Développement de la stimulation de l'adénylate cyclase par l'isoprotérénol et la corticotropine‐β(1–24) au cours de la conversion adipocytaire des cellules 3T3‐F442A en culture

Differentiation‐Dependent Development of Catecholamine and β (1–24) Corticotropin‐Stimulated Adenylate‐Cyclase Activity. Dissociated Effects of Insulin and Induction of β‐Receptors during Adipose Conversion of 3T3‐F442A Cells . 3T3‐F442A preadipocytes [Green, H. & Kehinde, O. (1976) Cell, 7 , 105–113] cloned from 3T3 mouse‐embryo fibroblasts, differentiate in monolayer culture into cells with morphological and biochemical characteristics of adipocytes when they pass from the growing to the resting state. The initiation of differentiation is not spontaneous: cells enter the differentiation program subsequent to prolonged maintenance in the confluent state with an adipogenic factor present in foetal calf serum [Kuri‐Harcuch, W. & Green, H. (1978) Proc. Natl Acad. Sci. USA, 75 , 6107–6109]. The present investigation compares the appearance of hormone sensitivity of the adenylate cyclase system and β‐adrenergic receptors in differentiating (3T3‐F442A) and nondifferentiating (3T3‐C2) cell lines. Concomitant with the changes in shape from very flattened with elongated processes (growing state) to spherical (resting state), exhibited at confluence by the susceptible 3T3‐F442A clone, there is an abrupt rise of isoproterenol‐stimulated adenylate‐cyclase activity which increases from 2‐fold to 15–20‐fold stimulation. This change occurs soon after confluence by day 1 or 2 when foci of rounded cells have appeared in clusters; then isoproterenol‐stimulated adenylate‐cyclase activity remains stable at this plateau of 15–20‐times the basal activity until day 24 when the cells have accumulated enough lipid droplets to gain all the characters of mature adipocytes. 3T3‐C2 cells, grown in 15% foetal‐calf serum, or 3T3‐F442A preadipocytes, grown in serum‐free medium, do not express either the adipocyte phenotype or isoproterenol coupling to their adenylate‐cyclase system. Thus, we conclude that the coupling of isoproterenol to the adenylate‐cyclase system is specific for the adipose conversion; in addition it is an early event which can be used as a primary marker of adipose differentiation. Specific [ 3 H]dihydroalprenolol binding sites increase 7‐fold when preadipocytes [25–30 fmol · (mg protein) −1 ] convert to adipocytes [200–220 fmol · (mg protein) −1 ] after long‐term exposure to 15% foetal‐calf serum in the absence or in the presence of 5 μg insulin/ml. When data are normalized to cells, preadipocytes exhibit only 1200–1600 β‐receptors per cell; during differentiation β‐receptor numbers increase dramatically to 30000–35000 sites per adipocyte. This considerable rise (20–30‐fold) in β‐receptor numbers is quite different from the small increase (only 60–70%) observed during adipose conversion of 3T3‐L1 cells [Lai, E., Rosen, O. M. & Rubin, Ch. S. (1981) J. Biol. Chem. 256 , 12866–12874]. In fact, the non‐converting 3T3‐C2 control cells grown in the presence of foetal calf serum exhibit a 60 % increase of β‐adrenergic sites when they pass from the growing to the resting state. Similarly, 3T3‐F442A and 3T3‐C2 cells, grown in serum‐free defined medium, both exhibit a 70–100% increase of β‐adrenergic receptors between the exponential growth phase and the post‐confluent stage. We conclude that the small increase of [ 3 H]dihydroalprenolol binding sites, observed when 3T3‐C2 or 3T3‐F442A cells reach confluence in a serum‐free medium, is related to the arrest of growth; it only concerns ‘spare binding sites’ which are unrelated to hormone‐stimulated adenylate‐cyclase activity. In contrast, the large increase of β‐adrenergic receptor numbers observed in differentiated cells is largely sufficient per se to explain the dramatic development of catecholamine‐stimulated adenylate‐cyclase activity occurring after differentiation of preadipocytes into adipocytes. Moreover, it seems unlikely that G/F protein exhibits differentiation‐dependent changes since 3T3‐F442A (as well as un‐differentiating 3T3‐C2 control cells) exhibit similar adenylate‐cyclase stimulation by guanine nucleotides and fluoride whatever the culture stages and conditions are. In addition, the number of β‐adrenergic hormone receptors appears to be increased in 3T3‐C2 (or 3T3‐F442A) serum‐free cells by approximately 30% over their counterpart grown in the presence of serum. This observation is attributed to the ‘down regulation’ of β‐adrenergic receptor numbers controlled by catecholamines associated with standard serums. In contrast to catecholamine‐sensitive adenylate‐cyclase activity, the appearance of β(1–24)‐corticotropin polypeptide coupling to the adenylate‐cyclase system of 3T3‐F442A cells is a delayed and progressive phenomenon reaching a maximum value by day 7 or 8 after confluence; at this time adenylate‐cyclase activity of adipocytes is stimulated 5–6‐fold by 10 μM β(1–24)‐corticotropin. Chronic exposure of 3T3‐F442A cell cultures to 5 μg insulin/ml led to a diminished maximal responsiveness of adenylate cyclase to β(1–24)‐corticotropin and prostaglandin E 1 (about a 50% decrease for both hormones); however, the sensitivity of the enzyme to catecholamines remains essentially unaffected as well as adenylate cyclase‐activities stimulated by fluoride and guanine nucleotides. These findings suggest that the setting of hormonal responsiveness of adenylate cyclase is independently programmed and controlled during adipose conversion of 3T3‐F442A cells.