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 a cids 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 l ipids. Both fatty acid and non-saponifiable lipid components of the to tal 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 stu dies). However, neither drug significantly affected ATP concentrations or the transport activity of Na+/K+-ATPase, two measures of cell viab ility. To establish the site of this inhibition, water-soluble C-14-la belled 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 con version of citrate into acetyl-CoA, catalyses an early rate-limiting s tep in fatty acid synthesis, levels of cytosolic citrate may be rate c ontrolling for de novo fatty acid and sterol synthesis. Indeed citrate concentrations were significantly reduced to 37+/-6% of control in he patocytes 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 fro m [1-C-14]pyruvate converted into citric acid cycle intermediates must come via pyruvate carboxylase (i.e. rather than pyruvate dehydrogenas e). Our findings indicate a role for carbonic anhydrase in hepatic de novo lipogenesis at the level of pyruvate carboxylation.