ATP CITRATE-LYASE AND GLYCOGEN-SYNTHASE KINASE-3-BETA IN 3T3-L1 CELLSDURING DIFFERENTIATION INTO ADIPOCYTES

ATP citrate-lyase (CL), acetyl-CoA carboxylase (ACC) and glycogen synt hase kinase-3 beta (GSK-3 beta) levels were measured in cytosol from 3 T3-L1 cells during differentiation from fibroblasts into fat-cells. Pr otein levels were estimated from immunoblots using specific antisera. Cytosol from confluent cells contain significant amounts of GSK-3 beta , which fell during differentiation of these cells into adipocytes. CL from confluent cells was found to be mostly in the form of a single p rotein band of apparent mass 110 kDa. Levels of CL and ACC increased d uring cell differentiation into adipocytes. During the first 3 days of differentiation, CL migration changed, and it was expressed as a comp lex of protein bands of apparent mass 110 kDa, 113 kDa and 115 kDa. At later stages of differentiation, when these cells had assumed the phe notype of fat-cells, they expressed CL mainly as protein bands of 110 and 113 kDa. When samples containing these bands were treated with alk aline phosphatase, the 113 kDa protein band collapsed into the 110 kDa species. This suggests that the slower-migrating species of CL is a h igher-order phosphorylation state of the same protein. Furthermore, wh en purified CL, mostly expressed as the 110 kDa species, was phosphory lated with cyclic AMP-dependent protein kinase alone or together with GSK-3 and resolved by SDS/PAGE, the phosphorylated CL now migrated mor e slowly as the 113 kDa and 115 kDa forms. CL phosphorylation was horm one-regulated, since, in samples from fat-cells that had the complex t wo-band pattern, when cultured in medium without serum or hormones, CL migration reverted to a single band of 110 kDa, similar to confluent cells. Treatment of these 'down-regulated' cells with insulin rapidly induced substantial amounts of the 113 kDa species, with a concomitant decrease in the 110 kDa species.