The role of carbonic anhydrase in de novo lipid synthesis was examined by measuring [1-14C]acetate incorporation into total lipids, fatty acids and non-saponifiable lipids in freshly isolated rat hepatocytes. Two carbonic anhydrase inhibitors, trifluoromethylsulphonamide (TFMS) and ethoxozolamide (ETZ) decreased incorporation of 14C into total lipids. Both fatty acid and non-saponifiable lipid components of the total lipid were inhibited to approximately the same extent by 100 microM 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 14C-labelled metabolites from perchloric acid extracts of the radiolabelled cells were separated by ion-exchange chromatography. TFMS inhibited 14C incorporation into citrate, malate, alpha-oxoglutarate and fumarate, but had no effect on incorporation of 14C 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 microM TFMS for 30 min. TFMS also inhibited the incorporation of 14C from [1-14C]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 14C from [1-14C]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.

The incorporation of radioactivity from 14C-labeled compounds into metabolic intermediates and total lipids was examined in 3T3 adipocytes. The heterocyclic sulfonamide carbonic anhydrase inhibitor (SCAI) 6-ethoxyzolamide (ETZ) caused a decrease (42+/-7% of control, IC50 = 2.2+/-1.1 x 10(-7) M) in the incorporation of [14C] bicarbonate into several Krebs cycle intermediates in 3T3-F442A adipocytes. This decrease in pyruvate carboxylase-mediated [14C] carbon fixation was associated with a reduction in fluorometrically determined [citrate] and [malate]. The ability of ETZ to decrease both the incorporation of radioactivity into and the concentrations of Krebs cycle intermediates was not of sufficient magnitude to lower [ATP], but was associated with a decrease in de novo lipogenesis from [14C]glucose. De novo lipogenesis was also inhibited to a similar extent by trifluormethanesulfonamide, an aliphatic SCAI, which suggests that the effects are mediated by carbonic anhydrase. ETZ did not inhibit de novo lipogenesis from [14C]glutamine (12.38+/-1.068 nmol/mg protein, ETZ; 12.5+/-0.846 nmol/mg protein, DMSO). This suggests that ETZ inhibition of lipogenesis involves an inhibitory effect on pyruvate carboxylase as opposed to acetyl CoA carboxylase, because the incorporation of glutamine into lipids does not involve pyruvate carboxylase. Decreased de novo lipogenesis was also observed by incubating cultures in media that contained 1 mM bicarbonate (atmosphere:100% humidified air) rather than 25 mM bicarbonate (atmosphere: 95% humidified air/5% CO2). This suggests that exogenous CO2/bicarbonate may be required to sustain maximal rates of de novo lipogenesis. Because these results implied that CA V, the mitochondrial isoform of carbonic anhydrase, might be present in adipocytes, CA V levels were measured by immunoblotting. Mitochondrial preparations of adipocytes and liver were found to contain similar concentrations of CA V. Unlike adipocyte CA III, CA V concentrations were not significantly different in lean and obese Zucker rats. However, CA V levels were ninefold higher in differentiated 3T3-F442A adipocytes compared to undifferentiated adipoblasts. Our data indicate that CA V is relatively abundant in adipocyte mitochondria and exhibits differentiation-dependent expression like pyruvate carboxylase and the cytosolic isozymes CA II and CA III. The possible roles of CA II and CA V in pyruvate carboxylation are discussed.

Primary rat adipocytes cultured in basement membrane component gels migrated and organized into large, three-dimensional, multicellular clusters. Gross morphological changes seen during this reorganization are described. The rate of cluster formation decreased with age of the rats and was stimulated by insulin in older, but not in younger rats. Echistatin, a disintegrin, partially inhibited the formation of multicellular clusters in a concentration-dependent fashion (50% inhibitory concentration approximately 10 nM). The original extracellular matrix was initially remodeled and eventually destroyed by the time large multicellular clusters were observed. This implied that one or more matrix-degrading protease(s) were being secreted. Adipocyte-conditioned medium was found to contain a divalent cation-sensitive gelatinase activity at approximately 72 and/or approximately 62 kDa. The elution profile of this activity from gelatin-Sepharose 4B was similar to matrix metalloproteinase 2 (MMP-2, a 72-kDa matrixin with a 62-kDa mature form), and the dimethyl sulfoxide eluant from these columns contained MMP-2 immunoreactivity. MMP-2 concentration and activity were greater in conditioned medium from young than from older animals; however, insulin did not affect the amount of MMP-2 in adipocyte-conditioned media. The matrixin inhibitor 1,10-phenanthroline not only blocked gelatinase activity in zymograms but also prevented extracellular matrix remodeling and destruction, as well as adipocyte migration and the formation of cell-cell contacts in adipocyte cultures. These observations are consistent with the hypothesis that the matrixin MMP-2 is secreted by adipocytes. Whereas matrixin activity alone may not be sufficient for the formation of multicellular clusters, the data indicate that it may have a requisite role in this process.

In peripheral tissues, carbonic anhydrase (CA) inhibition secondarily decreases the anaplerotic activity of pyruvate carboxylase activity leading to a decline in citric acid cycle intermediates and glutamate. In view of the important role of pyruvate carboxylase in the brain, we examined the effects of CA inhibition on pyruvate-carboxylase-mediated [¹⁴C]CO₂ fixation in cultured astrocytes from postnatal rat brains. Incubation with H[¹⁴C]O3̄ led to radiolabeling of metabolites found both in the cells and in the medium. These were separated by ion exchange chromatography for identification. Ethoxyzolamide (ETZ), a sulfonamide CA inhibitor (SCAT) with a heterocyclic side group, caused a 43–73% decrease in cell lysate [α-ketoglutarate] and ¹⁴C incorporation into major products of pyruvate carboxylation in the cell lysates and cell medium (i.e., released products). Half-maximal inhibition of [¹⁴C]CO₂ fixation was observed between 1 and 3 x l0^–7M. This is similar to the IC₅₀ value for ETZ inhibition of events in other cells that are thought to be mediated by CA. Inhibition was also observed with trifluor-methanesulfonamide, an aliphatic SCAI, providing further evidence that this effect is mediated by CA. Western blot analysis using isozyme-specific anti-sera indicated that astrocytes contain CA II, a cytosolic isozyme, but CA in, CA IV and CA V could not be detected. This finding is unusual since the effects of SCAIs on pyruvate carboxylation in other tissues have been attributed to inhibition of the intramitochondrial isozyme, CA V. [¹⁴C]CO₂ fixation was also decreased by lowering media [pyruvate] or by addition of 5 mM acetoacetate. It is hypothesized that SCAIs may inhibit pyruvate carboxylation in astrocytes by limiting the supply of bicarbonate to this enzyme while ketone bodies, by inhibiting glucose oxidation, may limit the supply of pyruvate. Interestingly, both SCAIs and ketogenic diets are used to treat adolescent forms of epilepsy. The possibility that these treatments might ultimately work by affecting anaplerotic pyruvate carboxylase activity in the brain is discussed.

Carbonic anhydrase (CA) was examined in two adipocyte cell lines, 3T3-L1 and 3T3-F442A. Both CA III and non-CA III activities, measured by 18O mass spectrometry, were present in 3T3-L1 and 3T3-F442A adipocytes; however, no CA activity was detected in 3T3 preadipocytes of either line. These observations were supported by immunoblot experiments employing CA III and CA II isoform-specific antisera. CA III, a major protein in rodent and murine adipocytes, and CA II, another isoform known to be present in adipose tissue, were observed only in the differentiated 3T3 adipocytes. The differentiation-dependent expression of these isozymes may imply an adipocyte-related role for CA. Compared with cultures maintained in the absence of insulin, 3T3 adipocytes maintained in the presence of insulin exhibited 65-90% lower concentrations of CA III. CA II was unaffected. This negative effect of insulin on CA III may explain the metabolic regulation of adipose CA III observed in vivo. After media changes, 3T3 adipocyte cultures rapidly lower media pH, which in turn lowers the bicarbonate/CO2 of bicarbonate/CO2-buffered media. Cultures maintained at low pH displayed 50-90% lower concentrations of CA II and CA III. Similarly, cultures maintained in a low bicarbonate/CO2 media (GibCO2-I medium containing 1 mM bicarbonate under an atmosphere of 100% humidified air) displayed 30-50% lower CA II and CA III concentrations. Thus CA II and CA III concentrations are influenced by pH and bicarbonate/CO2. Neither effect, the pH or the GibCO2-I media effect, was associated with changes in the concentration of pyruvate carboxylase or ATP citrate lyase (2 markers of adipocyte differentiation). Because the regulation by pH and bicarbonate/CO2 may be relatively selective for CA in adipocytes, a simple method for reducing the concentration/activity of CA in 3T3 adipocytes is described that may be a useful tool for studies on the physiological role of the enzyme.

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Ca2+-mobilizing and cAMP-dependent hormones rapidly increase sodium, potassium-dependent adenosine triphosphatase (Na+/K +-ATPase)-mediated transport in rat hepatocytes. To explore the possible role of protein phosphatases in these responses we used a protein phosphatase inhibitor, okadaic acid. Okadaic acid stimulation of ouabain-sensitive SrRb+-uptake was maximal between two and three minutes and displayed an ECs0 of 41 + 1 nM. Inhibition of Na÷/H + exchange with an amiloride analog abolished the response to insulin, but had no effect on okadaic acid-mediated stimulation of Na+/K÷-ATPase transport. In hepatocytes metabolically-radiolabeled with 32Pi, okadaic acid stimulated the incorporation of radioactivity into several 95 kDa peptides, one of which reacted with anti-LEAVE peptide antisera, that recognizes Na+/K÷-ATPase 0t-subunits. In other experiments Na÷/K+-ATPase was immunoprecipitated from detergent-solubilized membrane fractions of metabolically-radiolabeled cells with an antisera to purified rat kidney Na÷/K+-ATPase. A 95 kDa phosphoprotein was immunoprecipitated using anti-Na+/K÷-ATPase antisera, but not by preimmune serum. Okadaic acid stimulated incorporation of radioactivity into this band by 220 _+ 28%. These findings provide support for the hypothesis that rapid stimulation of hepatic Na+/K÷-ATPase by hormones may be related to protein kinase/phosphatase-mediated changes in the phosphorylation state of the Na+/K+-ATPase ct-subunit.

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