Fig 4. Analysis of receptor types and signal transduction involved in alamandine regulation of leptin expression.

(A) AT and (B) isolated adipocytes were pre-treated with A779, PD123177 (PD), D-Pro⁷Ang1-7 (DA1-7), or candesartan (Cand) for 1 h prior to 24 h treatment with alamandine and subsequent analysis of leptin mRNA expression. Control cells were treated with PBS for 24 h. (C) AT was pre-treated with pertussis toxin (PTX) for 3 h, YM2590, or U73122 for 1 h prior to 24 h treatment with alamandine and subsequent analysis of leptin mRNA expression. (D) AT was pre-treated with PP2, SB239063 (SB), BAY11-7082 (BAY), or AG490 (AG) for 1 h prior to alamandine (1 nM) treatment and measurement of leptin mRNA expression 24 h later. Leptin mRNA levels were normalized to β-actin. (E) c-Src, (F) p38 MAP kinase, and (G) IκBα activation over time in AT. AT was incubated with alamandine (1 nM) for the indicated times to measure protein phosphorylation by western blotting. The ratio of phospho-protein to total protein was calculated based on densitometric quantification of the bands. Each column and bar represents the mean ± SEM of three separate experiments. An asterisk (*) indicates P<0.05 vs. vehicle tissue. The leptin mRNA levels were normalized to β-actin.