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


Diabetes. 2015;64(11):3737-3750. 

In This Article

Research Design and Methods

Patient Recruitment

The study volunteers were a subset of RYGB surgery patients enrolled in a larger randomized controlled exercise trial[4] who completed the study and who underwent muscle biopsy (n = 101). Participants were recruited from two academic bariatric surgery practices in Pittsburgh, Pennsylvania, and Greenville, North Carolina. The study protocol was approved by the human ethics committees of the University of Pittsburgh and East Carolina University. All participants provided written informed consent to participate in the study. Male and female patients were eligible if they were between the ages of 21 and 60 years, had a BMI <55 kg/m2 and underwent RYGB surgery, were not diabetic, and volunteered to undergo muscle biopsy before and after the intervention. Other aspects of the study design, patient recruitment, and inclusion and exclusion criteria are described in detail elsewhere.[4]

Intervention Groups

Between 1 and 3 months after bariatric surgery, participants were randomized to a 6-month intervention of health education control (n = 51) (CON) or an exercise program (n = 50) (EX).[4] Study measurements were made over separate clinic visits before and after the 6-month interventions. The exercise program started after completion of baseline metabolic and body composition assessments and the initial muscle biopsy. Participants in the EX group were required to participate in three to five exercise sessions per week with at least one directly supervised session per week to assure that the target exercise intensity (60–70% maximum heart rate) and duration were achieved and to document progress. Participants progressed over 3 months to a minimum of 120 min/week of exercise, which was maintained for the final 3 months of the program. The CON group was asked to attend six health education sessions. The sessions were held once a month and included lectures, discussions, and demonstrations providing up-to-date information on health topics such as medication use, nutrition, and communicating with health-care professionals. Physical activity habits were also reported and documented at the health education session.

Intravenous Glucose Tolerance Test

A 3-h insulin-modified IVGTT was performed in the morning hours after an overnight fast and at least 48 h removed from the last exercise session to measure insulin action based on the Bergman minimal model calculations,[24] as previously described.[4]

Body Composition and Cardiorespiratory Fitness

Fat and lean mass were determined by DXA using a GE Lunar bone densiometer (GE Healthcare). Cardiorespiratory fitness (VO2peak) was measured by indirect calorimetry (Moxus Modular VO2 System, AEI Technologies, Inc.) during a 5–12-min graded exercise test on a cycle ergometer (Lode), as previously described.[4] Twelve-lead electrocardiogram recordings were monitored by the study physician and interpreted for contraindications to exercise. Body weight, waist circumference, blood pressure, and plasma lipid levels were measured by standard clinical protocols.

Muscle Biopsy and Specimen Collection Procedures

Percutaneous muscle biopsy samples were obtained after an overnight fast and at least 48 h removed from the last exercise session as described previously.[13] Briefly, muscle biopsy specimens were obtained under local anesthesia (2% buffered lidocaine) from the medial vastus lateralis 15 cm above the patella using a 5-mm Bergström muscle biopsy cannula with suction (Stille Surgical Instruments, Eskilstuna, Sweden). Immediately after the biopsy procedure, the specimen was blotted dry and trimmed of visible adipose tissue using a standard dissecting microscope (Leica EZ4; Leica Microsystems, Heerbrugg, Switzerland). Three portions of the specimen (~30 mg each) were snap frozen in liquid nitrogen and stored at −80°C for lipidomics analysis, immunoblotting, and assays of mitochondrial enzyme activity. A separate portion (~10 mg) was placed in relaxing and biopsy-preserving solution (BIOPS media; OROBOROS Instruments, Innsbruck, Austria) for high-resolution respirometry. For immunohistochemistry, a portion (~30 mg) was mounted on a small piece of cork with mounting medium (Shandon Cryochrome; Thermo Scientific, Pittsburgh, PA), frozen in isopentane cooled with liquid nitrogen for 2–3 min (−160°C), and then placed into liquid nitrogen. All frozen samples were stored at −80°C until analysis.

Mitochondrial Biochemistry

A portion of frozen muscle (20–30 mg) was used to measure NADH oxidase, citrate synthase, and creatine kinase activities and CL as described in the Supplementary Data.

High-resolution Respirometry

Immediately after the muscle biopsy procedure, muscle fiber bundles (~1–3 mg each) were prepared as described in the Supplementary Data. The fiber bundles were gently placed into the respirometer chambers (Oxygraph-2K; OROBOROS Instruments), and after a stable baseline was reached, two assay protocols were run in duplicate at 37°C and between 230–150 nmol/mL O2 in buffer Z with blebbistatin (25 μmol/L) (Supplementary Fig. 1). In protocol 1, complex I–supported LEAK (CI L or state 4) respiration was determined through the addition of glutamate (5 mmol/L) and malate (2 mmol/L). ADP (4 mmol/L) was added to elicit complex I–supported oxidative phosphorylation (OXPHOS) (CI P or state 3) respiration. Succinate (10 mmol/L) was then added to elicit complex I and II–supported OXPHOS (CI&II P ). Cytochrome c (10 μmol/L) was added to assess the integrity of the outer mitochondrial membrane. Finally, FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone) (2 μmol/L) was added to determine complex I and II–supported electron transfer system (ETS) capacity (CI&II E ) or maximal uncoupled respiration. In protocol 2, FAO-supported LEAK (FAO L ) respiration was determined through the addition of palmitoylcarnitine (25 μmol/L) and malate (2 mmol/L). ADP (4 mmol/L) was added to elicit FAO-supported OXPHOS (FAO P ). Glutamate (5 mmol/L) was then added to elicit CI and FAO P respiration. Succinate (10 mmol/L) was added to stimulate CI&II and FAO P respiration. Finally, cytochrome c (10 μmol/L) was added to assess mitochondrial integrity. After the assay protocols, the fiber bundles were retrieved from the respirometry chambers, dried, and weighed on an analytical balance (XS105; Mettler Toledo, Columbus, OH). Oxygen flux was normalized to dry weight of the fiber bundle.

Figure 1.

Exercise remodels CL profile without altering total CL or OXPHOS content in vastus lateralis after RYGB surgery. A: Total CL is not altered with RYGB surgery–induced weight loss with or without exercise (n = 21 EX, n = 20, CON). B: Exercise increases the relative proportion of CL-(C18:2)4, whereas CL-(C18:2)3(C18:1)1, CL-(C18:2)2(C18:1)2, and CL-(C18:2)3(C18:0)1 all decreased. C: Representative Western blot for five OXPHOS proteins and α-tubulin. D and E: OXPHOS content is not altered with RYGB surgery–induced weight loss with or without exercise. F: There was no difference between groups in the change in expression of OXPHOS proteins (n = 19 EX, n = 17 CON). The letters A and B denote significant differences between group/time points (P < 0.05, ANOVA). Data are mean ± SEM. AU, arbitrary unit; MWM, molecular weight marker; ww, wet weight.

Analysis of Sphingolipid and DAG Species

Intramuscular sphingolipids and DAGs were quantified by high-performance liquid chromatography-tandem mass spectrometry as described previously[25] and in the Supplementary Data. Intramuscular DAG and ceramide content was normalized to tissue wet weight (pmol/mg tissue).

Fiber Type and Intramyocellular Triglyceride Content

Determination of intramyocellular triglyceride (IMTG) content was performed using a modified version of methods previously used in our laboratory[13] and as described in the Supplementary Data. Oil red O staining intensity and cross-sectional area was determined in type I and type II myocytes. Analysis is based on >200 fibers/section.

Protein Content by Immunoblot

A portion of frozen muscle (~30 mg) was prepared for immunoblot analysis for GLUT protein expression as described in the Supplementary Data. Gel-to-gel variation was controlled by using a standardized sample on each gel. Protein loading was controlled by normalizing bands of interest to α-tubulin.

Statistical Analysis

Group differences in baseline characteristics were determined using two-sample Student t test (two-tailed) or χ2 or Fisher exact tests. Any variables with high skew were log- or square root–transformed to achieve a normal distribution. The general linear mixed model with repeated measures (PROC MIXED) was performed to detect group and time effects in the outcome variables. Group, time, and group * time were treated as fixed effects and subjects nested within each group as random effects. Age, sex, and race were covariates. P values for post hoc tests were adjusted by false discovery rate (FDR). P < 0.05 was considered significant. Analyses were performed using SAS 9.1 or JMP Pro 11 for Macintosh (SAS Institute Inc.).