Muscle uncoupling protein 3 overexpression mimics endurance training and reduces circulating biomarkers of incomplete β-oxidation

Abstract

Exercise substantially improves metabolic health, making the elicited mechanisms important targets for novel therapeutic strategies. Uncoupling protein 3 (UCP3) is a mitochondrial inner membrane protein highly selectively expressed in skeletal muscle. Here we report that moderate UCP3 overexpression (roughly 3-fold) in muscles of UCP3 transgenic (UCP3 Tg) mice acts as an exercise mimetic in many ways. UCP3 overexpression increased spontaneous activity (∼40%) and energy expenditure (∼5-10%) and decreased oxidative stress (∼15-20%), similar to exercise training in wild-type (WT) mice. The increase in complete fatty acid oxidation (FAO; ∼30% for WT and ∼70% for UCP3 Tg) and energy expenditure (∼8% for WT and 15% for UCP3 Tg) in response to endurance training was higher in UCP3 Tg than in WT mice, showing an additive effect of UCP3 and endurance training on these two parameters. Moreover, increases in circulating short-chain acylcarnitines in response to acute exercise in untrained WT mice were absent with training or in UCP3 Tg mice. UCP3 overexpression had the same effect as training in decreasing long-chain acylcarnitines. Outcomes coincided with a reduction in muscle carnitine acetyltransferase activity that catalyzes the formation of acylcarnitines. Overall, results are consistent with the conclusions that circulating acylcarnitines could be used as a marker of incomplete muscle FAO and that UCP3 is a potential target for the treatment of prevalent metabolic diseases in which muscle FAO is affected.

Keywords: acylcarnitines; exercise mimetic; fatty acid oxidation; mitochondria; oxidative stress.

Figure 1.

Summary of the study design (A) and the training and indirect calorimetry protocol (B), using WT and UCP3 Tg mice. During wk 1, trained mice were acclimated to the treadmill equipment: they ran 10 min the first day from 8 to 10 m/min. Subsequently, mice ran an extra 10 min/d to reach 50 min at the end of the first week. Treadmill speed was increased every day (1 m/min/d) to reach 14 m/min at the end of the first week. Mice were then trained for an additional 4 wk for 5 d/wk at 1 h/d and 14 m/min, with a 5% slope. Indirect calorimetry measurements were done over 24 h, in the fed state at the end of the fourth week of training, with 40 h of rest for the trained mice (after the last training session, to avoid a confounding effect of the last training session).

Figure 2.

Endurance training induced similar weight loss in WT and muscle-specific UCP3 Tg mice. A) Top panel: representative Western blots of UCP3 expression in isolated mitochondria. Complex III was used as a loading control. Bottom panel: quantification of UCP3 expression by density analysis. Body weight is expressed in grams (absolute values). ^††LFDR < 20%, n = 7–11. B, C) Evolution of mouse body weight during the study. B) Absolute body weight. C) Body weight is represented as percentage of initial body weight (d 1 of study). No-EB and EB mice were pooled; n = 17–21. Data are means ± sem. ^#P < 0.05, ^##P < 0.01, ^###P < 0.0001: training effect; ^∧∧∧P < 0.001: genotype effect.

Figure 3.

Muscle-specific UCP3 Tg mice have increased whole-body energy expenditure and oxidative muscle fibers after endurance training. A) Locomotor activity in untrained and trained WT and UCP3 Tg mice. Genotype × training effect: P < 0.05 for the light phase. n = 6–9. ^#P < 0.05: training effect; ^∧P < 0.05: WT trained (TR) vs. UCP3 Tg untrained (noTR). B) Wheel counts in untrained and trained WT and UCP3 Tg mice. n = 6–9. ^#P < 0.05, ^###P < 0.001: training effect. C) Time to exhaustion on a treadmill. n = 6. D) Fatigue was elicited in FDB with 1 tetanic contraction/s for 3 min. Peak tetanic force was calculated as the difference between the force just before contraction and the maximum force during contraction. n = 6. E) Energy expenditure measured during resting period in untrained and trained WT and UCP3 Tg mice. n = 4–6. ^∧P < 0.05: genotype effect; ^#P < 0.05: training effect. F) RER measured during resting period in untrained and trained WT and UCP3 Tg mice. G, H) Top panel: representative immunohistochemical detection of fiber typing on serial sections of TA (G) and soleus (H) muscles. Pink, type I; blue, type IIa; brown, type IIb; blue/brown, type IIa/b. Bottom panel: quantification of fiber typing of tibialis anterior (G) and soleus (H) muscles. Scale bar = 200 μm. Data are means ± sem. Genotype × training effect: P < 0.05 for type I fiber. n = 6. ^∧P < 0.05: genotype effect; ^#P < 0.05: training effect.

Figure 4.

Endurance training and muscle-specific UCP3 overexpression in mice promotes muscle complete fatty acid oxidation. A) Left panel: total FAO measured in isolated mitochondria. Center panel: complete FAO (¹⁴CO₂ production), expressed as percentage of total FAO. Right panel: incomplete FAO (¹⁴C ASP production), expressed as percentage of total FAO. Training × genotype effect: P = 0.07 for percentage complete and incomplete FAO. n = 12–14. ^##P < 0.01: training effect. B) Skeletal muscle mitochondrial content. n = 16–19. ^###P < 0.0001: training effect. C) Left panel: estimated whole-muscle FAO. Right panel: estimated whole-muscle complete FAO. NoEB and EB mice were pooled. noTR, untrained; TR, trained. Training × genotype effect: P = 0.05 for whole-muscle complete FAO. n = 12–14. ^##P < 0.01, ^###P < 0.0001: training effect; ^∧P < 0.05: WT TR vs. UCP3 Tg TR. D) Total FAO measured in mouse primary myotubes. ***P < 0.0001: treatment effect. E) Complete FAO (¹⁴CO₂ production) measured in mouse primary myotubes. n = 4–5. ***P < 0.0001: treatment effect; ^∧P < 0.05: genotype effect. F) Incomplete FAO (¹⁴C-labeled ASPs) measured in mouse primary myotubes. ¹⁴C-labeled ASPs produced in medium (M) and in cell lysate (L) were measured. n = 4–5. **P < 0.01: treatment effect. Data are means ± sem.

Figure 5.

Muscle-specific overexpression of UCP3 protects against oxidative stress similar to endurance training in mice. A) Top panel: representative Western blots of MnSOD in isolated mitochondria. Bottom panel: quantification of MnSOD protein by density analysis. n = 4–6. B) Top panel: representative Western blots of GRx2 in isolated mitochondria. Bottom panel: quantification of GRx2 protein by density analysis. n = 4. Complex III was used as a loading control. C) 4-HNE adducts measured in isolated mitochondria. n = 4–6. D) Left panel: representative Western blot of protein carbonyls measured in TA muscle. Middle panel: Ponceau staining (loading control). Right panel: quantification of protein carbonyl by density analysis. n = 4. LFDR estimate computed by the complementary LFDR estimation method for 4-HNE; noEB vs. EB in WT untrained mice, is >50%. See Supplemental Data. nTR, untrained; TR, trained. Data are means ± sem. ^†LFDR < 50%, ^††LFDR < 20%; ^†††LFDR < 5%.

Figure 6.

Muscle-specific UCP3 overexpression in mice results in a trained-like phenotype regarding plasma acylcarnitine levels. A−D) Amounts of plasma free carnitine (A), plasma acetylcarnitine (B), plasma short-chain acylcarnitines (C), and plasma long-chain acylcarnitines (D) are shown. Plasma acylcarnitine levels were measured by Q-TOF LC/MS. n = 6–8. LFDR estimates computed by the complementary LFDR estimation method for C16:2 and C16:1, untrained (noTR) vs. trained (TR) Tg noEB mice, and for C16:0, noEB vs. EB in WT untrained mice, are >50%. See Supplemental Data. ^†LFDR < 50%, ^††LFDR < 20%, ^†††LFDR <5%. E) Palmitoylcarnitine formation catalyzed by CPT1 or CPT2 measured in isolated mitochondria from untrained and trained WT and UCP3 Tg mice. ^###P < 0.0001: training effect. n = 5–9. F) CrAT activity in isolated mitochondria from untrained and trained WT and UCP3 Tg mice. Genotype × training effect: P = 0.01. n = 10–14, each measurement done in duplicate. Data are means ± sem. ^#P = 0.05: training effect; ^∧∧P < 0.01: WT noTR vs. UCP3 Tg noTR.