Abstract
Insulin-like growth factor-1 (IGF-1) and insulin are known to prevent apoptosis. The signaling network of IGF-1 and insulin occurs via multiple pathways involving different insulin receptor substrates (IRSs). To define their roles in the anti-apoptotic function of IGF-1 and insulin, we established brown pre-adipocyte cell lines from wild-type and IRS knockout (KO) animals. In response to 16 h of serum deprivation, IRS-1-deficient cells showed a significant decrease in response to IGF-1 protection from apoptosis, whereas no changes were observed in the IRS-2, IRS-3, or IRS-4 KO cells. Five hours after serum withdrawal, cells already began to undergo apoptosis. At this early time point, IGF-1 and insulin were able to protect both wild-type and IRS-1 KO cells from death by 85-90%. After a longer period of serum deprivation, the protective ability of insulin and IGF-1 was decreased, and this was especially reduced in the IRS-1 KO cells. Reconstitution of these cells with IRS-1, IRS-2, IRS-3, or IRS-1/IRS-2 chimeras restored the anti-apoptotic effects of IGF-1, whereas overexpression of IRS-4 had no effect at long time points and actually reduced the effect of IGF-1 at the short time point. The biochemical basis of the defect in anti-apoptosis was not dependent on phosphorylation of mitogen-activated protein kinase; whereas phosphoinositide 3-kinase activity was decreased by 30% in IRS-1 KO cells. Akt phosphorylation was slightly reduced in these cells. Phosphorylation of the transcription factors cAMP response element-binding protein and FKHR by IGF-1 and insulin was markedly reduced in IRS-1 KO cells. In addition, both IGF-1 and insulin prevented caspase-3 cleavage in the wild-type cells, and this effect was greatly reduced in the IRS-1-deficient cells. These findings suggest that the IRS proteins may play differential roles in the anti-apoptotic effects of IGF-1 and insulin in brown pre-adipocytes, with IRS-1 being predominant, possibly acting through caspase-3-, CREB-, and FKHR-dependent mechanisms.
Highlights
The pleiotropic effects of IGF-11 and insulin on metabolism, mitogenesis, and cell survival are mediated by a complex net
We have investigated the role of the insulin receptor substrates (IRSs) proteins in the anti-apoptotic function of Insulin-like growth factor-1 (IGF-1) and insulin by using KO cell lines derived from all four IRS KO mice
IRS-1 Plays a Critical Role in the Anti-apoptotic Function of IGF-1—To determine if the brown pre-adipocyte cell lines were sensitive to serum withdrawal-induced apoptosis, we grew the wild-type cells in media deprived from serum in the absence or presence of IGF-1 or insulin for various times (Fig. 1A)
Summary
Apoptosis is a form of programmed cell death, which plays a critical role in controlling cell number and eliminating misplaced cells, and is characterized by chromatin condensation, cytoplasmic blebbing, and DNA fragmentation [10]. Multiple mechanisms have been proposed by which these factors protect cells from apoptosis, including the PI3K/ Akt pathway [12,13,14], the Ras/MAPK pathway [13], Jun Nterminal kinase [15], and p38 MAPK [16]. These pathways lead to phosphorylation of several downstream proteins involved in apoptosis, such as the Bcl-2 family member Bad [17], caspase-9 [18], the forkhead transcription factors [19], CREB [20], NF-B [21], and others. Caspase-3 and the transcription factors CREB and FKHR appear to be downstream mediators of IRS-1, which may mediate the anti-apoptotic function of IGF-1 and insulin
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