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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2004-1995
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 5 2888-2897
Copyright © 2005 by The Endocrine Society

Sympathetic Modulation by Carvedilol and Losartan Reduces Angiotensin II-Mediated Lipolysis in Subcutaneous and Visceral Fat

Aderville Cabassi, Pietro Coghi, Paolo Govoni, Elisa Barouhiel, Ester Speroni, Stefania Cavazzini, Anna Maria Cantoni, Renato Scandroglio and Enrico Fiaccadori

Laboratory of Clinical Physiology, Hypertension Unit (A.C., P.C., E.B., E.F.) and Toxicology Unit (S.C.), Department of Internal Medicine, Nephrology, and Health Sciences; Section of Histology and Embryology, Department of Experimental Medicine (P.G., R.S.), and Veterinary Pathology Unit (A.M.C.), Department of Animal Health, University of Parma Faculty of Veterinary Medicine, 43100 Parma, Italy; and Department of Pharmacology (E.S.), Faculty of Pharmacy, University of Bologna, 40126 Bologna, Italy

Address all correspondence and requests for reprints to: Aderville Cabassi, M.D., Department of Internal Medicine, Nephrology and Health Sciences, University of Parma, Via Gramsci 14, 43100 Parma, Italy. E-mail: cabassia{at}unipr.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Advanced heart failure is characterized by increased activation of the renin-angiotensin system and the development of cachexia. Angiotensin II (Ang II) has been proposed as a lipid metabolism regulator. The effects of exogenous Ang II (osmotic minipump, 525 ng/kg/min for 12 d) on interstitial sc glycerol and norepinephrine levels, indexes of lipolysis, and sympathetic activation, respectively, were measured in Sprague Dawley rats by consecutive microdialysis performed in vivo in white adipose tissue. Higher sustained interstitial glycerol and norepinephrine levels were found after 7 and 12 d of Ang II infusion. Triglyceride to DNA content ratio and adipocyte diameter were reduced in sc and visceral (retroperitoneal and epididymal) fat tissues of Ang II-infused rats, whose body weight was lower and blood pressure higher. Losartan, an Ang II receptor 1 blocker, and carvedilol, an {alpha}1-nonselective-ß1,2,3-adrenergic blocker, but not doxazosin, an {alpha}1-selective-adrenergic blocker, lowered glycerol and norepinephrine levels, preventing lipolysis and weight loss. Our results indicate that Ang II stimulates lipolysis in sc and visceral adipocytes by sympathetic activation and ß-adrenergic-receptor stimulation. Nonselective-ß-adrenergic and Ang II-receptor1 blockade markedly attenuated the rise of norepinephrine, preventing catabolic effects. The metabolic benefits of carvedilol and losartan, in addition to recognized protective cardiovascular effects, may be relevant in cachectic patients with advanced heart failure.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LOSS OF LEAN and fat body mass are common features in patients with advanced heart failure who develop cachexia (1). Cardiac cachexia, which represents an independent predictor of mortality in heart failure patients (2), involves neurohormonal and inflammatory mechanisms including the activation of renin-angiotensin and sympathetic nervous systems (SNS) (1). Their pharmacological antagonism is widely used in patients with congestive heart failure (3, 4, 5). Whereas the role of angiotensin II (Ang II) and its SNS interaction on neural and vascular tissues is widely known (6, 7), the in vivo effect of Ang II on adipose tissue metabolism is less understood and still controversial. In the last two decades, a full renin-angiotensin system has been demonstrated in adipose tissue (8), but so far most of the data on the metabolic action of Ang II have been obtained from in vitro (9) or ex vivo studies (10, 11). Recent studies addressing the in vivo effects of Ang II reported contrasting results on lipolysis and adipose blood flow (12, 13, 14). Concordant are the results on body weight loss in rats after chronic Ang II infusion, even at low doses (15, 16, 17, 18, 19). Ang II-induced weight loss has been explained by an anorexigenic (17, 19) effect, by the reduction of plasma leptin levels (18), by an increased expression of uncoupling protein-1 in interscapular brown adipose tissue (IBAT) (20) leading to higher thermogenesis and energy expenditure (19), and by a decrease of IGF-I, which enhances protein degradation and loss of skeletal muscle mass (17, 21).

Still controversial is the contribution of lipid mobilization and fat loss to Ang II-induced cachexia. Both unchanged epididymal white adipose tissue (EWAT) (18, 20, 21) and reduced retroperitoneal white adipose (RWAT) (18) fat mass have been reported in Ang II-infused rats, suggesting some site-specific effect of Ang II. However, norepinephrine (NE), released in white adipose tissue from sympathetic nerve endings, and epinephrine, reaching the adipose tissue from the circulation (22, 23), are considered to be the main regulators of peripheral lipid metabolism. Yet the actual contribution of the SNS to the Ang II-mediated wasting effect has not been clearly defined. The Ang II-infused rat model used in the present study mimics the increased activation of the renin angiotensin system observed in advanced heart failure. On this basis, the purpose of our study was to determine the in vivo effect of a high-dose chronic administration of Ang II on both lipid metabolism, by measuring interstitial and plasma glycerol concentrations, and local SNS activity, by determining interstitial NE release and tyrosine hydroxylase (TH) activity in sc dorsal white adipose tissue (DWAT). Lipolysis was also investigated in DWAT, inguinal adipose tissue (IWAT), visceral RWAT, and EWAT and IBAT by evaluating the triglyceride (TG) to DNA content ratio, the activity of hormone-sensitive lipase (HSL), and the adipocyte size distribution analysis.

The present study confirmed the wasting effect on body mass of high doses of Ang II and demonstrates a marked lipolytic action on both sc and visceral adipose tissues. The lipolytic effect of chronic Ang II is mainly dependent on angiotensin II receptor 1 (AT1) receptor-mediated activation of adipose and systemic SNS. Carvedilol, an {alpha}l-nonselective-ß1,2,3-adrenergic receptor blocker (24), and losartan, an AT1 receptor blocker, but not the selective {alpha}l-adrenergic blocker doxazosin attenuated the catabolic effect of Ang II on white adipose tissue preventing fat and body weight loss.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

All the experimental procedures were approved by the local institutional animal ethics committee. Male Sprague Dawley rats (430–480 g) (Charles River Laboratories, Calco, Italy) were implanted sc, in the dorsal region, with osmotic minipumps (Alzet 2002 model, Alza, Palo Alto, CA) delivering either Ang II at high doses (525 ng/kg/min) or the vehicle for 12 d. Five concurrent groups of rats were evaluated: 1) vehicle-infused rats (CTR rats, n = 7); 2) Ang II-infused rats (Ang II rats, n = 9), Ang II rats receiving by oral gavage from the day before the minipump implantation until the 12th day of Ang II infusion; 3) losartan (LST, 25 mg/kg/d, n = 5); 4) carvedilol (CVD, 90 mg/kg/d, n = 4); or 5) doxazosin (DXZ, 30 mg/kg/d, n = 4). A sixth group of CTR rats (pair-feeding group, n = 5) was started 1 d later and given the same amount of food as that eaten by the Ang II rats on the previous day: because no differences in daily food intake was found after 12 d between CTR and Ang II rats, only data on the concurrent groups are presented. The dose for the antihypertensive drugs were based on equipotent hypotensive effects in previous personal dose-finding experiments (data not shown). Rats were kept individually in metabolic cages in a room with 12-h light, 12-h dark cycles and controlled temperature (between 23 and 25 C) and fed standard laboratory rat chow (0.54% of sodium chloride) and tap water ad libitum. Body weight as well as food and water intake was measured daily. Food intake was measured as a difference between the weight of the daily given pellets and the remaining amount of pellets and crumbs collected at the bottom of the cage after being air dried if urine contaminated. Systolic arterial pressure was measured every 3 d in conscious rats using a tail-cuff plethysmographic method and recorded on a polygraph Maclab/8 system (AD Instruments Ltd., Castle Hill, New South Wales, Australia) (25). Data values for each rat were taken as an average of at least four stable readings.

Subcutaneous adipose tissue microdialysis procedure

Under light anesthesia with ether, a flexible polycarbonate concentric microdialysis probe with membrane of 10 mm length, 0.5 mm outside diameter, and molecular weight cut-off 20 kDa (CMA/20, CMA/Microdialysis AB, Stockholm, Sweden) was inserted in the sc DWAT of the parascapular region, allowing sampling from the interstitial space of adipose tissue and measuring in the collected dialysate the interstitial concentration of glycerol, as an index of TGs hydrolysis, and NE, as an index of sympathetic activity, as we previously described (26). After the basal microdialysis procedure was performed 2 d before the Ang II-minipump implantation, rats were housed again and the experiment repeated on the same animals after 7 and 12 d of Ang II infusion. A bioptic sample taken at the time of probe insertion was weighed and divided into two portions. One was used for histological confirmation of the position in DWAT by cryostatic sections and the other for determination of TH activity, the rate-limiting enzyme of catecholamine synthesis (27). The microdialysis probe was perfused (microperfusion pump, CMA/100) with a Ringer’s solution added with ethanol (50 mM), to calculate the ethanol outflow to inflow probe ratio estimating the local blood flow (28), which can strongly influence interstitial glycerol levels. At the beginning of each microdialytic procedure, the in vivo recovery of glycerol and NE was obtained by plotting the measured dialysate levels at five successive rates against the perfusion rates (0.50, 1.25, 2.00, 2.75, 3.50 µl/min) allowing the calculation at zero flow (dialysate glycerol at 2.0 µl/min to zero flow ratio: 66 ± 3%; dialysate NE at 2.0 µl/min to zero flow ratio: 74 ± 3%). At the end of the calibration time, the perfusion rate was fixed at 2.0 µl/min and dialysates were collected at 30-min intervals over a period of 120 min (basal period), obtaining four consecutive dialysate samples for each rat.

In a subset of experiments, three CTR rats and four Ang II rats after 7 d of Ang II infusion underwent an extended microdialytic procedure to differentiate the contribution of TG hydrolysis and adipose blood flow changes on Ang II-mediated interstitial glycerol changes. Therefore, after the basal collection period (120 min), hydralazine (60 µM) perfusion was started through the microdialysis probe for 90 min to counterbalance the Ang II-induced vasoconstriction observed after 7 d in DWAT, and the dialysates were collected at 30-min intervals for glycerol and ethanol measurements.

Blood sampling

After each microdialytic procedure, three arterial blood aliquots of 0.4, 0.5, and 1.2 ml were collected from the tail artery to measure plasma levels of glycerol, NE, and Ang II, respectively. Blood samples for NE were collected and stored as previously described (25), whereas those for Ang II were collected in a refrigerated glass tube (4 C) containing 250 µl of mixed protease inhibitor solution (146 µmol/liter pepstatin, 50 mmol/liter 1,10-phenanthroline, 125 mmol/liter EDTA, 2 g/liter neomycin sulfate, 2% dimethyl sulfoxide, and 2% ethanol in water), centrifuged, and the plasma frozen at –80 C until the analysis.

Adipose histologic studies, TGs/DNA ratio content, HSL activity

After 12 d of Ang II infusion, rats were killed by decapitation. sc DWAT and IWAT, and visceral RWAT, EWAT, and IBAT were carefully removed, dried, and weighed, and an aliquot was immersed in 4% formaldehyde, dehydrated in ethanol, transitioned in xylene, and embedded in paraffin. Five-micrometer-thick sections were stained with hematoxylin and eosin. Optical microscopy images were digitally captured with Image-Pro Plus 4.5.1. software (Media Cybernetics, Wokingham, UK), and the adipocyte size was measured. Three aliquots from DWAT, RWAT, and EWAT were immediately frozen and used to measure HSL activity, the major enzyme involved in TG hydrolysis (29), TG to DNA content ratio, and tissue NE levels.

Analytical procedures

Glycerol concentration in dialysate was measured by an enzymatic assay kit (TR0100, Sigma, St. Louis, MO). For adipose TG analysis, fat samples were extracted with saturated CHCl3-MeOH (1:2), homogenized, and after two subsequent centrifugations (3000 rpm for 30 min at 4 C) separated by washing with 500 µl ice-cold phosphate buffer saline, and then 1.5 ml H2SO4 was added to the tubes, vortexed, and further centrifuged at 1000 rpm for 20 min at 4 C. The upper phase was then discarded and 75 mg Na2S2O3 were added to the lower phase. After the tube was vortexed and centrifuged for 5 min (at 1000 rpm at 4 C) and the upper phase removed again, the lower phase was dried under nitrogen. The pellet was then dissolved in isopropanol (70%) for 10 sec, and the duplicate TG measurements were performed using a Sigma TG kit. Tissue TG concentration was normalized to the DNA content measured by the DNeasy tissue kit (QIAGEN Italy, Milan, Italy). Ethanol was determined by an enzymatic fluorometric method (30).

HSL activity was measured in fat samples according to the method by Langfort et al. (31). Fat was homogenized at maximum speed, on ice, in 10 volumes of 0.25 M sucrose, 1 mM dithioerythritol, 40 mM ß-glycerophosphate, 10 mM sodium pyrophosphate, 310 nM okadaic acid, 20 µg ml–1 leupeptin, 20 µg ml–1 antipain, and 6.5 µg ml–1 pepstatin (pH 7.0). The crude homogenate was centrifuged at 15,800 x g in an Eppendorf tube at 4 C for 75 sec. The infranatant was recovered and stored at –80 C until analyzed. HSL activity was determined by measuring the release of [3H]oleic acid from tri[3H]olein, representing the activity of the phosphorylated activated form of HSL, which has a much higher activity toward the triglyceride substrate than the diglyceride substrate. Tri[3H]olein substrates were emulsified with phospholipids by sonication and BSA used as a fatty acid acceptor. Fourteen-microliter samples of infranatant were incubated for 30 min at 37 C with 100 µl 5 mmol/liter tri[3H]olein substrate (1.25 x 106 cpm) and enzyme dilution buffer to a total volume of 200 µl. Hydrolysis was stopped by the addition of 3.25 ml methanol-chloroform-heptane (10:9:7 by volume) followed by 1.1 ml 0.1 mmol/liter K2CO3/0.1 mol/liter H3BO3 (pH 10.5). The mixture was vortexed vigorously for 10 sec and centrifuged at 1100 x g for 20 min. One milliliter of supernatant containing the released fatty acids was mixed with 10 ml of scintillation liquid. Radioactivity was determined in a scintillation counter (Packard Instrument Co., Meriden, CT).

Dialysate NE levels, TH activity in DWAT, and plasma NE were analyzed before and after 7 and 12 d of Ang II infusion by HPLC-electrochemical detection as previously described (27). Tissue NE levels after 12 d of Ang II infusion were also evaluated in DWAT, RWAT, and EWAT. Eluates from tissue homogenates, obtained after extraction of NE, were injected in a HPLC system as previously described (25). Protein concentration was determined with a protein assay (Bio-Rad Laboratories, Hercules, CA) to normalize tissue NE and HSL activity. Plasma Ang II was measured by a commercially available competitive RIA (Ang II RIA, IBL, Hamburg, Germany) in CTR rats (n = 5), Ang II rats (n = 5), and antihypertensive-treated groups (n = 3 for each group).

Statistical analysis

Values are presented as mean ± SD. Statistical analysis was based on a two-way ANOVA model for repeated measures, in which the dependent variable (body weight, water and food intake, arterial pressure, plasma Ang II, plasma and interstitial glycerol, NE level, ethanol ratio, or TH activity) represents the same measurement taken at various time during the experimental procedure. The ANOVA model was applied to the CTR rats, Ang II rats, and antihypertensive-treated Ang II rats with the treatment representing the between-group factor, whereas time was the within-group factor. The Bonferroni post hoc test was used to compare group means when ANOVA showed a significant effect for the factor. In histological studies, cell diameter was compared by using the Kruskal-Wallis rank-sum test followed by Dunn’s posttest because of nonnormal distribution. Relations between interstitial glycerol and NE, tissue NE, and HSL activity in DWAT, RWAT, and EWAT were analyzed by linear regression analysis using Pearson correlation coefficients. P < 0.05 denotes statistical significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Body weight, blood pressure, heart rate, food and water intake, and plasma Ang II

ANOVA analysis of the body weight time course revealed an Ang II-mediated weight loss over time (P < 0.05), with a significant effect of the treatment (P < 0.001) and also a significant interaction between the time and the treatment (P < 0.001) (Fig. 1AGo). The body weight loss in Ang II-rats, compared with CTR rats, started from d 5 (P < 0.05) and increased until the end of the experiment (–13% after 12 d, P < 0.001). Weight loss was completely prevented in Ang II rats LST and Ang II rats CVD but not in Ang II rats DXZ (Fig. 1AGo). Systolic arterial pressure was significantly increased in Ang II rats, compared with CTR rats, from d 3 (P < 0.01) until the end of the experiment. Ang II-induced arterial hypertension was completely prevented in antihypertensive-treated Ang II groups (Fig. 1BGo). ANOVA on blood pressure measurements showed a significant effect for the time (P < 0.001), the treatment (P < 0.001), and their interaction (P < 0.001) (Fig. 1BGo). Analysis of heart rate data indicated an expected effect for the treatment (P < 0.001) and an interaction between the time and the treatment (P < 0.001) because a 10% reduction in heart rate was found in Ang II rats CVD, compared with Ang II rats, starting from d 3 (P < 0.01).



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  		FIG. 1. Body weight, blood pressure, food, and water intake in treated and untreated antihypertensive Ang II-infused rats. CTR rats (n = 7), Ang II rats (525 ng/kg/min for 12 d) (n = 9), Ang II rats LST pretreated (from the day before starting Ang II infusion) by oral gavage (25 mg/kg/d, n = 5), Ang II rats DXZ (30 mg/kg/d, n = 4), or Ang II rats CVD (90 mg/kg/d, n = 4) had daily body weight (A), food (C), and water (D) intake measurements, whereas systolic blood pressure (B) was taken every 3 d. Vertical line indicates the starting of 12-d Ang II infusion through the minipump. Data are expressed as mean ± SD. A, *, P < 0.05 Ang II rats vs. CTR rats, vs. Ang II rats LST, and vs. Ang II rats CVD; B, *, P < 0.05 Ang II rats vs. CTR rats, vs. Ang II rats LST, vs. Ang II rats DXZ, and vs. Ang II rats CVD; D, *, P < 0.05 CTR rats vs. Ang II rats.

 
No difference in food intake was found among the experimental groups (Fig. 1CGo). Water intake slightly increased in all groups over time (P < 0.001) but with no difference among groups except for a higher intake in Ang II rats, compared with CTR rats, for the days around the microdialytic procedures (d 1, 7, and 8) (Fig. 1DGo). No significant difference was found in hematocrit values between the groups (data not shown). Plasma Ang II was 7- to 8-fold higher in Ang II rats, compared with CTR rats after 7 d (175.7 ± 31.6 vs. 22.9 ± 4.0 pg/ml, P < 0.001) and 6- to 7-fold after 12 d (151.0 ± 19.3 vs. 24.2 ± 3.1 pg/ml, P < 0.001). Ang II rats LST further increased Ang II plasma levels after 7 (+24%, P < 0.05) and 12 d (+29%, P < 0.05), compared with Ang II rats, whereas no significant differences were observed in Ang II rats CVD and Ang II rats DXZ.

Interstitial and plasma glycerol levels

DWAT interstitial glycerol concentration (average of four consecutive samples for each rat) data showed a significant effect over time of Ang II (P < 0.001), an effect of the treatment (P < 0.001), and an interaction between the treatment and the time (P < 0.05) (Fig. 2AGo). Interstitial glycerol in DWAT was markedly increased after 7 (+71%, P < 0.001) and 12 d (+63%, P < 0.001) in Ang II rats, compared with CTR rats. Furthermore, Ang II increased the ethanol outflow to inflow ratio (+12% after 7 d, P < 0.01, and +6% after 12 d, P < 0.05), indicating an Ang II-mediated DWAT vasoconstriction (Fig. 2CGo) that could influence interstitial glycerol levels by reducing its clearance from the tissue. In the subset of experiments performed in Ang II rats (n = 4) and CTR rats after 7 d, hydralazine in the former group induced a vasodilation in DWAT (–11% of ethanol outflow to inflow ratio) counterbalancing Ang II-mediated vasoconstriction. The 71% increase of interstitial glycerol levels after 7 d of Ang II infusion was reduced by 19% because of the hydralazine-mediated vasodilation but was still higher (net difference +52%, P < 0.001) when compared with CTR rats before hydralazine perfusion. These results clearly indicate the major contribution of the increased DWAT lipolysis induced by Ang II independent from its vascular effect. Hydralazine-perfused CTR-rats showed a reduction in ethanol outflow to inflow ratio (–23%, P < 0.01), indicating a vasodilation with concomitant reduction of interstitial glycerol (–33%), compared with vehicle-perfused CTR rats.



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  		FIG. 2. Glycerol levels and adipose blood flow in treated and untreated antihypertensive Ang II-infused rats. Subcutaneous interstitial (A) and plasma (B) glycerol levels and ethanol outflow to inflow ratio (C) were evaluated in the same animals 2 d before and after 7 and 12 d of Ang II infusion in CTR rats (n = 7), Ang II rats (n = 9), Ang II rats LST (n = 5), Ang II rats DXZ (n = 4), and Ang II rats CVD (n = 4). The vertical line indicates the starting of 12-d Ang II infusion through the minipump. Data are expressed as mean ± SD. A, *, P < 0.05, Ang II rats vs. CTR rats, vs. Ang II rats LST, vs. Ang II rats CVD; B, *, P < 0.05, Ang II rats vs. CTR rats; C, *, P < 0.05 Ang II rats vs. CTR rats, vs. Ang II rats-CVD.

 
Plasma glycerol levels were higher in Ang II rats after 7 (+43%, P < 0.001) and 12 d (+32%, P < 0.05), compared with CTR rats (Fig. 2BGo). LST markedly lowered interstitial glycerol levels after 7 (–31%, P < 0.01) and 12 d (–29%, P < 0.05) of Ang II infusion without affecting local blood flow, compared with Ang II rats. Even CVD significantly reduced interstitial glycerol levels after 7 (–34%, P < 0.01) and 12 d (–33%, P < 0.01) of Ang II infusion. CVD increased local blood flow and counteracted Ang II-mediated DWAT vasoconstriction after 7 d (P < 0.05) but not after 12 d (Fig. 2CGo). LST and CVD caused no significant changes in plasma level glycerol, even if a reduction trend was evident (Ang II rats LST: –20% after 7 d, P = 0.076; Ang II rats CVD –21% after 12 d, P = 0.067) (Fig. 2BGo). DXZ did not show any significant effect on DWAT interstitial and plasma glycerol concentrations and on adipose blood flow (Fig. 2Go, A–C).

Plasma, interstitial, and tissue NE and TH activity

DWAT interstitial sc NE data (average of four consecutive samples for each rat) showed a significant effect over time of Ang II (P < 0.001) of the treatment (P < 0.001) and an interaction between the treatment and the time (P < 0.001) (Fig. 3AGo). Interstitial NE was increased after 7 (+47%, P < 0.001) and 12 d (+66%, P < 0.001) in Ang II rats as compared with CTR rats. TH activity showed a 3.1-fold rise in Ang II rats after 7 d (P < 0.001) and a 2.0-fold rise after 12 d (P < 0.001), compared with CTR rats (Fig. 3BGo). ANOVA revealed a significant effect of time (P < 0.001) of the treatment (P < 0.001) and their interaction (P < 0.001) (Fig. 3BGo).



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  		FIG. 3. SNS level in treated and untreated antihypertensive Ang II-infused rats. Subcutaneous DWAT interstitial NE (A), TH activity (B), and plasma NE (C) and in CTR rats (n = 7), Ang II rats (n = 9), Ang II rats LST (n = 5), Ang II rats DXZ (n = 4), and Ang II rats CVD (n = 4). The vertical line indicates the starting of 12-d Ang II infusion through the minipump. DWAT, RWAT, and EWAT tissue NE levels (D) were measured in all groups after 12 d of Ang II infusion. Data are expressed as mean ± SD. A, *, P < 0.05 Ang II rats vs. CTR rats, vs. Ang II rats LST, vs. Ang II rats CVD; B, *, P < 0.05 Ang II rats vs. CTR rats, vs. Ang II rats LST and vs. Ang II rats CVD; C, *, P < 0.05 Ang II rats vs. Ang II rats LST, vs. Ang II rats DXZ, vs. Ang II rats CVD; D, in DWAT and EWAT: *, P < 0.05 Ang II rats vs. CTR rats, vs. Ang II rats LST, and vs. Ang II rats CVD; RWAT: *, P < 0.05 Ang II rats vs. CTR rats, vs. Ang II rats CVD, vs. Ang II rats LST.

 
Interstitial glycerol and NE levels in DWAT (four samples for each rat) were not correlated before Ang II infusion (basal time, Fig. 4AGo), but a positive linear relationship was found after 7 (Fig. 4BGo) and 12 d (Fig. 4CGo) of Ang II infusion. The correlation was maintained for each treated and untreated individual group of rats infused with Ang II after 7 (Ang II rats, r = 0.599, P < 0.001, n = 36; Ang II rats LST, r = 0.495, P < 0.05, n = 20; Ang II rats DXZ, r = 0.602, P < 0.05, n = 16; Ang II rats CVD, r = 0.627, P < 0.01, n = 16) as well as after 12 d (Ang II rats, r = 0.586, P < 0.001, n = 36; Ang II rats LST, r = 0.493, P < 0.05, n = 20; Ang II rats DXZ, r = 0.546, P < 0.05, n = 16; Ang II rats CVD, r = 0.659, P < 0.01, n = 16).



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  		FIG. 4. Relation of interstitial glycerol and NE in sc DWAT. Correlation of interstitial glycerol and NE in CTR rats (n = 7), Ang II rats (n = 9), Ang II rats LST (n = 5), Ang II rats DXZ (n = 4), and Ang II rats CVD (n = 4) 2 d before (A) and after 7 (B) and 12 d (C) of Ang II infusion. Four consecutive observations were determined for each rat.

 
Plasma NE levels in Ang II rats were increased 62 and 71%, respectively, after 7 (P < 0.001) and 12 d (P < 0.001) over their basal values and 49 and 61%, respectively, after 7 (P < 0.001) and 12 d (P < 0.001) as compared with CRT rats (Fig. 3CGo).

DWAT, RWAT, and EWAT tissue NE concentrations after 12 d of Ang II infusion were greater than in CTR-rats (Fig. 3DGo). A positive correlation was found between tissue NE and HSL activity in DWAT (Fig. 5AGo), RWAT (Fig. 5BGo), and EWAT (Fig. 5CGo) when the results from all the groups were combined, but there was no correlation for any individual small group except for the Ang II rats group in EWAT (r = 0.826, P = 0.006, n = 9).



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  		FIG. 5. Relation of HSL activity and tissue NE in sc and visceral fat pads. Correlation of NE and HSL in DWAT (A), RWAT (B), and EWAT (C) white adipose tissues in CTR rats (n = 7), Ang II rats (n = 9), Ang II rats LST (n = 5), Ang II rats DXZ (n = 4), and Ang II rats CVD (n = 4).

 
After 7 and 12 d of Ang II infusion, LST significantly lowered the interstitial NE levels (–22%, P < 0.05; –36%, P < 0.001, respectively) and TH activity (–48%, P < 0.001; –32%, P < 0.001, respectively) (Fig. 3Go, A and B). After 7 and 12 d of Ang II infusion, CVD but not DXZ reduced the interstitial NE levels (–24%, P < 0.05; –32%, P < 0.001, respectively) and TH activity (–33%, P < 0.001; –24%, P < 0.001, respectively) (Fig. 3Go, A and B). After 12 d, all the antihypertensive drugs lowered plasma NE (Ang II rats LST: –20%, P < 0.05; Ang II rats DXZ: –23%, P < 0.05; Ang II rats CVD: –27%, P < 0.01), compared with Ang II rats. LST and CVD were able to reduce tissue NE only in DWAT, RWAT, and EWAT (Fig. 3DGo).

TG to DNA ratio content, HSL activity, fat pad weight, and adipocyte diameter

ANOVA showed a significant difference in TG to DNA content ratio among groups (DWAT, P < 0.05; RWAT, P < 0.001; EWAT, P < 0.01) (Fig. 6AGo). Ang II rats (n = 6) showed a reduced TG to DNA ratio, indicating a lipolytic effect in sc DWAT (–36%, P < 0.01) and visceral RWAT (–40%, P < 0.01) and EWAT (–33%, P < 0.01), compared with CTR rats (n = 6, Fig. 6AGo). Moreover, HSL activity was slightly but significantly higher in Ang II rats, compared with CTR rats in DWAT (+18%, P < 0.05), RWAT (+31%, P < 0.05), and EWAT (+27% P < 0.05), without significant changes in antihypertensive-treated groups. A loss of relative fat mass (corrected for body weight) of RWAT and EWAT was found in Ang II rats, compared with CTR rats without changes in IBAT (Fig. 6BGo).



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  		FIG. 6. Lipid content and fat pad weight in treated and untreated ANG II-infused rats. TG to DNA content ratio (A) and adipose organ weight (B) in DWAT, RWAT, and EWAT were measured in CTR rats, Ang II rats, Ang II rats LST, Ang II rats DXZ, and Ang II rats CVD after 12 d of Ang II infusion. Data are expressed as mean ± SD. A, in DWAT: *, P < 0.05 Ang II rats LST and Ang II rats CVD and CTR rats vs. Ang II rats; RWAT: *, P < 0.05 Ang II rats CVD and Ang II rats LST and CTR rats vs. Ang II rats; EWAT: *, P < 0.05 Ang II rats CVD and CTR rats vs. Ang II rats; B, in RWAT: *, P < 0.05 Ang II rats vs. CTR rats vs. Ang II rats LST and Ang II rats CVD; EWAT: *, P < 0.05 Ang II rats vs. CTR rats.

 
As shown in Table 1Go, adipocyte diameter showed a significant reduction in Ang II rats, compared with CTR rats in DWAT (P < 0.01) as well as RWAT (P < 0.01) and EWAT (P < 0.01). Kruskall-Wallis one-way ANOVA did not reveal significant differences in cell diameter among groups in IBAT (P = 0.26) and IWAT (P = 0.12). The percentage of adipocytes with a diameter exceeding the 90th percentile was almost 7 times less in Ang II rats (2.6%) than in CTR rats (17.8%) in DWAT, 25 times less in RWAT (1.3% in Ang II rats vs. 32.0% in CTR rats) and 2 times less EWAT (6.8% in Ang II rats vs. 14.7% in CTR rats).


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  		TABLE 1. Cell diameter and percentage above 90th percentile in brown and white sc and visceral adipose tissues in control, untreated, and antihypertensive-treated Ang II-infused rats

 
LST and CVD but not DXZ protected against Ang II-induced RWAT weight loss (P < 0.01, Fig. 6BGo) and significantly attenuated the Ang II-mediated TG to DNA ratio (Fig. 6AGo) and cell size reductions in DWAT, RWAT, and EWAT (Table 1Go).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study demonstrates that a chronic high dose of systemically infused Ang II induces lipolysis in both sc (DWAT) and visceral (RWAT, EWAT) fat, proving a major role for fat loss in the development of cachexia. Moreover, a marked increase in adipose and systemic sympathetic activation, expressed by higher interstitial, tissue, and plasma NE and an increased TH activity, parallels the Ang II-mediated lipolytic effect. In addition, equipotent antihypertensive drugs differently affect the metabolic actions of Ang II, with carvedilol, an {alpha}1-nonselective-ß1,2,3-adrenoceptor blocker, and losartan, an AT1 receptor blocker, but not doxazosin, an {alpha}1-selective-adrenergic receptor blocker, significantly lowering interstitial glycerol and preventing lipolysis and weight loss through the attenuation of Ang II-induced SNS activation.

Our results could be particularly relevant to understand cardiac cachexia mechanisms because the high dose of Ang II infused brought 6- to 8-fold increases in plasma Ang II levels, which are in the same concentration range in human advanced heart failure (32).

The present results confirm previous reports (16, 17, 18, 19, 20) showing a marked body weight loss after chronic systemic Ang II infusion. Based on our results, body weight loss appears to be independent from anorexigenic effects, which contrasts with reports citing it as being one of the main mechanisms (11, 17, 19, 20). Furthermore, our results do not support significant influences of water intake on body weight changes as indicated by similar hematocrit levels among groups. Our findings agree with those of Cassis et al. (18), who did not find a dipsogenic effect of Ang II at high doses, probably because of a rapid and sustained development of tachyphylaxis and desensitization of central Ang II receptors (33), even if an increase in water intake has been reported in other studies (11, 19) with Ang II infused at lower doses.

The in vivo demonstration of a greater interstitial glycerol concentration in sc DWAT after chronic high-dose systemic infusion of Ang II suggests a sustained stimulation of lipolysis, which could contribute to Ang II-mediated weight loss. However, higher interstitial glycerol levels could reflect either an increased TG hydrolysis or an impaired tissue clearance due to reduced local blood flow. In the present study, Ang II infusion induced a sustained reduction in local blood flow in DWAT (–12% after 7 d, –6% after 12 d) as estimated by ethanol dilution technique (30). Adipose hydralazine perfusion through the microdialysis probe antagonized the Ang II-mediated vasoconstriction after 7 d and permitted a separate estimate of adipose blood flow changes from TG hydrolysis on interstitial glycerol levels. From our results, more than two thirds of the increase in interstitial glycerol seems to be related to the Ang II-stimulated TG hydrolysis. Ang II-induced lipolytic effect was not affected by the slight and recently reported lipolytic effect of hydralazine (14) because in hydralazine-perfused CTR rats, a marked reduction of glycerol levels was instead observed.

Contrasting results are reported on the in situ acute effect of Ang II on lipolysis and adipose blood flow (12, 13, 14), whereas no data, until now, have been available on the effect of chronic Ang II infusion. In fact, in situ Ang II administration has been found to either stimulate lipolysis without changes in blood flow (13) or inhibit lipolysis with reduction of adipose blood flow (12). It has been proposed that Ang II could have antilipolytic effects at physiological concentrations but lipolytic actions at supraphysiological concentrations (14).

Higher interstitial glycerol levels in sc DWAT were also found after 12 d of Ang II infusion. This finding was associated with a reduced TG to DNA content ratio (–36%) and a shift in cell distribution toward smaller adipocytes in histologic studies (seven times lower percentage of cells over the 90th percentile), thereby clearly supporting an Ang II-mediated increase of lipolysis. Lipolysis was even more evident in visceral RWAT and EWAT with a marked reduction of TG to DNA ratio (–40 and –33%, respectively) a lower mean adipocyte size, with a smaller percentage of larger adipocytes, and a significant reduction in fat mass.

Our results agree with reports showing a reduced RWAT fat mass (18) after Ang II infusion but contrast with those observing unchanged EWAT fat mass (18, 21), even if in the latter studies Ang II was infused for a shorter time and at a 3 times lower doses. Changes in EWAT mass have been reported to be closely related to total fat mass under conditions of cachexia in uremia (34). In the present study, regional differentiation of Ang II-mediated lipolysis results from the similar fat cell size and weight in IWAT and IBAT and significant differences in DWAT, EWAT, and RWAT. Therefore, lipolysis is not extended to all fat pads but is sufficiently diffused to determine greater plasma glycerol levels after Ang II infusion.

In the present study, an in vivo increase of peripheral sympathetic traffic to sc DWAT, expressed by greater interstitial NE concentrations, is closely associated with the lipolytic effect of chronic Ang II infusion. In DWAT, local SNS hyperactivity is also supported by the sustained increase in TH activation and the higher NE tissue content. Presynaptic reuptake inhibition by Ang II could be a contributor of increased NE nerve release as reported in IBAT (10, 11).

The SNS activation after chronic administration of Ang II is not limited just to DWAT but is extended to other fat pads as expressed by elevated NE content in visceral RWAT and EWAT and systemically increased plasma NE levels. Our results contrast with those reported recently by Cassis et al. (35), who did not find any difference in NE content in EWAT by infusing Ang II at a 3-fold lower dose and for almost half the time.

A positive linear relationship between tissue NE and active HSL, the major lipolytic enzyme (29), is found in sc DWAT but more pronounced in visceral RWAT and EWAT. Ang II could induce the activation of HSL by its phosphorylation probably through the sympathetic stimulation of ß-adrenergic cAMP-dependent protein kinase (36) and as recently demonstrated, by direct Ang II stimulation of MAPKs (37).

From our results it is clear that SNS activation represents the primary mediator of Ang II effects on body weight loss and peripheral lipid metabolism. In fact, the present study, for the first time, provides in vivo evidence of antagonizing actions of CVD, an {alpha}1-nonselective-ß1,2,3-adrenergic blocker, on Ang II-mediated metabolic effects, preventing body weight loss, attenuating interstitial DWAT glycerol rise, TG to DNA ratio reduction, and the shift toward smaller cells in sc DWAT as well as visceral RWAT and EWAT. The {alpha}1-selective-adrenergic blocker DXZ was unable to produce these effects, indicating the stimulation of ß-receptors as a central mechanism of lipid regulation when high doses of Ang II are infused.

Lipolysis in white adipose tissue depends on the balance between the interstitial NE levels and the relative amounts of antilipolytic {alpha}2- (38, 39) and lipolytic ß-adrenoceptors (40, 41), which are predominantly ß3-adrenoceptors in rodents, in which they represent almost 90%, whereas in humans they are less than 15–20% (40). Both CVD and LST markedly reduced adipose SNS activation as expressed by the reduction of DWAT interstitial NE and TH activation and RWAT and EWAT tissue NE diminution. The inhibitory effect of prejunctional receptors (ß2-adrenoceptor for CVD and AT1-receptor for LST) in adipose sympathetic nerve endings (6, 7) could contribute to reduce interstitial NE levels and lower stimulation of ß-adrenergic receptors on fat cell surfaces. Furthermore, when interstitial NE is high, as in the present study, or when ß1-adrenoceptors are desensitized by prolonged sympathetic stimulation, ß3-adrenoceptors become the main lipolytic receptors (40, 41), suggesting that the nonselective ß-adrenergic blocker, CVD, could also act by antagonizing NE-induced stimulation of lipolytic ß3-adrenergic receptors (24) and allowing NE to stimulate the antilipolytic {alpha}2-adrenoceptors (38, 39). SNS activation could also contribute to Ang II-induced weight loss by an increased energy expenditure through the expression of uncoupling protein-1 in IBAT (42), the stimulation of adaptive thermogenesis, or lowering leptin secretion (18, 19). From our results, LST was able to reduce Ang II-induced sympathetic activation as well as its lipolytic effect but was incapable of reversing Ang II-mediated adipose vasoconstriction; even CVD lost its ability to reverse Ang II-induced adipose vasoconstriction after 12 d. These data suggest that adipose vasoregulation after Ang II chronic infusion seems far less dependent on a direct AT1-receptor stimulation or sympathetic-mediated constriction. Rather, other hormonal and metabolic factors such as insulin, unbound nonesterified fatty acid, prostacyclin, prostaglandin, and nitric oxide (43) may influence local vascular tone.

Besides experimental studies, there are clinical evidences showing a major role for neurohormonal activation, involving especially the SNS and renin-angiotensin-aldosterone systems in the development of cachexia in heart failure patients (1, 2). This hypothesis is supported by reports from multicenter randomized (44, 45) and small clinical trials on heart failure patients (46), indicating that long-term treatment with ß-blockers, either ß1-selective such as bisoprolol and metoprolol or the nonselective {alpha}1-ß1,2,3 blocker, CVD, could prevent and reverse the development of cachexia (44, 45, 46) by reducing plasma NE and increasing plasma leptin, which reflects the amount of fat mass (46). ß-Blockers have been also shown to reduce energy expenditure, thermogenesis, and exacerbate insulin resistance and weight gain in hypertensive patients (47). Moreover, in heart failure patients in the Studies of Left Ventricular Dysfunction Study, an angiotensin-converting enzyme inhibitor, enalapril, also showed the ability to reduce the risk of developing cachexia (48) by suggesting a favorable effect on catecholamines.

In summary, our study demonstrates that chronic Ang II infusion induces an in vivo lipolytic effect on both sc and visceral adipose tissue through increased local and general sympathetic activity, contributing to fat mass and body weight loss and the development of cachexia. This lipolytic effect involves not only visceral adipose tissue but also sc DWAT, whose importance in increasing the risk of cardiovascular progression has been repositioned alongside the better-known contribution of visceral tissue (49). Independent of blood pressure levels, CVD, the nonselective {alpha}1-ß1,2,3 blocker, and LST, the AT1 receptor blocker, but not the {alpha}1-selective adrenergic blocker, DXZ, prevented Ang II-mediated lipolytic effects and body weight loss, attenuating the adipose SNS activation. These observations could have important clinical implications for understanding the pathophysiology of cachexia associated with the chronic activation of the renin-angiotensin system, as occurs in heart failure, because a part of the beneficial effects of CVD and LST on cardiovascular morbidity and mortality in heart failure patients (4, 5) could be ascribed to the metabolic effects reported in the present study.


   Acknowledgments
 
Expert technical laboratory assistance of Sabine Halpern, Martin Theullet, Maria Giovanna Fedeli (for RIAs), and Antonio Ferrarini is greatly acknowledged.


   Footnotes
 
This work was supported by the Progetto di Ateneo di Ricerca D’Eccellenza (CV0199-04) Research Project of Excellence and FIL 60006-2004 of the University of Parma.

First Published Online March 1, 2005

Abbreviations: Ang II, Angiotensin II; AT1, angiotensin II receptor 1; CTR, vehicle infused; CVD, carvedilol; DWAT, dorsal white adipose tissue; DXZ, doxazosin; EWAT, epididymal white adipose tissue; HSL, hormone-sensitive lipase; IBAT, interscapular brown adipose tissue; IWAT, inguinal white adipose tissue; LST, losartan; NE, norepinephrine; RWAT, retroperitoneal white adipose; SNS, sympathetic nervous system; TG, triglyceride; TH, tyrosine hydroxylase.

Received October 11, 2004.

Accepted February 18, 2005.


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 Introduction
 Materials and Methods
 Results
 Discussion
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