Endocrine Abstracts

The insulin-like growth factors (IGFs) are important regulators of cellular function, with effects on growth, survival and metabolism mediated primarily through the type 1 IGF receptor (IGFIR). Recent work suggested that localisation of IGFIR to a subset of lipid rafts, known as caveolae, may be important for IGF function. In this study we have investigated whether these membrane domains were involved in IGF-I-mediated cell survival by comparing signalling in caveolae-positive fibroblasts (NWTb3) and caveolae-negative hepatocytes (HepG2).

Subcellular fractionation (n=3) and co-immunoprecipitation (n=3) of NWTb3 cells demonstrated that the IGFIR associated with caveolin, a marker for caveolae. Assessment of nuclear morphology demonstrated that 5nM IGF-I was able to protect NWTb3 cells from apoptosis induced by 24hr serum withdrawal, with a reduction from 41.6 plus/minus 1.6% to 6.1 plus/minus 0.4% apoptosis in the presence of IGF-I (p<0.05, n=4). When caveolae were disrupted, using 5mM methyl-cyclodextrin (MCD) to sequester membrane cholesterol, IGF-I-stimulated cell survival was significantly impaired (IGF-I 6.1 plus/minus 0.4%; IGF-I, MCD 27.1 plus/minus 1.0%; n=4), even though methyl-cyclodextrin alone had no effect.

Treatment with LY294002, a PI-3K inhibitor suggested that IGF-induced cell survival was mediated through the PI-3K/PKB pathway (p<0.05, n=4). Immunoblot analysis demonstrated a reduction in IGF-I-mediated activation of PKB in cells pre-treated with methyl-cyclodextrin (n=3), which suggests that methyl-cyclodextrin may impair IGF-induced cell survival by inhibiting activation of PKB.

When experiments were conducted in cells shown to be caveolin-deficient (HepG2), methyl-cyclodextrin had the same effect on IGF-induced PKB phosphorylation (n=3) and cell survival (IGF-I 1.3 plus/minus 0.2%; IGF-I, MCD 2.6 plus/minus 0.2%, n=4) as observed in caveolin-positive NWTb3 cells. This suggests that whilst IGFIR associates with caveolin, the interaction between IGFIR and caveolin may not be obligatory for IGF signalling.