Transfer of carbon and hydrogen across the mitochondrial membrane in the control of gluconeogenesis.

Summary According to current concepts, the membrane of rat liver mitochondria is relatively impermeable to oxalacetate, and carbon or hydrogen equivalents for gluconeogenesis are thought to be transported out of the mitochondria as malate and aspartate. Present work is concerned with 1) attempts to elucidate under which metabolic conditions malate or aspartate serve as major carriers of carbon flux for gluconeogenesis in perfused rat liver; 2) the nature of the factors controlling the anion transport systems; and 3) the influence of anion concentration gradients across the mitochondrial membrane on the control of gluconeogenesis. Three different experimental approaches have been employed; 1) addition of specific inhibitors of transport or enzyme systems to the perfused liver; 2) tracer studies with (1- 14 C) lactate, (1- 14 C) pyruvate, and (2- 14 C) acetate; and 3) calculations of the intracellular distribution of metabolites from assumptions of near equilibrium of glutamate-pyruvate and glutamate-oxalacetate transaminases and lactate, malate and s-hydroxybutyrate dehydrogenases. Results of the latter calculations predict a predominantly cytosolic distribution for oxalacetate and α-ketoglutarate and a predominately mitochondrial distribution for aspartate and glutamate. Malate distributions depended on the nature of the gluconeogenic precursor. Malate concentration gradients from mitochondria to cytosol were predicted to be low with lactate but high with pyruvate as substrate. A high malate concentration gradient under the latter conditions is consistent with it being the carrier of both carbon and hydrogen atoms for gluconeogenesis. Addition of butylmalonate, which inhibits a malate:phosphate exchange in isolated mitochondria, resulted in a diminished rate of glucose production from pyruvate, an increased malate concentration gradient from mitochondria to cytosol, an oxidation of the cytosolic NAD-system and an inhibitory interaction in the gluconeogenic sequence at glyceraldehyde-P-dehydrogenase. Gluconeogenesis from lactate and lactate uptake were almost completely inhibited by 0.2 mM amino-oxyacetic acid (an inhibitor of glutamate oxaloacetate transaminase), while gluconeogenesis from pyruvate, and pyruvate uptake were not affected. Since amino-oxyacetic acid does not inhibit lactate dehydrogenase, these results establish that aspartate efflux from mitochondria is associated with gluconeogenesis from lactate while with pyruvate as substrate a transaminase step is not obligatory for gluconeogenesis. Steady state isotope tracer studies revealed equilibration between intermediates within ten minutes. However, isotope equilibration was not achieved between the mitochondrial and cytosolic pools of malate and aspartate. Mitochondrial oxaloacetate did not equilibrate equally between mitochondrial malate and mitochondrial aspartate. The present work supports the concept that exchange of anions across the mitochondrial membrane is an integral feature of gluconeogenesis in rat liver, and that malate transport can be a rate-controlling step in the overall sequence.