Resistance to Apoptosis in Preadipocytes with Knockout of Both the Insulin and IGF-1 Receptors.

Boucher, Jeremie; Macotela, Yazmin; Kriauciunas, Kristina; Kahn, C. R.

Insulin and IGF-1 are potent inducers of cell proliferation, growth and survival. Despite this, studies in C. elegans and Drosophila, as well as mice with heterozygous whole body knockout (KO) of the IGF-1 receptor or fat specific KO of the insulin receptor, show that disruption of insulin/IGF1 signaling can result in increased lifespan and resistance to various apoptotic stressors. To better understand the role of these receptors in the apoptosis pathway, we created immortalized brown preadipocytes cells with knockout of both the insulin receptor (IR) and the IGF-1 receptor (IGF1R) by expression of adenoviral Cre in preadipocytes from IR and IGFR lox/lox mice. Surprisingly, IR/IGF1R double KO cells (DKO) did not display any decrease in proliferation rate (doubling time = 9.9 ± 0.8 h for WT cells and 10.1± 0.7 h for DKO cells). DKO cells also showed significant resistance to apoptosis in under both normal and stress conditions. Under standard culture conditions with 10% fetal growth serum, only 3.4 ± 0.7% of DKO cells were apoptotic compared to 6.1 ± 1.0% of WT cells as determined by FACS analysis of cells stained with propidium iodine and annexin V. ELISA assay of mono and oligonucleosomes also revealed an almost 90% reduction in DNA fragmentation in DKO compared to WT cells. With serum deprivation, there was a time dependent increase in apoptosis in both cell lines, but with an 80% lower rate of apoptosis in DKO cells. DKO cells also showed a dramatic reduction in activation of caspase-3, the main executioner of apoptosis. This was due to reduction in both of the major pathways involved in caspase activation, i.e., the extrinsic pathway induced by activation of death-inducing receptors such as TNFα and the intrinsic pathway induced by growth factor deprivation, DNA damage, oxidative stress, and hypoxia. Western blot analysis revealed that DKO cells also had a two fold decrease in the expression of the pro-apoptotic protein Bax and a 2 fold increase in the anti-apoptotic protein Bc12. In summary, disruption of insulin and IGF1 receptor signaling has a profound effect to protect against apoptosis, and this may play an important role in the increased stress resistance and lifespan observed with these pathways.

APOPTOSIS; FAT cells; INSULIN receptors; INSULIN-like growth factor-binding proteins; LABORATORY mice; CELL proliferation

Diabetes, 2007, Vol 56, pA511

0012-1797

Academic Journal