Insulin-Mediated Down-Regulation of the Anti-Angiogenic Protein PEDF is mTOR Dependent.

Wang, Binhui; Atherton, Philip J.; Manning, Gillian; Donnelly, Richard

Pigment Epithelium Derived Factor (PEDF) is a potent anti-angiogenic protein that is inversely associated with diabetic microangiopathy. Decreased expression of PEDF has been implicated in proliferative diabetic retinopathy, and we have previously shown that insulin down-regulates expression of PEDF mRNA but the underlying pathways and mechanisms involved are unclear. Human umbilical artery smooth muscle cells were cultured to confluence, serum starved and quiesced in low-serum medium for 24hrs. Cells were then exposed to Insulin 100 nM alone or insulin + inhibitors of PI3K (wortmannin 100nM), MEK/ERK (PD98056 100uM) or mTOR (rapamycin 100nM) for 24hr. Following cell lysis, total RNA was extracted and a cDNA pool created for analysis of PEDF mRNA by real time RT-PCR on a Bio-Rad iCycler. In comparison with untreated cells, when normalized to the housekeeping gene beta2-microglobulin (B2MG), insulin reduced the expression of PEDF mRNA by 39% (n= 12, p<0.001). Neither wortmannin or PD98059 significantly influenced the inhibitory effect of insulin on PEDF mRNA expression: PEDF mRNA relative to B2MG was decreased by 23% (n=8, p=0.021) and 35% (n=8, p=0.04), respectively. In contrast, insulin-mediated suppression of PEDF was abolished in the presence of rapamycin (n=12, p=0.005). Insulin-mediated down-regulation of PEDF expression is potentially important in the development of proliferative retinopathy, and this study has shown that the effect is substantially mediated by the mTOR intracellular signaling pathway.

EPITHELIUM; VASCULAR endothelial growth factors; DIABETES complications; DIABETIC retinopathy; MESSENGER RNA; INSULIN; RAPAMYCIN

Diabetes, 2007, Vol 56, pA212

0012-1797

Academic Journal