IGF-I inhibits apoptosis induced by serum withdrawal, but potentiates TNF-alpha-induced apoptosis, in 3T3-L1 preadipocytes

We have previously shown that human preadipocytes in primary culture underg o apoptosis in response to serum deprivation and addition of tumour necrosi s factor alpha (TNF-alpha), and have proposed that regulation of preadipocy te apoptosis in vivo may contribute to the overall control of adipose mass. In the present study we have investigated both pro- and anti-apoptotic fac tors, and the signalling pathways by which they act, in murine 3T3-L1 pread ipocytes. Apoptotic indices (fraction of cells undergoing apoptosis) were d etermined by microscopic examination of acridine orange-stained cells, fluo rescence-activated cell sorting of propidium iodide-stained cells, or phase -contrast video microscopy. Murine 3T3-L1 cells were more susceptible to ap optosis than human preadipocytes. In medium containing 10% newborn calf ser um, the basal apoptotic index was very low (<2%), but the number of apoptot ic cells increased significantly following serum withdrawal (10% after 24 h ). Addition of TNF-<alpha> (6 nM) stimulated apoptosis in both serum-contai ning and serum-free media (apoptotic indices of 12% and 20% respectively af ter 24 h). IGF-I inhibited by approximately 50% the apoptosis induced by se rum withdrawal, but increased by 25% the apoptosis induced by TNF-alpha in serum-free medium. It was shown by using specific inhibitors of lipid and p rotein kinases (LY294002, rapamycin, PD98059, SB203580) that both phosphoin ositide 3-kinase and MAP kinase pathways contribute to the anti-apoptotic a ction of IGF-I on serum-starved cells, while phosphoinositide 3-kinase but not MAP kinase activity is required for the paradoxical pro-apoptotic actio n of IGF-I in the presence of TNF-alpha. We conclude that, in addition to i ts previously described anti-apoptotic action, IGF-I can also be pro-apopto tic in 3T3-L1 cells in the presence of TNF-alpha, and that both the anti- a nd pro-apoptotic effects of IGF-I require the activation of phosphoinositid e 3-kinase.