ROLE OF HEPATIC CARBONIC-ANHYDRASE IN DE-NOVO LIPOGENESIS

The role of carbonic anhydrase in de novo lipid synthesis was examined by measuring [1-C-14]acetate incorporation into total lipids, fatty acids and non-saponifiable lipids in freshly isolated rat hepatocytes. Two carbonic anhydrase inhibitors, trifluoro-methylsulphonamide (TFMS) and ethoxozolamide (ETZ) decreased incorporation of C-14 into total lipids. Both fatty acid and non-saponifiable lipid components of the total lipid were inhibited to approximately the same extent by 100 mu M TFMS (29+/-0.3% and 35+/-0.3% of control respectively in replicate studies). However, neither drug significantly affected ATP concentrations or the transport activity of Na+/K+-ATPase, two measures of cell viability. To establish the site of this inhibition, water-soluble C-14-labelled metabolites from perchloric acid extracts of the radiolabelled cells were separated by ion-exchange chromatography. TFMS inhibited C-14 incorporation into citrate, incorporation of C-14 into acetoacetate. Since ATP citrate-lyase, the cytosolic enzyme that catalyses the conversion of citrate into acetyl-CoA, catalyses an early rate-limiting step in fatty acid synthesis, levels of cytosolic citrate may be rate controlling for de novo fatty acid and sterol synthesis. Indeed citrate concentrations were significantly reduced to 37+/-6% of control in hepatocytes incubated with 100 mu M TFMS for 30 min. TFMS also inhibited the incorporation of C-14 from [1-C-14]pyruvate into malate, citrate and glutamate, but not into lactate. This supports the hypothesis that TFMS inhibits pyruvate carboxylation, i.e. since- all of the C-14 from [1-C-14]pyruvate converted into citric acid cycle intermediates must come via pyruvate carboxylase (i.e. rather than pyruvate dehydrogenase). Our findings indicate a role for carbonic anhydrase in hepatic de novo lipogenesis at the level of pyruvate carboxylation.