DIFFERENTIATION-DEPENDENT EXPRESSION OF CARBONIC ANHYDRASE-II AND ANHYDRASE-III IN 3T3 ADIPOCYTES

Carbonic anhydrase (CA) was examined in two adipocyte cell lines, 3T3- Ll and 3T3-F442A. Both CA III and non-CA III activities, measured by O -18 mass spectrometry, were present in 3T3-Ll and 3T3-F442A adipocytes ; however, no CA activity was detected in 3T3 preadipocytes of either line. These observations were supported by immunoblot experiments empl oying CA III and CA II isoform-specific antisera. CA III, a major prot ein in rodent and murine adipocytes, and CA II, another isoform known to be present in adipose tissue, were observed only in the differentia ted 3T3 adipocytes. The differentiation-dependent expression of these isozymes may imply an adipocyte-related role for CA. Compared with cul tures 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 viv o. 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 concentrat ions of CA II and CA III. Similarly, cultures maintained in a low bica rbonate/CO2 media (GibCO2-I Medium containing 1 mM bicarbonate under a n atmosphere of 100% humidified air) displayed 30-50% lower CA II and CA III concentrations. Thus CA II and CA III concentrations are influe nced by pH and bicarbonate/CO2. Neither effect, the pH or the GibCO2-I media effect, was associated with changes in the concentration of pyr uvate carboxylase or ATP citrate lyase (2 markers of adipocyte differe ntiation). Because the regulation by pH and bicarbonate/CO2 may be rel atively selective for CA in adipocytes, a simple method for reducing t he concentration/activity of CA in 3T3 adipocytes is described that ma y be a useful tool for studies on the physiological role of the enzyme .