Overfeeding reduces insulin sensitivity and increases oxidative stress, without altering markers of mitochondrial content and function in humans

Abstract

Background: Mitochondrial dysfunction and increased oxidative stress are associated with obesity and type 2 diabetes. High fat feeding induces insulin resistance and increases skeletal muscle oxidative stress in rodents, but there is controversy as to whether skeletal muscle mitochondrial biogenesis and function is altered.

Methodology and principal findings: Forty (37 ± 2 y) non-obese (25.6 ± 0.6 kg/m(2)) sedentary men (n = 20) and women (n = 20) were overfed (+1040 ± 100 kcal/day, 46 ± 1% of energy from fat) for 28 days. Hyperinsulinemic-euglycemic clamps were performed at baseline and day 28 of overfeeding and skeletal muscle biopsies taken at baseline, day 3 and day 28 of overfeeding in a sub cohort of 26 individuals (13 men and 13 women) that consented to having all 3 biopsies performed. Weight increased on average in the whole cohort by 0.6 ± 0.1 and 2.7 ± 0.3 kg at days 3 and 28, respectively (P<0.0001, without a significant difference in the response between men and women (P = 0.4). Glucose infusion rate during the hyperinsulinemic-euglycemic clamp decreased from 54.8 ± 2.8 at baseline to 50.3 ± 2.5 µmol/min/kg FFM at day 28 of overfeeding (P = 0.03) without a significant difference between men and women (P = 0.4). Skeletal muscle protein carbonyls and urinary F2-isoprostanes increased with overfeeding (P<0.05). Protein levels of muscle peroxisome proliferator-activated receptor gamma coactivator-1α (PGC1α) and subunits from complex I, II and V of the electron transport chain were increased at day 3 (all P<0.05) and returned to basal levels at day 28. No changes were detected in muscle citrate synthase activity or ex vivo CO(2) production at either time point.

Conclusions: Peripheral insulin resistance was induced by overfeeding, without reducing any of the markers of mitochondrial content that were examined. Oxidative stress was however increased, and may have contributed to the reduction in insulin sensitivity observed.

Trial registration: ClinicalTrials.gov NCT00562393.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Flow of participants in the study.

Figure 2. Urinary F2-isoprostane content and skeletal muscle protein carbonyls in response to overfeeding.

Urine F2-isoprostane (A), skeletal muscle protein carbonyls quantification (B) and representative blots (C) and the association between protein carbonyls and peripheral insulin resistance at end of overfeeding (D). Data are expressed as mean±SEM at baseline (white), day 3 (striped) and day 28 (black) of overfeeding. Difference by RM-ANOVA *P<0.05, **P = 0.01, urinary F2-isoprostanes data is based on n = 37 (18 men and 17 women) and skeletal muscle protein carbonyl on n = 18 (8 men and 10 women).

Figure 3. Skeletal muscle protein expression and enzyme activities in response to overfeeding.

Skeletal muscle complexes of the electron transport chain (a), Mn-superoxide dismutase (SOD), uncoupling protein-3 (UCP3), PPAR-coactivator 1α (PGC1α), and carnitine palmitoyltransferase (CPT1b) proteins and representative samples of the Western blots (b) and skeletal muscle citrate synthase (CS), hydroxyacyl-CoA dehydrogenase (βHAD), hexokinase and phosphofructokinase (PFK) activities (c). Data expressed relative to baseline at day 3 (striped) and day 28 (black) of overfeeding (n = 26; 13 men and 13 women). Difference by one-way t-test *P<0.05, **P<0.01.