Atp Citrate-Lyase and Glycogen-Synthase Kinase-3-Beta in 3t3-L1 Cells During Differentiation into Adipocytes

Benjamin, W. B., Pentyala, S. N., Woodgett, J. R., Hod, Y., Marshak, D. (1994) Atp Citrate-Lyase and Glycogen-Synthase Kinase-3-Beta in 3t3-L1 Cells During Differentiation into Adipocytes. Biochemical Journal, 300. pp. 477-482. ISSN 0264-6021

URL: http://www.ncbi.nlm.nih.gov/pubmed/7911658
DOI: 10.1042/bj3000477

Abstract

ATP citrate-lyase (CL), acetyl-CoA carboxylase (ACC) and glycogen synthase kinase-3 beta (GSK-3 beta) levels were measured in cytosol from 3T3-L1 cells during differentiation from fibroblasts into fat-cells. Protein 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 protein band of apparent mass 110 kDa. Levels of CL and ACC increased during cell differentiation into adipocytes. During the first 3 days of differentiation, CL migration changed, and it was expressed as a complex of protein bands of apparent mass 110 kDa, 113 kDa and 115 kDa. At later stages of differentiation, when these cells had assumed the phenotype of fat-cells, they expressed CL mainly as protein bands of 110 and 113 kDa. When samples containing these bands were treated with alkaline phosphatase, the 113 kDa protein band collapsed into the 110 kDa species. This suggests that the slower-migrating species of CL is a higher-order phosphorylation state of the same protein. Furthermore, when purified CL, mostly expressed as the 110 kDa species, was phosphorylated with cyclic AMP-dependent protein kinase alone or together with GSK-3 and resolved by SDS/PAGE, the phosphorylated CL now migrated more slowly as the 113 kDa and 115 kDa forms. CL phosphorylation was hormone-regulated, since, in samples from fat-cells that had the complex two-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.

Item Type: Paper
Uncontrolled Keywords: DEPENDENT PROTEIN-KINASE RABBIT SKELETAL-MUSCLE RAT ADIPOSE-TISSUE ACETYL-COA CARBOXYLASE FATTY-ACID SYNTHESIS PHOSPHORYLASE-KINASE S6 KINASE INSULIN PURIFICATION SEQUENCE
Subjects: organs, tissues, organelles, cell types and functions > cell types and functions > cell functions > cell differentiation
organs, tissues, organelles, cell types and functions > organs types and functions > metabolism
CSHL Authors:
Communities: CSHL labs
Depositing User: Matt Covey
Date Deposited: 26 Aug 2015 20:15
Last Modified: 26 Aug 2015 20:15
PMCID: PMC1138187
Related URLs:
URI: http://repository.cshl.edu/id/eprint/31409

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