UBC Theses and Dissertations
Mechanisms of gluconeogenic activation in the rainbow trout liver Suarez, Raul Kamantigue
Experiments were conducted using isolated mitochondria, enzyme (pyruvate carboxylase), and cells from rainbow trout liver to study some of the mechanisms which may be involved in the regulation of gluconeogenesis in this animal. On the basis of these studies, the following were found: 1. Mitochondria prepared from rainbow trout liver consistently display high respiratory control and ADP/O ratios. These appear to possess a complete Krebs cycle, since pyruvate, palmitoyl L-carnitine, citrate, and various Krebs cycle intermediates can all be oxidized. Rapid oxidation of pyruvate and palmitoyl L-carnitine requires the presence of malate. Oxidation of palmitoyl L-carnitine appears to inhibit pyruvate oxidation. Malate stimulates alphaketoglutarate oxidation while aspartate inhibits glutamate oxidation, indicating the presence of malate-alphaketoglutarate and glutamate-aspartate carriers. 2. Palmitoyl DL-carnitine inhibits ¹⁴CO₂ production from 1-[¹⁴C]-pyruvate and from 1-[¹⁴C]-alanine by mitochondria from rainbow trout liver. The inhibitory effect occurs in both respiratory states III and IV. Fixation of H¹⁴CO₃ into acid-stable products by intact mitochondria requires pyruvate and ATP and is inhibited by sodium arsenite. This inhibitory effect is completely abolished by acetyl DL-carnitine. It is proposed that under these conditions. oxidation of palmitoyl DL-carnitine results in inhibition of pyruvate dehydrogenase while oxidation of acetyl DL-carnitine results in activation of pyruvate carboxylase in intact rainbow trout liver mitochondria. 3. Pyruvate carboxylase from rainbow trout liver has a pH optimum of about 8.0, possesses an absolute requirement for activation by acetylCoA, and prefers MgATP over other nucleoside triphosphates. K⁺ causes a decrease in the apparent Km for HCO₃⁻. AcetylCoA activation shows positive cooperativity with Ka = 0.072 mM and nH = 1.78 at pH 7.7, 2.5 mM free Mg²⁺, 100 mM K⁺, and saturating concentrations of substrates. A high acetylCoA concentration causes a decrease in the apparent Km values for MgATP and HCO₃⁻, and a biphasic double reciprocal plot with pyruvate as the varied substrate. MgADP and AMP are competitive inhibitors with respect to MgATP. The enzyme shows a "U-type" response to the adenylate energy charge and retains considerable activity throughout a wide range of energy charge values. It is proposed that intramitochondrial acetylCoA concentration and the adenylate energy charge control,the rate of pyruvate carboxylation in vivo. 4. Isolated hepatocytes from rainbow trout liver are capable of net synthesis of glucose from U-[¹⁴C]-lactate. Gluconeogenesis from lactate is stimulated by palmitate, glucagon, and cAMP, and inhibited by 3-mercaptopicolinic acid. Treatment of hepatocytes with glucagon and cAMP at the concentrations which stimulate gluconeogenesis from lactate results in inhibition of pyruvate kinase activity. Although maximum enzyme activity is unaffected, the ratios of activity at low PEP concentration to activity at high PEP concentration are depressed by such treatments. These results confirm those of previous studies which indicate that fatty acid oxidation may stimulate gluconeogenesis in rainbow trout liver. cAMP may act as the intracellular messenger of glucagon; gluconeogenic activation by this hormone probably involves pyruvate kinase inactivation in vivo which may occur as a result of a cAMP-dependent phosphorylation of the enzyme. A regulatory scheme is proposed in which gluconeogenic activation results from inhibition of pyruvate oxidation and stimulation of pyruvate carboxylation by fatty acid oxidation and inhibition of futile cycling between pyruvate and PEP by glucagon through a cAMP-mediated inactivation of pyruvate kinase.
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