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

The recently cloned uncoupling protein homolog UCP3 is expressed primarily in muscle and therefore may play a significant role in the regulation of en ergy expenditure and body weight. However, investigation into the regulatio n of uncoupling protein has been hampered by the inability to assess its ac tivity 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 C-13 NMR to measure rates of mitochondrial substrat e oxidation with P-31 NMR to assess unidirectional ATP synthesis flux. Thes e combined P-31/C-13 NMR measurements were performed in control, 10-day tri iodo-L-thyronine (T-3)-treated (model of increased UCP3 expression), and ac ute 2,4-dinitrophenol (DNP)-treated (protonophore and mitochondrial uncoupl er) 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 gastrocne mius muscle, C-13 NMR measurements of tricarboxylic acid cycle flux as an i ndex of mitochondrial substrate oxidation were 61 +/- 21, 148 +/- 25, and 3 10 +/- 48 nmol/g per min in the control, T-3 and DNP groups, respectively. P-31 NMR saturation transfer measurements of unidirectional ATP synthesis f lux 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 no rmalized to the control group, suggest that acute administration of DNP (mi tochondrial uncoupler) and chronic administration of T-3 decrease energy co upling by approximate to 80% and approximate to 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 regulati on of uncoupling protein activity in vivo and elucidating its role in energ y metabolism and obesity.