Substrate dependence of the mitochondrial energy status in the isolated working rat heart.

Here we show for the first time that insulin increases the efficiency of the working heart by 35% through an increase in the free energy of the NADH dehydrogenase reaction, an equivalent increase in the cytosolic free energy of ATP hydrolysis and a decrease in O2 consumption. These changes in turn are linked to changes in the components of the tricarboxylic acid cycle and the potential and pH gradient between the cytosolic and mitochondrial phases. As the insulin effects can be duplicated by ketone bodies, we suggest that insulin acts on mitochondria via control of substrate levels and cofactor ratios. To investigate the mechanism underlying the insulinor ketonemediated increase in hydraulic efficiency of the working heart, we used a combination of 3lP NMR spectroscopic, enzymatic and HPLC methods to measure cytoplasmic ATP, PCr and Pi concentrations and pH, TCA cycle components and tissue [HCO3-] Isolated working rat hearts were perfused with Krebs Henseleit buffer containing 10 mM glucose or 10 mM glucose with the ketone bodies, 1 mM acetoacetate and 4 mM d-P-hydroxybutyrate, in the presence or absence of insulin. Hearts were perfused via the left atrium with a preload of 20 cm H20 and an arterial afterload of 60 mmHg. The coronary venous effluent [COz] was measured using a blood gas analyser. 31P NMR spectroscopy was used to measure cytoplasmic ATP, PCr and Pi concentrations and pH. The pH was estimated from the Pi chemical shift relative to PCr, calibrated against solutions of known free [Mg2+] . The cytoplasmic [HCOy] was calculated using the cytoplasmic pH and the coronary venous effluent [CO,]. In a parallel series of experiments, hearts were perfused under similar conditions and then freeze-clamped and enzymatic (Masuda et al., 1990) and HPLC methods were used to measure the tissue metabolite concentrations. The relative volumes of the intraand extracellular H 2 0 spaces were estimated using 3H2O and 14C-mannitol (Masuda er al., 1990). The free mitochondrial [NAD+]/NADH] was estimated from the measured components of the glutamate dehydrogenase reaction (Williamson et al . , 1967). The cytoplasmic free energy of ATP hydrolysis. A G A ~ , was calculated for the appropriate free [Mg2+] from 31P NMR spectroscopic measurements of PCr, ATP and Pi and enzymatically measured creatine. Estimation of the potential between the cytoplasmic and mitochondrial phases, Em,,, was made using the stoichiometry of 4H+ moved from mitochondrial to cytoplasmic phases by NADHCoQ reductase and CoQ-cytC reductase (Alexandre er al., 1978), according to the equation: where R, the gas constant, is 8.315 J/deg/mol, T , the absolute temperature, is 311 K, and F, the Faraday constant, is 96.48 J/moVmV. The distribution of the measured tissue NH4+ was calculated, assuming that NH3 is permeant across the mitochondrial membrane.