The molecular mechanisms which control the terminal differentiation of mature fat cells remain largely unknown. Various studies have shown that the signalling molecule cyclic AMP (CAMP) can modulate the growth of cells. It has been reported that i n vivo sympathetic innervation exerts an inhibitory influence on the proliferation of rat preadipocytes [l] and that i n v i t r o CAMP promotes the differentiation of primary cultures of rat adipocytes [2]. However studies using preadipocyte cell lines have produced conflicting results showing that CAMP either has no effect upon [3], promotes [4] or inhibits [5] differentiation. In order to investigate this apparent anomaly we have carried out a thorough study of the effect of CAMP on the differentiation of 3T3-F442A preadipocytes. 3T3-F442A fibroblasts were grown to confluence in Dulbecco’s modified Eagle‘s medium (DMEM) containing 10% calf serum. 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 (~90% 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). Similar effects were observed upon the differentiation of cells in DDM. Thus, forskolin (50 pM) , cholera toxin (10 ng/ml) and CPT-CAMP (0.25 mM) decreased the GPDH activity of differentiated cells by ~ 7 0 % and IBMX increased GPDH activity by 21%. These results suggest that a small increase in intracellular CAMP levels, as occurs in the presence of IBMX, promotes differentiation while the larger increases in CAMP levels achieved with the forskolin, cholera toxin and CPT-CAMP treatments inhibit the differentiation process. To confirm that CAMP exerts differential effects upon differentiation, cells were incubated with FCS/insulin medium in the 0.0 L I . . I I . . .d . . .,,.,.1 . ..,,,,.I , ,,I... d , ,.I... * . . .,,d
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Apr 1, 1997
Yannick P Maneuf + 1
Open Access
