C-13/P-31 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 skelet al muscle and change in uncoupling protein 3 (UCP3) expression during the t ransition from the fed to fasted state, we used a novel noninvasive P-31/C- 13 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 fa st. Compared with fed levels, UCP3 mRNA and protein levels in the gastrocne mius increased 1.7- (p < 0.01) and 2.9-fold (p < 0.001), respectively, foll owing a 48-h fast. Tricarboxylic acid cycle flux measured using C-13 NMR as an index of mitochondrial substrate oxidation was 212 +/- 23 and 173 +/- 2 5 nmol/g/min (p not significant) in the fed and 48-h fasted groups, respect ively 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 gr oups, respectively. Mitochondrial energy coupling as expressed by the ratio of ATP synthesis to tricarboxylic acid cycle flux was not different betwee n the fed and fasted states. To test the hypothesis that UCP3 may be involv ed in the translocation of long chain free fatty acids (FFA) into the mitoc hondrial matrix under conditions of elevated FFA availability, [U-C-13]palm itate/albumin was administered in a separate group of rats with (+) or with out (-) etomoxir (an inhibitor of carnitine palmitoyltransferase I). The ra tio of glutamate enrichment ((+) etomoxir/(-) etomoxir) in the hind limb mu scles 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 inc rease in UCP3 mRNA and protein expression in skeletal muscle during the tra nsition from the fed to fasted state, mitochondrial energy coupling does no t change, Furthermore, UCP3 does not appear to have a major role in FFA tra nslocation into the mitochondria, The physiological role of UCP3 following a 48-h fast in skeletal muscle remains to be elucidated.