Fig 1.
Effects of Ang1-7 in vitro and in vivo.
Ang1-7 dose-response of leptin expression in AT (A, B) and isolated adipocytes (C, D). AT and isolated adipocytes were incubated with Ang1-7 for 24 h. Leptin mRNA expression (A, C) and secreted leptin (B, D) were measured as described in the Materials and Methods. Ang1-7 was administered intra-peritoneally over a 2-day period. Blood serum and peri-renal AT were collected 24 h later. Serum leptin levels (E) and leptin content in peri-renal AT (F) were measured as described in the Materials and Methods. Rat body weights (BW) at the time of blood sampling (G). Each column and bar represents the mean ± SEM of three separate experiments. An asterisk (*) indicates P<0.05 vs. vehicle. Secretion levels of leptin were normalized to total adipocyte protein, and expression of leptin mRNA was normalized to that of β-actin. AT leptin content was normalized to body weight.
Fig 2.
Analysis of receptor types involved in high- and low-dose Ang1-7 regulation of leptin expression.
(A, C) AT and (E) isolated adipocytes were pre-treated with A779, PD123177, D-Pro7Ang1-7 (DA1-7), or candesartan (Cand) for 1 h prior to Ang1-7 addition. Control cells were treated with PBS for 24 h. The cells were incubated for 24 h prior to measuring leptin mRNA expression. (B, D) isolated adipocytes were pre-treated with A779 for 1 h prior to Ang1-7 addition, and incubated for 24 h prior to measuring leptin secretion. Secreted leptin was measured as described in the Materials and Methods of the supplemental data. Each column and bar represents the mean ± SEM of three separate experiments. An asterisk (*) indicates P<0.05 vs. vehicle. Leptin secretion levels were normalized to total adipocyte protein, and expression of leptin mRNA was normalized to that of β-actin.
Fig 3.
Effects of alamandine in vitro and in vivo.
Alamandine dose-response of leptin expression in AT (A, B) and isolated adipocytes (C, D). AT and isolated adipocytes were incubated with alamandine for 24 h and leptin mRNA expression (A, C) and secreted leptin (B, D) were measured as described in the Materials and Methods. Alamandine was administered intra-peritoneally over a 2-day period. Serum leptin levels (E) and leptin content in peri-renal AT (F) were measured as described in the Materials and Methods. Rat body weights (BW) at the time of blood sampling (G). Each column and bar represents the mean ± SEM of three separate experiments. An asterisk (*) indicates P<0.05 vs. vehicle. Secretion levels of leptin were normalized to total adipocyte protein, and the expression of leptin mRNA was normalized to that of β-actin. Leptin content in AT was normalized to body weight.
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.
Fig 5.
iNOS and NO mediate the effect of alamandine on leptin expression.
(A) AT was pre-treated with NG-nitro-arginine methyl ester hydrochloride (L-NAME; 1 mM) for 1 h prior to alamandine or S-nitroso-L-glutathione (GSNO) and incubated for 24 h prior to measuring leptin mRNA expression. (B) AT was pre-treated with 1400w for 1 h prior to alamandine and incubated for 24 h prior to measuring leptin mRNA expression. (C) Isolated adipocytes were cultured with or without alamandine for 24 h prior to measuring iNOS expression by western blotting. The ratio of iNOS to β-actin was calculated based on densitometric quantification of the bands. (D) Isolated adipocytes were cultured with or without alamandine for 24 h prior to measuring NO. NO was measured as described in the Materials and Methods. Levels of NO were normalized to total adipocyte protein. Leptin and iNOS mRNA levels were normalized to β-actin. Each column and bar represents the mean ± SEM of three separate experiments. An asterisk (*) indicates P<0.05 vs. vehicle tissue.
Fig 6.
Alamandine induces PAI-1 expression.
Alamandine dose-response of PAI-1 mRNA expression in AT (A) and of PAI-1 protein expression in isolated adipocytes (B). (A) AT was cultured with or without alamandine for 24 h prior to measuring PAI-1 mRNA expression. (B) Isolated adipocytes were cultured with or without alamandine for 24 h prior to measuring PAI-1 protein expression. PAI-1 mRNA and protein levels were normalized to β-actin. Each column and bar represents the mean ± SEM of three separate experiments. An asterisk (*) indicates P<0.05 vs. vehicle tissue.
Fig 7.
Postulated regulatory mechanism by alamandine of leptin expression in AT.
Alamandine inhibits leptin expression, which is mediated by Gq/11 and cytotoxic signal transduction pathways, ultimately resulting in the induction of NO expression. See text for more detail.