Operation and energy dependence of the reducing-equivalent shuttles during lactate metabolism by isolated hepatocytes.
The participation and energy dependence of the malate-aspartate shuttle in transporting reducing equivalents generated from cytoplasmic lactate oxidation was studied in isolated hepatocytes of fasted rats. Both lactate removal and glucose synthesis were inhibited by butylmalonate, aminooxyacetate or cycloserine confirming the involvement of malate and aspartate in the transfer of reducing equivalents from the cytoplasm to mitochondria. In the presence of ammonium ions the inhibition of lactate utilization by butylmalonate was considerably reduced, yet the transfer of reducing equivalents into the mitochondria was unaffected, indicating a substantially lesser role for butylmalonate-sensitive malate transport in reducing-equivalent transfer when ammonium ions were present. Ammonium ions had no stimulatory effect on uptake of sorbitol, a substrate whose oxidation principally involves the alpha-glycerophosphate shuttle. The role of cellular energy status (reflected in the mitochondrial membrane electrical potential (delta psi) and redox state), in lactate oxidation and operation of the malate-aspartate shuttle, was studied using a graded concentration range of valinomycin (0-100 nM). Lactate oxidation was strongly inhibited when delta psi fell from 130 to 105 mV whereas O2 consumption and pyruvate removal were only minimally affected over the valinomycin range, suggesting that the oxidation of lactate to pyruvate is an energy-dependent step of lactate metabolism. Our results confirm that the operation of the malate-aspartate shuttle is energy-dependent, driven by delta psi. In the presence of added ammonium ions the removal of lactate was much less impaired by valinomycin, suggesting an energy-independent utilization of lactate under these conditions. The oxidizing effect of ammonium ions on the mitochondrial matrix apparently alleviates the need for energy input for the transfer of reducing equivalents between the cytoplasm and mitochondria. It is concluded that, in the presence of ammonium ions, the transport of lactate hydrogen to the mitochondria is accomplished by malate transfer that is not linked to the electrogenic transport of glutamate across the inner membrane, and, hence, is clearly distinct from the butylmalonate-sensitive, energy-dependent, malate-aspartate shuttle.
Berry
M N
MN
Department of Medical Biochemistry, School of Medicine, Flinders University of South Australia, Adelaide.
Phillips
J W
JW
Gregory
R B
RB
Grivell
A R
AR
Wallace
P G
PG
D000641:Ammonia / Q000494:pharmacology; D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004734:Energy Metabolism; D005943:Gluconeogenesis; D007773:Lactates / Q000378:metabolism*; D019344:Lactic Acid; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008293:Malates / Q000378:metabolism; D008297:Male; D008930:Mitochondria, Liver / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D010168:Palmitates / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D014634:Valinomycin / Q000494:pharmacology
D007773:Lactates; D008293:Malates; D010168:Palmitates; D009243:NAD; D014634:Valinomycin; D001224:Aspartic Acid; D019344:Lactic Acid; D000641:Ammonia; C030298:malic acid