Long-term angiotensin II AT1 receptor inhibition produces adipose tissue hypotrophy accompanied by increased expression of adiponectin and PPARgamma

Abstract

To clarify the mechanism of the effects of angiotensin II AT(1) receptor antagonists on adipose tissue, we treated 8 week-old male Wistar Kyoto rats with the angiotensin II AT(1) receptor antagonist Candesartan cilexetil (10 mg/kg/day) for 18 weeks. Candesartan cilexetil reduced body weight gain, decreased fat tissue mass due to hypotrophy of epididymal and retroperitoneal adipose tissue and decreased adipocyte size without changing the number of adipocytes. Candesartan cilexetil decreased serum leptin levels and epididymal leptin mRNA, increased serum adiponectin levels and epididymal adiponectin mRNA, decreased epididymal tumor necrosis factor alpha (TNFalpha) mRNA, and increased fatty acid synthase mRNA. Considered free of peroxisome proliferator-activated receptor gamma (PPARgamma) agonist activity, Candesartan cilexetil increased epididymal expression of PPARgamma mRNA. The effects of Candesartan cilexetil on adipokine production and release may be attributable to PPARgamma activation and/or decrease in adipocyte cell size. In addition, Candesartan cilexetil treatment increased the expression of epididymal angiotensin II AT(2) receptor mRNA and protein and decreased the expression of renin receptor mRNA. These results suggest that Candesartan cilexetil influences lipid metabolism in adipose tissue by promoting adipose tissue rearrangement and modulating adipokine expression and release. These effects are probably consequences of local angiotensin II AT(1) receptor inhibition, angiotensin II AT(2) receptor stimulation, and perhaps additional angiotensin II-independent mechanisms. Our results indicate that the activity of local renin-angiotensin system plays an important role in adipose tissue metabolism. The decrease in the pro-inflammatory cytokine TNFalpha and the increase in the anti-inflammatory adipokine adiponectin indicate that Candesartan cilexetil may exert significant anti-inflammatory properties.

Figures

FIG. 1. Effect of long-term angiotensin II AT1 receptor blockade on body weight, food and water intake

Two groups of 10 male Wistar Kyoto rats were treated with either angiotensin II AT1 receptor blocker, Candesartan cilexetil or vehicle for 18 weeks. During the treatment the animals were housed two per cage. Body weight, food and water intake were evaluated once a week. A. Body weight expressed in g as mean ± S.E.M., n = 10 for each group. B. Food intake expressed in g per kg of body weight per day. Food intake was measured individually for each cage housing two rats and normalized for body weight. Results are expressed as mean ± S.E.M., n = 5 for each group. C. Water intake expressed in ml per kg of body weight per day. Water intake was measured individually for each cage housing two rats and normalized for body weight. Results are expressed as mean ± S.E.M., n = 5 for each group. * P<0.05

FIG. 2. Effect of long-term angiotensin II AT1 receptor blockade on adiposity index and diameter of adipocytes

Two groups of 10 male Wistar Kyoto rats were treated with either Candesartan cilexetil or vehicle for 18 weeks. At the end of treatment the rats were sacrificed by decapitation and epididymal and retroperitoneal fat was used for adipocyte isolation. A. Adiposity index for epididymal and retroperitoneal adipose tissue expressed as a percent of adipose tissue weight relative to body weight. B. Diameter of adipocytes isolated from epididymal fat pads. The adipocytes were isolated from epididymal adipose tissue by collagenase digestion and evaluated by light microscopy. The diameter of at least 100 cells from each rat was used to calculate average value. C. Representative pictures of adipocytes isolated from control and Candesartan cilexetil-treated animals. Scale bar = 100 μm. Results are expressed as mean ± S.E.M., n=10 for each group. *** P < 0.001

FIG. 3. Effect of long term angiotensin II AT1 receptor blockade on leptin and adiponectin plasma levels and gene expression in white adipose tissue

Two groups of 10 male Wistar Kyoto rats were treated with either Candesartan cilexetil or vehicle for 18 weeks. Animals were sacrificed by decapitation and their serum was used for determination of leptin (A) and adiponectin (C) levels by RIA. Results are expressed as mean ± S.E.M. in ng/ml and μg/ml for leptin and adiponectin, respectively. Epididymal fat pads were used for RNA isolation and determination of leptin (B) and adiponectin (D) gene expression by RT-PCR. Results from RT-PCR are normalized to the expression of GAPDH mRNA and expressed as mean ± S.E.M., n=10 for each experimental group. * P < 0.05, *** P < 0.001

FIG. 4. Effect of long-term angiotensin II AT1 receptor blockade on gene and protein expression of angiotensin II AT1 and AT2 receptors in white adipose tissue

Two groups of 10 male Wistar Kyoto rats were treated with either Candesartan cilexetil or vehicle for 18 weeks. At the end of treatment the rats were sacrificed by decapitation and epididymal fat was used for RT-PCR and Western Blot analysis. Expression of angiotensin II AT1 (A) and AT2 receptor mRNA (B) was normalized to the level of GAPDH mRNA. Protein levels of angiotensin II AT1 (C) and AT2 (D) receptors were normalized to the level of β-actin. Results are expressed as mean ± S.E.M., n = 10 for each experimental group. * P < 0.05

FIG. 5. Effect of long-term angiotensin II AT1 receptor blockade on expression of Renin and (Pro)Renin receptor genes in white adipose tissue

Two groups of 10 male Wistar Kyoto rats were treated with either Candesartan cilexetil or vehicle for 18 weeks. At the end of treatment the rats were sacrificed by decapitation and epididymal fat was used for real time PCR analysis. Expression of Renin (A) and (Pro)Renin receptor (B) was normalized to the expression of GAPDH mRNA. Results are expressed as mean ± S.E.M., n = 10 for each experimental group. *P < 0.05

FIG. 6. Effect of long-term angiotensin II AT1 receptor blockade on expression of TNFα, PPARγ, FAS and HSL genes in white adipose tissue

Two groups of 10 male Wistar Kyoto rats were treated with either Candesartan cilexetil or vehicle for 18 weeks. At the end of treatment the rats were sacrificed by decapitation and epididymal fat was used for RT-PCR analysis. Expression of TNFα (A), PPARγ (B), FAS (C), and HSL (D) mRNA was normalized to the expression of GAPDH mRNA. Results are expressed as mean ± S.E.M., n = 10 for each experimental group. * P < 0.05