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

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

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

PLCβ4 expression is regulated by feeding/fasting cycle in VAT.

<p>VAT was isolated every 6 h (A, B, D, and E), or after fasting for 12 h from 1 a.m. to 1 p.m. (in the right panel of A, C, D, and E). (A-C) Daily change and starvation effects on PLCβ4 expression in VAT. Total RNA (A) or protein (B, C) was extracted for measurement of mRNA and protein expression of PLCβ4, respectively. (D, E) Effects of PLCβ4 expression levels on the LPL response to AngII in VAT. VAT was treated with the indicated doses of AngII. After 12 h incubation, LPL mRNA levels (D) and, after 18 h incubation, total LPL protein levels (E) were measured. Each column and bar represents the mean ± SEM of 4 values from 2 separate experiments for mRNA measurement (A and D) and 3 values from 3 separate experiments for western blotting experiments. An asterisk (*) indicates <i>p</i><0.05 vs. control or without AngII. In panel B, C, and E, the ratio of LPL or PLCβ4 protein to β-actin was calculated based on densitometric quantification of the bands. In panels A and D, mRNA levels were normalized to β-actin mRNA.</p

Analysis of types of AngII receptors and G proteins involved in AngII regulation of LPL expression.

<p>(A) VAT or SAT was pre-treated with 10 μM of candesartan (Cand), 5 μM of PD123177, or vehicle (Cont) for 1 h prior to AngII (1 μM) addition. (B) VAT or SAT was pre-treated with 100 nM YM25490 for 1 h or 100 ng/mL of PTX for 12 h prior to AngII (1 μM) addition. Control cells were treated with 12 h with PBS. After 24 h treatment with AngII, LPL mRNA expression was measured. 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 mRNA levels were normalized to β-actin.</p

PLCβ4 is highly expressed in VAT and visceral adipocytes.

<p>Total protein (A, C) and total RNA (B, D) was extracted for western blot analysis of PLCβ4 protein and for qRT-PCR analysis of PLCβ4 mRNA, respectively (A and B, adipose tissue; C and D; adipocytes). Duplicated samples in each group were processed for western blotting. Each column and bar represents the mean ± SEM of 6 values from three separate experiments. An asterisk (*) indicates <i>p</i><0.05 vs. VAT or visceral adipocytes. In panel A and C, the ratio of PLCβ4 to β-actin was calculated based on densitometric quantification of the bands. In panel B and D, PLCβ4 mRNA levels were normalized to β-actin.</p

PLCβ4 is critical for the inhibition of LPL expression in visceral adipocytes.

<p>(A) siRNA specific to PLCβ4 reduced PLCβ4 expression in visceral adipocytes. Visceral adipocytes were transfected with PLCβ4-siRNA or scrambled siRNA, and incubated for 12 h to measure the enzyme expression levels by western blot analysis. (B, C) AngII reduced LPL expression via PLCβ4 in visceral adipocytes. After transfection with PLCβ4-siRNA or of scrambled siRNA, adipocytes were incubated with AngII (1 μM), PMA (10 or 100 nM), or vehicle (untreated). After 12 h incubation total protein (B) and total RNA (C) was extracted for measurement of LPL protein and mRNA levels, respectively. Each column and bar represents the mean ± SEM for three separate experiments. An asterisk (*) indicates <i>p</i><0.05 vs. control. In panel A and B, the ratio of PLCβ4 or LPL protein to β-actin was calculated based on densitometric quantification of the bands. In panel C, LPL mRNA levels were normalized to β-actin.</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

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

Sequential Nodal activity in left-right asymmetry at the mouse node.

<p>Summary of the symmetry-breaking events that occurs in the node during early somitogenesis. <i>Nodal</i> expression is represented in light red oval, and <i>Cerl2</i> expression in light green oval. The dynamic behavior of Cerl2 protein is illustrated in green triangles, and the readout of nodal signaling, pSmad2, is indicated in red pentagons. The asymmetric expression of <i>Nodal</i> in the left-LPM of mouse embryos is represented by the filled red oval. Dashed to thicker lines indicate increase in intensity. At 2-somite stage, Cerl2 protein (green triangles) localizes and prevents the activation of <i>Nodal</i> genetic circuitry on the right side of the embryo (dashed red oval). Later, due to nodal flow, Cerl2 right-to-left translocation shutdowns Nodal activity in the node and consequently affects the activity of Nodal in the LPM (dashed red oval). The arrows represent the nodal signal transfer across the node. a) 1-somite stage, b) 2-somite stage, c) 4-somite stage, d) 6-somite stage.</p