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Inhibition of Gluconeogenesis by Butylmalonate in Perfused Rat Liver

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      The role of mitochondrial anion exchange reactions in the control of gluconeogenesis in perfused livers from fasted rats was investigated by using 2-n-butylmalonate to inhibit malate transport across the mitochondrial membrane. Glucose production from pyruvate or lactate was inhibited 50% to 80% by 5 mm butylmalonate. Oleate (0.5 or 1.5 mm) increased glucose production from pyruvate from 82 to 218 µmoles per 100 g, body wt, per hour, while butylmalonate decreased the rate of gluconeogenesis observed in the presence of oleate to 130 µmoles per 100 g, body wt, per hour. Uptake of substrates and oxygen consumption by the liver were only slightly inhibited, while production of ketone bodies was stimulated both in the presence and absence of oleate. Measurements of the tissue contents of intermediates of the gluconeogenic sequence with pyruvate as substrate identified sites of interaction at the pyruvate carboxylase, glyceraldehyde-3-P dehydrogenase and phosphofructokinase steps.
      Calculated estimates of the intracellular distribution of malate were consistent with the hypothesis that the primary effect of butylmalonate was inhibition of malate transport, resulting in an increased concentration gradient of malate between mitochondria and cytosol. Since malate egress from the mitochondria provides both the carbon and reducing equivalents required for glucose production from pyruvate, diminished malate transport results directly in a decreased rate of gluconeogenesis. A relative deficiency of reducing equivalents in the cytosol caused by competition between lactate dehydrogenase and glyceraldehyde-P dehydrogenase for the available NADH in the cytosol accounts for the inhibitory site of interaction at the latter step. The activational interaction at phosphofructokinase is interpreted as due to a redistribution of citrate into the mitochondria accompanying the altered malate concentration gradient. It is postulated that an increased rate of recycling between fructose diphosphate and fructose-6-P, resulting in a wasteful use of ATP, accounts for the maintenance of high oxygen consumption rates despite decreased net glucose formation after butylmalonate addition.
      Flux through the citric acid cycle in livers perfused with pyruvate was calculated to be 230 µmoles per 100 g, body wt, per hour. This rate was unchanged by addition of butylmalonate in the absence of oleate. In the presence of 1.5 mm oleate, flux through the mitochondrial malate dehydrogenase was in the direction of malate synthesis, and the activity of the citric acid cycle was calculated to be 320 µmoles per 100 g, body wt, per hour. Further addition of butylmalonate decreased citric acid cycle flux to 210 µmoles per 100 g, body wt, per hour.
      The present work provides substantial support to 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 over-all sequence.

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