Contribution of the G-3-P shuttle to submaximal respiration in mitochondria from type IIb skeletal muscle

It has been hypothesized that cytosolic reducing equivalents may assist mitochondrial oxidative phosphorylation. In support, addition of 10mM glycerol-3-phosphate (G-3-P) to type IIb mitochondria (MITO) respiring in the presence of 1mM pyruvate + 1mM malate (P+M), resulted in a doubling of the maximal respiration rate while reducing the ADP/O ratio to 2.15. Thus, under maximal conditions, addition of G-3-P resulted in partial displacement of P+M oxidation, and accounted for roughly 70% of the total oxygen consumption. In this study we evaluated the contribution of the G-3-P shuttle during submaximal respiration. MITO were isolated from the gracilis (99% type IIb) muscle of rabbit (n=5). Respiration was measured polarographically in the presence of a total adenylate pool of 5mM and constant 5mM Pi. Substrate provided was P+M. with or w/o 10mM G-3-P. The [ADP], ATP/ADP, and deltaG_ATP of the respiration medium were clamped to various levels using creatine kinase + a 50mM total creatine pool. Under all substrate conditions, respiration was found to be linear with deltaG_ATP. In comparison, respiration was significantly greater in the presence of P+M+G-3-P. Slopes of the P+M and P+M+G-3-P vs. deltaG_ATP regression lines were similar, suggesting that over the submaximal range investigated, the contribution of G-3-P is constant and independent of [ADP], ATP/ADP and deltaG_ATP. Collectively, we have demonstrated that the potential contribution of extramitochondrial reducing equivalents to respiration within MITO from type IIb muscle is substantial. Moreover, under submaximal conditions this contribution appears constant and independent of the energetic demand.