Chronic activation of AMP kinase results in NRF-1 activation and mitochondrial biogenesis.

The underlying mechanism by which skeletal muscle adapts to exercise training or chronic energy deprivation is largely unknown. To examine this question, rats were fed for 9 wk either with or without beta-guanadinopropionic acid (beta-GPA; 1% enriched diet), a creatine analog that is known to induce muscle adaptations similar to those induced by exercise training. Muscle phosphocreatine, ATP, and ATP/AMP ratios were all markedly decreased and led to the activation of AMP-activated protein kinase (AMPK) in the beta-GPA-fed rats compared with control rats. Under these conditions, nuclear respiratory factor-1 (NRF-1) binding activity, measured using a cDNA probe containing a sequence encoding for the promoter of delta-aminolevulinate (ALA) synthase, was increased by about eightfold in the muscle of beta-GPA-fed rats compared with the control group. Concomitantly, muscle ALA synthase mRNA and cytochrome c content were also increased. Mitochondrial density in both extensor digitorum longus and epitrochlearis from beta-GPA-fed rats was also increased by more than twofold compared with the control group. In conclusion, chronic phosphocreatine depletion during beta-GPA supplementation led to the activation of muscle AMPK that was associated with increased NRF-1 binding activity, increased cytochrome c content, and increased muscle mitochondrial density. Our data suggest that AMPK may play an important role in muscle adaptations to chronic energy stress and that it promotes mitochondrial biogenesis and expression of respiratory proteins through activation of NRF-1.

In vivo effects of uncoupling protein-3 gene disruption on mitochondrial energy metabolism.

To clarify the role of uncoupling protein-3 (UCP3) in skeletal muscle, we used NMR and isotopic labeling experiments to evaluate the effect of UCP3 knockout (UCP3KO) in mice on the regulation of energy metabolism in vivo. Whole body energy expenditure was determined from the turnover of doubly labeled body water. Coupling of mitochondrial oxidative phosphorylation in skeletal muscle was evaluated from measurements of rates of ATP synthesis (using (31)P NMR magnetization transfer experiments) and tricarboxylic acid (TCA) cycle flux (calculated from the time course of (13)C enrichment in C-4 and C-2 of glutamate during an infusion of [2-(13)C]acetate). At the whole body level, we observed no change in energy expenditure. However, at the cellular level, skeletal muscle UCP3KO increased the rate of ATP synthesis from P(i) more than 4-fold under fasting conditions (wild type, 2.2 +/- 0.6 versus knockout, 9.1 +/- 1.4 micromol/g of muscle/min, p < 0.001) with no change in TCA cycle flux rate (wild type, 0.74 +/- 0.04 versus knockout, 0.71 +/- 0.03 micromol/g of muscle/min). The increased efficiency of ATP production may account for the significant (p < 0.05) increase in the ratio of ATP to ADP in the muscle of UCP3KO mice (5.9 +/- 0.3) compared with controls (4.5 +/- 0.4). The data presented here provide the first evidence of uncoupling activity by UCP3 in skeletal muscle in vivo.

13C/31P NMR assessment of mitochondrial energy coupling in skeletal muscle of awake fed and fasted rats. Relationship with uncoupling protein 3 expression.

To examine the relationship between mitochondrial energy coupling in skeletal muscle and change in uncoupling protein 3 (UCP3) expression during the transition from the fed to fasted state, we used a novel noninvasive (31)P/(13)C NMR spectroscopic approach to measure the degree of mitochondrial energy coupling in the hind limb muscles of awake rats before and after a 48-h fast. Compared with fed levels, UCP3 mRNA and protein levels in the gastrocnemius increased 1.7- (p < 0.01) and 2.9-fold (p < 0.001), respectively, following a 48-h fast. Tricarboxylic acid cycle flux measured using (13)C NMR as an index of mitochondrial substrate oxidation was 212 +/- 23 and 173 +/- 25 nmol/g/min (p not significant) in the fed and 48-h fasted groups, respectively. Unidirectional ATP synthesis flux measured using (31)P NMR was 79 +/- 15 and 57 +/- 9 nmol/g/s (p not significant) in the fed and 48-h fasted groups, respectively. Mitochondrial energy coupling as expressed by the ratio of ATP synthesis to tricarboxylic acid cycle flux was not different between the fed and fasted states. To test the hypothesis that UCP3 may be involved in the translocation of long chain free fatty acids (FFA) into the mitochondrial matrix under conditions of elevated FFA availability, [U-(13)C]palmitate/albumin was administered in a separate group of rats with (+) or without (-) etomoxir (an inhibitor of carnitine palmitoyltransferase I). The ratio of glutamate enrichment ((+) etomoxir/(-) etomoxir) in the hind limb muscles was the same between groups, indicating that UCP3 does not appear to function as a translocator for long chain FFA in skeletal muscle following a 48-h fast. In summary, these data demonstrate that despite a 2-3-fold increase in UCP3 mRNA and protein expression in skeletal muscle during the transition from the fed to fasted state, mitochondrial energy coupling does not change. Furthermore, UCP3 does not appear to have a major role in FFA translocation into the mitochondria. The physiological role of UCP3 following a 48-h fast in skeletal muscle remains to be elucidated.

Assessment of mitochondrial energy coupling in vivo by 13C/31P NMR.

The recently cloned uncoupling protein homolog UCP3 is expressed primarily in muscle and therefore may play a significant role in the regulation of energy expenditure and body weight. However, investigation into the regulation of uncoupling protein has been hampered by the inability to assess its activity in vivo. In this report, we demonstrate the use of a noninvasive NMR technique to assess mitochondrial energy uncoupling in skeletal muscle of awake rats by combining (13)C NMR to measure rates of mitochondrial substrate oxidation with (31)P NMR to assess unidirectional ATP synthesis flux. These combined (31)P/(13)C NMR measurements were performed in control, 10-day triiodo-l-thyronine (T(3))-treated (model of increased UCP3 expression), and acute 2,4-dinitrophenol (DNP)-treated (protonophore and mitochondrial uncoupler) rats. UCP3 mRNA and protein levels increased 8.1-fold (+/- 1.1) and 2.8-fold (+/- 0.8), respectively, in the T(3)-treated vs. control rat gastrocnemius muscle. (13)C NMR measurements of tricarboxylic acid cycle flux as an index of mitochondrial substrate oxidation were 61 +/- 21, 148 +/- 25, and 310 +/- 48 nmol/g per min in the control, T(3), and DNP groups, respectively. (31)P NMR saturation transfer measurements of unidirectional ATP synthesis flux were 83 +/- 14, 84 +/- 14, and 73 +/- 7 nmol/g per s in the control, T(3), and DNP groups, respectively. Together, these flux measurements, when normalized to the control group, suggest that acute administration of DNP (mitochondrial uncoupler) and chronic administration of T(3) decrease energy coupling by approximately 80% and approximately 60%, respectively, and that the latter treatment correlates with an increase in UCP3 mRNA and protein expression. This NMR approach could prove useful for exploring the regulation of uncoupling protein activity in vivo and elucidating its role in energy metabolism and obesity.