate-derived substrates. Exercise improved FAO L respiration and CI&II & FAO P respiration. There was also an increase for both groups (time effect) for FAO P respiration and CI&II and FAO P respiration (Fig. 2B), suggesting an independent effect of RYGB surgery–induced weight loss. A benefit of the mitochondrial respiration protocol is that ratios of oxygen flux in various respiratory states can be examined. Flux control ratios provide an internal normalization and qualitative assessment of coupling and substrate control independent of mitochondrial content, quality of the fiber bundle preparation, and inadvertent variation in assay conditions. We found an increase in both groups (time effect) for the CI phosphorylation system control ratio (Fig. 2C). This ratio is an expression of the limitation of CI ETS capacity by the phosphorylation system that increases with increasing capacity of the phosphorylation system. Similarly, there was an increase in both groups for the CI&II phosphorylation system control ratio (Fig. 2D). On examining the ratio of CI P /CI&II P (CI control ratio), we found an increase for both groups (Fig. 2E). This observation indicates that the relative contribution of CI to maximal OXPHOS respiration increases after RYGB surgery–induced weight loss, with or without exercise. There was no change in the LEAK control ratio with either intervention (Fig. 2F), indicating no change in inner mitochondrial membrane uncoupling. Finally, the cytochrome C response for both groups was <10%, indicating maintenance of mitochondrial membrane integrity.

Figure 2.

Exercise improves mitochondrial respiratory capacity, whereas RYGB surgery–induced weight loss improves the phosphorylation system control ratio. A: Exercise improves ADP (4 mmol/L)-stimulated respiration supported by CI (5 mmol/L glutamate and 2 mmol/L malate), CI&II (10 mmol/L succinate), and ETS capacity (2 μmol/L FCCP). B: Exercise improves LEAK respiration and ADP (4 mmol/L)-stimulated respiration supported by FAO and CI&II (25 μmol/L palmitoylcarnitine, 2 mmol/L malate, 5 mmol/L glutamate, and 10 mmol/L succinate). There is also a time effect for ADP (4 mmol/L)-stimulated respiration supported by FAO (25 μmol/L palmitoylcarnitine, 2 mmol/L malate) and FAO and CI&II (25 μmol/L palmitoylcarnitine, 2 mmol/L malate, 5 mmol/L glutamate, and 10 mmol/L succinate). C: There was an increase in both groups (time effect) for the CI phosphorylation system control ratio. D: There was an increase in both groups (time effect) for the CI&II phosphorylation system control ratio. E: There was an increase in both groups (time effect) for the CI control ratio. F: No change was seen in the LEAK control ratio (n = 30 EX, n = 41 CON). The letters A and B denote significant differences between groups (P < 0.05, ANOVA). Data are mean ± SEM. *P < 0.05, group × time interaction; **P < 0.05, time effect. Cyto C, cytochrome c.

Mitochondrial Enzyme Assays

Activities of both succinate dehydrogenase and citrate synthase, enzyme complexes of the tricarboxylic acid cycle, tended to increase in the EX but not in the CON group (Table 2). NADH oxidase, representing the activity of the entire ETC, also tended to be higher in the EX group. Moreover, the ratio of NADH oxidase to CL significantly increased in the EX group, reflecting an exercise-induced increase in the ETC activity per unit of mitochondrial mass. Creatine kinase activity did not change with the interventions (Table 2).

IMTG and DAG Content

There was no change in fiber type proportion or fiber cross-sectional area (Table 3). IMTG content in type I myofibers was decreased in the CON group but not in the EX group, indicating an exercise-induced preservation of higher IMTG during weight loss (Fig. 3B). The exercise effect was not seen in type IIA fibers for which both groups showed decreased (time effect) IMTG content (Fig. 3C). We found no significant change in any species or total content of DAG in either group.

Figure 3.

RYGB surgery reduces IMTG content in type I and type II myofibers without altering whole-muscle DAG content. A: Representative images of immunofiber typing and oil red O staining generated from a muscle biopsy specimen. B: The CON group but not the EX group had reduced IMTG in type I myofibers. C: IMTG content was significantly reduced in type IIA myofibers for both groups (time effect). D: There was no change in IMTG content in type IIX myofibers (n = 34 EX, n = 33 CON). EG: There were no EX or CON group effects on any DAG species quantified (n = 20 EX, n = 16 CON). The letters A and B denote significant differences between group/time points (P < 0.05, ANOVA). Data are mean ± SEM. *P < 0.05, group × time interaction; **P < 0.05, time effect. AU, arbitrary unit; ORO, oil red O; ww, wet weight.

Intramyocellular Sphingolipid

We quantified a profile of sphingolipid species, purported lipotoxic mediators of insulin resistance. We found a significant decrease for the EX group and a time effect for total sphingolipid, total ceramide, and total unsaturated ceramide. There was also a group effect for total sphingolipid, ceramide, and saturated and unsaturated ceramide largely driven by the higher content in the EX group preintervention (Fig. 4A). Similar observations were made for many other ceramide species (Fig. 4B and C). Finally, there were no intervention effects on sphingosine species (Fig. 4D).

Figure 4.

Ceramide content in muscle decreases with RYGB surgery–induced weight loss. Exercise may further reduce content of certain molecular species of ceramide. A: Total sphingolipid, ceramide, and unsaturated ceramide all decreased in both groups (time effect), with the decreases being more pronounced in the EX group. Ceramide levels before intervention were higher in the EX than in the CON group. B: C16, C18:1, and C24:1 ceramides were decreased in both groups (time effect), with the decreases being more pronounced in the EX group. There is also a group effect for C18 and C18:1 influenced mainly by higher levels of ceramide at EX preintervention. C: There was a number of group effects (higher in EX) for many of the low-abundance ceramides. D: There were no EX or CON effects on any sphingosine species quantified (n = 20 EX, n = 16 CON). The letters A and B denote significant differences between group/time points (P < 0.05, ANOVA). Data are mean ± SEM. *P < 0.05, group × time interaction; **P < 0.05, time effect; #P < 0.05, group effect. ww, wet weight.

GLUT Expression

We measured protein expression of insulin dependent (GLUT4) and independent (GLUT1 and 12) GLUTs as potential mediators of improved SI after the interventions. No change in expression of GLUT was found in either the EX or the CON group (Fig. 5).

Figure 5.

GLUT protein expression did not change with the EX or CON groups. A: Representative Western blots for GLUT1, GLUT4, GLUT12, and the housekeeping protein α-tubulin. GLUT1 (B), GLUT4 (C), and GLUT12 (D) protein expression in muscle did not change with either the CON or the EX interventions (n = 19 EX, n = 17 CON). Data are mean ± SEM. AU, arbitrary unit.

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