Induction of Adenosine 3′,5′-Monophosphate-Dependent Protein Kinase Subunits during Adipogenesisin Vitro*

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Abstract
Fatty acid metabolism in adipocytes is known to be regulated by the intracellular transducer cAMP. This study was undertaken to determine the temporal and hormonal regulation of cAMP-dependent protein kinase during the differentiation of preadipocyte mesenchymal cells to adipocytes. For this we have used a stable cell line (TAl) in which the undifferentiated preadipocyte acquires adipocyte functions and morphology after growth to confluence. We observed that synthesis of type I and II cAMP-dependent protein kinases was induced during the adipogenic conversion of growth-arrested TAl cells. In preconfluent cells, neither mRNAs encoding regulatory subunits (RI, RIIβ) and catalytic subunit (Cα) nor the peptides themselves were detectable. Within several days of growth arrest at high cell density, mRNAs for RI, RIIβ, and Cα were detectable in total RNA extracted from cell populations. The subunits themselves were detectable in some, but not all, of the cells by indirect immunofluorescence. Immunoblotting of cytosolic extracts indicated that RI and the β-isoform of RII (mol wt = 52,000) were expressed in these cells. Analysis of subunit presence or absence in single cells by immunofluorescence also indicated that kinase subunit expression preceded the accumulation of lipid droplets within the cells. Further, the subunits were predominantly associated with a reticular cytoplasmic structure (Golgi apparatus?) abutting the nucleus. Conversion of TAl cells to adipocytes can be accelerated by indomethacin (125 μM) or dexamethasone (1 μM) treatment, compounds that also enhanced the accumulation of RIIβ, and Cα mRNAs. Within 2–3 days of addition of indomethacin to confluent cultures, RIIβ message content is increased about 20-fold, and protein content is increased about 5-fold relative to those in untreated cultures. Cα mRNA content is increased about 5- fold relative to that in untreated cells. The response to dexamethasone requires 6–7 days, and changes in RIIβ message levels were the most pronounced. We also observed the induction of. mRNAs for the functionally relevant mRNA lipoprotein lipase in indomethacin-treated cells. In addition to this apparent transcriptional regulation of kinase subunit expression, we provide evidence for regulation at the posttranscriptional level. Within a differentiated culture, there exist stem cells that can be selected, will repopulate the dish, and will again differentiate into adipocytes upon growth arrest at high cell density. In preconfluent populations of these stems cells, unlike the preconfluent TAl cells originally plated, both RIIβ and Cα messages were present. However, the subunits themselves were not detectable until after growth arrest at confluence. Therefore, before confluence, kinase subunit messages either were not translated or the peptides themselves were rapidly degraded. During differentiation of these cells, the message content of RIIβ and Cα was increased by dexamethasone and indomethacin, as was observed for adipocytes derived from previously undifferentiated TAl cells. We conclude that transcription of genes for RIIβ, RI, and Cα precedes morphological differentiation of adipocytes and can be regulated by conditions facilitating the differentiation process (Endocrinology123: 2408–2418,1988)