Insulin and angiotensin II are additive in stimulating TGF-β1 and matrix mRNAs in mesangial cells

Insulin and angiotensin II are additive in stimulating TGF-β1 and matrix mRNAs in mesangial cells. Angiotensin II (Ang II) and insulin are implicated in the mesangial cell hypertrophy and excessive accumulation of mesangial matrix seen in glomerulosclerosis. Therefore, the effects of Ang II with and without insulin on mRNA levels of several important extracellular matrix genes and transforming growth factor beta-1 (TGF-β1) were examined. Ang II alone (1 µM) added to quiescent, murine mesangial cells in serum-free, insulin-free media slightly but not significantly increased TGF-β1, fibronectin, collagen I, collagen IV and laminin message levels. The slight elevations in message expression were reversed by losartan, suggesting that these modest effects are mediated by the AT-1 receptor. Ang II alone also had no significant effects on TGF-β1 and extracellular matrix message levels in quiescent rat mesangial cells. In contrast, significant increases in mRNA for collagen I (6-fold), collagen IV (4-fold), fibronectin 1 (4-fold) and TGF-β1 (2-fold) were seen with insulin alone (10−6 M) in rat mesangial cells, and a dose-response effect could be demonstrated for insulin (10−9 to 10−6 M). Ang II plus insulin further significantly increased collagen I (9-fold), collagen IV (9-fold), fibronectin 1 (5-fold) and TGF-β1 (3-fold) message expression. These effects were partially reversed in the presence of losartan. The Northern analyses were supported by measurements of active and total TGF-β1 activity (pg/ml/5 × 106 cells): 1145 ± 76 and 1960 ± 199, serum free control; 1121 ± 92 and 1932 ± 214, Ang II (10−6 M); 4589 ± 103 (P < 0.001 vs. control) and 11071 ± 1952 (P < 0.01 vs. control), insulin (10−6 M); and 6881 ± 183 (P < 0.001 vs. control) and 16626 ± 1435 (P < 0.01 vs. control), insulin plus Ang II. These results suggest that insulin, itself, significantly increases TGF-β1 and extracellular matrix gene expression in rat mesangial cells. Ang II alone has modest effects, while Ang II and insulin have additive effects. To explain the mechanism of these additive effects, we investigated the action of Ang II on insulin signaling and the effect of insulin on Ang II AT1 receptor mRNA expression. Ang II did not enhance insulin-induced insulin receptor substrate-1 (IRS-1) phosporylation or phosphatidylinositol3 (PI-3) kinase activity, but did enhance insulin-induced mitogen activated protein (MAP) kinase activity. Insulin increased message levels of AT1 receptor by twofold. These results suggest that enhancement of MAP kinase activity and AT1 receptor regulation by insulin may contribute to the additive effects of insulin and Ang II in mesangial cells