Angiotensin II Reduces Lipoprotein Lipase Expression in Visceral Adipose Tissue via Phospholipase C β4 Depending on Feeding but Increases Lipoprotein Lipase Expression in Subcutaneous Adipose Tissue via c-Src

<div><p>Metabolic syndrome is characterized by visceral adiposity, insulin resistance, high triglyceride (TG)- and low high-density lipoprotein cholesterol-levels, hypertension, and diabetes—all of which often cause cardiovascular and cerebrovascular diseases. It remains unclear, however, why visceral adiposity but not subcutaneous adiposity causes insulin resistance and other pathological situations. Lipoprotein lipase (LPL) catalyzes hydrolysis of TG in plasma lipoproteins. In the present study, we investigated whether the effects of angiotensin II (AngII) on TG metabolism are mediated through an effect on LPL expression. Adipose tissues were divided into visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) for comparison. AngII accelerated LPL expression in SAT but, on the contrary, suppressed its expression in VAT. In both SAT and VAT, AngII signaled through the same type 1 receptor. In SAT, AngII increased LPL expression via c-Src and p38 MAPK signaling. In VAT, however, AngII reduced LPL expression via the G_q class of G proteins and the subsequent phospholipase C β4 (PLCβ4), protein kinase C β1, nuclear factor κB, and inducible nitric oxide synthase signaling pathways. PLCβ4 small interfering RNA experiments showed that PLCβ4 expression is important for the AngII-induced LPL reduction in VAT, in which PLCβ4 expression increases in the evening and falls at night. Interestingly, PLCβ4 expression in VAT decreased with fasting, while AngII did not decrease LPL expression in VAT in a fasting state. In conclusion, AngII reduces LPL expression through PLCβ4, the expression of which is regulated by feeding in VAT, whereas AngII increases LPL expression in SAT. The different effects of AngII on LPL expression and, hence, TG metabolism in VAT and SAT may partly explain their different contributions to the development of metabolic syndrome.</p></div

Effects of alamandine <i>in vitro</i> and <i>in vivo</i>.

<p>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.</p

PKCβ1 and p38 MAP kinase activation by AngII in VAT and SAT, respectively.

<p>(A) PKCβ1 activation over time in VAT. VAT was incubated with AngII (1 μM) for the indicated times to measure PKCβ1 phosphorylation. The ratio of phospho-PKCβ1 to total PKCβ1 was calculated based on densitometric quantification of the bands. (B) p38 MAP kinase activation over time in SAT. SAT was incubated with AngII (1 μM) for the indicated times to measure p38MAP kinase. The ratio of phospho-p38 to total p38 was calculated based on densitometric quantification of the bands. Each column and bar represents the mean ± SEM for three separate experiments. An asterisk (*) indicates <i>p</i><0.05 vs. time 0.</p

Postulated regulatory mechanism by AngII of LPL expression in VAT and SAT.

<p>AngII stimulates LPL expression in SAT and, conversely, inhibits its expression in VAT. In both cases, the AngII-induced actions are mediated by the same ATR1 but different G proteins and intracellular signaling pathways. In VAT, PLCβ4 expression is regulated by feeding/fasting cycle and is responsible for the inhibitory role of AngII on LPL expression. See text for more detail.</p

Analysis of receptor types involved in high- and low-dose Ang1-7 regulation of leptin expression.

<p>(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.</p

PKC, NFκB, and iNOS mediate the effect of AngII on LPL expression in VAT.

<p>(A) IκBα phosphorylation over time in VAT. VAT was cultured with AngII (1 μM) for the indicated times to measure IκBα phosphorylation with western blotting. The ratio of phospho IκBα to total IκBα was calculated based on densitometric quantification of the bands. (B, C) iNOS expression in VAT and visceral adipocytes. VAT and isolated visceral adipocytes were cultured with or without AngII (1 μM) for 24 h to measure iNOS expression by western blotting. Duplicate samples in each group were processed for western blotting. The ratio of iNOS to β-actin was calculated based on densitometric quantification of the bands (B, VAT; C, visceral adipocytes). (D) PKC is upstream of iNOS in LPL regulation in VAT. VAT was pre-treated with L-N^G-nitroarginine Methyl Ester (L-NAME) (1 mM) or 1400w (10 nM) for 1 h prior to phorbol 12-myristate 13-acetate (PMA) (10 nM) addition. After 24 h of AngII (1 μM) or PMA treatment, LPL mRNA expression was measured. The mRNA levels were normalized with β-actin. Each column and bar represents the mean ± SEM for three separate experiments. An asterisk (*) indicates <i>p</i><0.05 vs. time 0 or without AngII.</p

Effects of Ang1-7 <i>in vitro</i> and <i>in vivo</i>.

<p>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.</p

AngII has opposite effects on LPL expression in VAT and SAT.

<p>AngII dose-response of LPL expression in adipose tissues. VAT (A1, B1, and C1) or SAT (A2, B2, and C2) was incubated with AngII (vehicle, 10, 100, or 1000 nM) for 24 h and secreted LPL activity (A), LPL protein expression (B), and LPL mRNA expression (C), respectively, were measured as described in Materials and Methods. Each column and bar represents the mean ± SEM for three separate experiments. An asterisk (*) indicates <i>p</i><0.05 vs. without AngII. The LPL activity levels were normalized with total protein, and expression levels of LPL protein and mRNA were normalized to those of β-actin.</p

iNOS and NO mediate the effect of alamandine on leptin expression.

<p>(A) AT was pre-treated with N^G-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.</p

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

<p>(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) I<i>κ</i>Bα 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.</p