Changes in the expression of guanine nucleotide‐binding proteins during differentiation of 3T3‐F442A cells in a hormonally defined medium

Abstract

Using specific antisera, the expression of the G protein α subunits, G8, gi1, Gi2, Gi3, and G0, were determined in 3T3‐F442A cells during their differentiation to adipocytes in a hormonally defined medium. Differentiation caused distinct increases in the expression of two Gs isoforms and decreases in the expression of both Gi2 and Gi3. Differentiation also resulted in a 2‐ to 4‐fold increase in forskolin‐stimulated adenylyl cyclase activity and a 15‐fold increase in the response of cells to a β‐adrenergic agonist. The increase in Gs expression was also observed, to a lesser degree, in cells maintained at confluence under conditions where morphological conversion was negligible and the decreased expression of Gi2 and Gi3 and the increased β‐adrenergic responsiveness did not occur.

Growth Hormone-dependent Differentiation of 3T3-F442A Preadipocytes Requires Janus Kinase/Signal Transducer and Activator of Transcription but Not Mitogen-activated Protein Kinase or p70 S6 Kinase Signaling

“…Previous work has shown that differentiation of 3T3-F442A preadipocytes can be induced in a serum-free medium (6,13). GH, insulin, and EGF are essential components of this medium, with other factors exerting modulatory influences.…”

“…Confluent cultures were washed three times in phosphate-buffered saline and incubated in a defined differentiation medium (DDM) (13). Under these conditions, at least 70% of the cells exhibited adipocyte morphology and were positive for Oil Red O staining after 6 -10 days.…”

Subcellular localization of G-proteins in primary-cultured mouse preadipocytes and adipocytes

“…In mouse adipocyte membranes, the major G-proteins identified by Western blotting, ADP ribosylation with bacterial toxins, and Northern blotting are the long and short forms of G s a and G i a1, -2, and -3 [Bégin-Heick, 1990, 1992Gettys et al, 1991Gettys et al, , 1995. By contrast, in the clonal adipocyte lines HGFu, Ob17 [McFarlane-Anderson et al, 1993] and 3T3-F442A cells [Kilgeour and Anderson, 1993], G i a1 was not detected.…”

Cyclic Amp Modulates Adipogenesis in 3t3-F442a Cells

“…Confluent cultures were induced to differentiate by replacing the growth medium with either DMEM containing 10% foetal calf serum (FCS) and 5 pg/ml insulin or with a defined differentiation medium (DDM) containing growth hormone ( 2 nM), insulin (1.8 pM) , T, (0.1 ng/ml) , EGF (50 ng/ml) and other factors as previously described [6] along with the drugs being tested. After three days the drugs were removed and after a further three days lipid filling of the cells was assessed by staining with Oil Red 0 and the activity of a-glycerophosphate dehydrogenase (GPDH), a marker for adipocyte differentiation, was measured [6].Initial studies demonstrated that, during differentiation of cells with FCS/insulin, raising intracellular CAMP levels with forskolin ( 5 0 pM)' cholera toxin (10 ng/ml) or CPT-CAMP (0.25 mM) severely attenuated lipid filling and GPDH activity (~9 0 % decrease relative to control cells).Dideoxyforskolin (50 pM) , an inactive analogue, had no effect on the extent of differentiation. In contrast, inclusion of the phosphodiesterase inhibitor isobutylmethyl xanthine (IBMX, 500 pM) in the FCS/insulin medium promoted differentiation (88% increase in GPDH activity).…”

“…Confluent cultures were induced to differentiate by replacing the growth medium with either DMEM containing 10% foetal calf serum (FCS) and 5 pg/ml insulin or with a defined differentiation medium (DDM) containing growth hormone ( 2 nM), insulin (1.8 pM) , T, (0.1 ng/ml) , EGF (50 ng/ml) and other factors as previously described [6] along with the drugs being tested. After three days the drugs were removed and after a further three days lipid filling of the cells was assessed by staining with Oil Red 0 and the activity of a-glycerophosphate dehydrogenase (GPDH), a marker for adipocyte differentiation, was measured [6].…”