Angiotensin II Regulates Body Weight Through Sympathetic Neurotransmission

Lisa Cassis, Vicki English, Marc Helton and Robert Lodder

Division of Pharmaceutical Sciences, College of Pharmacy

University of Kentucky, Lexington, KY 40536

Running title: Angiotensin Regulates Body Weight

Corresponding author:

Lisa A. Cassis, Ph.D.

Professor

Division of Pharmaceutical Sciences

Room 434, College of Pharmacy

University of Kentucky

Lexington, KY 40536-0082

Phone: 606-257-4749

FAX: 606-257-7564

e-mail: lcassis@pop.uky.edu

Number of text pages total, 29, including references and abstract

Number of tables, 4.

Number of figures, 5.

Number of references, 22.

Number of words in abstract, 246.

Number of words in Introduction, 554.

Number of words in the Discussion, 1915.

Abbreviations: Angiotensin, AngII; norepinephrine, NE; interscapular brown adipose tissue, ISBAT; epididymal fat, EF; left ventricle, LV; retroperitoneal fat, RPF, nisoxetine, NIS;

Abstract

Previous studies in our laboratory suggested a local renin-angiotensin system in adipose tissue, with adipose Angiotensin II (AngII) important in the regulation of body weight. Angiotensin II facilitates the activity of the sympathetic nervous system through increased release of norepinephrine (NE), regulation of NE uptake, and of NE synthesis. To test the hypothesis that AngII regulates body weight by facilitating sympathetic neurotransmission, we examined the effect of chronic AngII infusion on ligand binding of the NE uptake transporter and the turnover of NE in tissues with cardiovascular (left ventricle, LV; kidney) and metabolic (interscapular brown adipose tissue, ISBAT; epididymal fat, EF) relevance. Chronic infusion of AngII dose-dependently increased the density for [³H]nisoxetine (NIS) binding in ISBAT, but not in LV. Increased NE uptake transporter density in ISBAT occurred at doses of AngII that reduced body weight. Alpha-methyl-para-tyrosine (AMPT) was injected into rats following 7 and 14 days of saline or AngII infusion to determine NE turnover in ISBAT, LV, kidney and EF. At a dose of AngII that reduced body weight, NE turnover was increased in all tissues. Endogenous NE content was reduced in LV and kidney following AngII, and increased in ISBAT. In rats administered the -adrenergic receptor antagonist, propranolol, AngII reductions in body weight, food intake and water were totally eliminated. These data support the hypothesis that AngII regulates body weight through sympathetic neurotransmission. Moreover, in ISBAT, a thermogenically active tissue important in energy expenditure, chronic AngII infusion increased NE uptake transporter density and NE content, indicative of an increased density of sympathetic innervation.

Angiotensin II (AngII), the primary peptide of the renin-angiotensin system with biologic activity, is important in the regulation of arterial pressure and fluid and electrolyte balance. On the basis of a variety of evidence, including demonstration of components necessary for AngII synthesis, AngII receptor localization, detectable quantities of immunoreactive AngII, and functional responsiveness to AngII, local tissue renin-angiotensin systems have been proposed (Phillips et al., 1983). The majority of proposed sites for tissue renin-angiotensin systems exhibit a relationship to cardiovascular control. Previous studies in our laboratory demonstrated a high level of angiotensinogen mRNA expression (Cassis et al., 1988a,b), renin-like activity (Shenoy and Cassis, 1997), AngII receptor localization (Cassis et al., 1996, 1998), immunoreactive AngII (Shenoy and Cassis, 1997), and AngII regulation of sympathetic neurotransmission (Cassis and Dwoskin, 1992; English and Cassis, 1999) in rat adipose tissue. These results suggest adipose tissue as a potential site for a local tissue renin-angiotensin system.

Potential functions for adipose AngII production have been suggested by studies demonstrating that exogenous and endogenous AngII facilitates the evoked release of NE from ISBAT slices (Cassis et al, 1992, 1993, 1994, 1999). Additionally, previous studies have demonstrated a potential role for AngII in cold-induced thermogenesis of brown adipose tissue (Cassis, 1993; Cassis et al., 1998a). Alterations in AngII production and function have been demonstrated in brown adipose tissue from Zucker genetically obese (fa/fa) rats compared to lean controls (Cassis, 1994). Recent studies in our laboratory demonstrated that AngII regulates body weight (Cassis et al, 1998; English and Cassis, 1999). Chronic infusion of AngII to rats decreased body weight in a dose-dependent manner (Cassis et al, 1998b). Reductions in body weight following AngII infusion were associated with decreased food intake, increased water intake, and site-specific reductions in adipose tissue mass. Importantly, AngII decreased body weight when blood pressure was normalized by vasodilator treatment, demonstrating that AngII regulation of body weight was independent of elevations in blood pressure. Additional studies suggested that increases in sympathetic neurotransmission may contribute to AngII regulation of body weight (Cassis et al., 1998b; English and Cassis, 1999). Reductions in body weight following chronic AngII infusion were associated with increases in NE release from sympathetic nerve terminals innervating ISBAT (English and Cassis, 1999), a thermogenically active tissue contributing to peripheral energy expenditure. Morever, thermal infrared imaging demonstrated an increase in the abdominal surface temperature of AngII-infused rats (Cassis et al., 1998), indicative of elevations in energy expenditure.

We hypothesized that AngII regulates body weight by increasing sympathetic neurotransmission. The present study utilized the chronic AngII infusion model to determine if AngII regulation of body weight resulted from enhanced sympathetic neurotransmission. Initial studies focused on the regulation of the NE neuronal uptake transporter at sympathetic terminals innervating ISBAT and LV following the infusion of different doses of AngII. Results from these studies demonstrated that chronic infusion of AngII regulated the NE uptake transporter, an integral component of the peripheral sympathetic nervous system. In follow-up studies, the effect of chronic AngII infusion on NE turnover was determined in tissues with metabolic (ISBAT, EF) and cardiovascular (LV, kidney) relevance to the effects of AngII. To mechanistically determine if chronic AngII infusion regulated body weight through enhanced sympathetic neurotransmission at the site of -adrenergic receptors, the effects of the non-selective -adrenergic receptor antagonist, propranolol, on AngII-regulation of body weight were determined.

Methods.

AngII infusion model. Male, Sprague Dawley rats (350 - 400 g; Harlan Laboratories, IN) were used in all studies. All rats were housed 2/cage in an approved animal facility for one week before use under a 12 h light/dark cycle and were given free access to food and water. During each experimental protocol rats were housed individually in cages for the daily (10:00 a.m.) measurement of body weight, food and water intake. Baseline measurements of food intake, water intake, and blood pressure were performed on all rats in an individual study for a minimum of three days preceding each experimental protocol. All studies were reviewed and approved by the Institutional Animal Care and Use Committee.

For AngII infusion, rats were anesthetized with diethyl ether, shaved in the interscapular region, and osmotic mini-pumps (Model 2001 for 7 day infusion; Model 2002 for 14 day infusion, Alza Corporation, CA) were implanted subcutaneously. Mini-pumps contained either AngII (200 - 600 ng/kg/min; Sigma, St. Louis, MO) or sterile saline (sham-surgery) and were primed according to the manufacturer's instructions preceding implantation to assure immediate subcutaneous delivery of AngII. The skin overlaying the mini-pump was closed with surgical staples, and the rats were allowed to recover on warmed heating pads.

[³H]NIS binding in ISBAT, LV. [³H]NIS binding was measured according to previously published methods (King et al., 1999). The uptake inhibitor, nisoxetine was used as a selective ligand for the NE uptake transporter site (Tejani-Butt, 1992). Tissues were removed, placed in ice-cold buffer (5 ml; 50 mM Tris, 120 mM NaCl, 5 mM KCl, pH 7.4) and homogenized three times for 10 sec with a polytron (Kinematica GmbH). Homogenates were diluted to a total volume of 30 ml with ice cold membrane buffer and centrifuged at 1,100 X g for 15 minutes at 4C. The supernatant was re-suspended with 30 ml of buffer and centrifuged at 40,000 X g for 10 min at 4C. The centrifugation procedure was repeated and the final membrane pellet (1 - 2.5 mg protein/ml buffer) was re-suspended in binding buffer (50 mM Tris, 300 mM NaCl, 5 mM KCl, pH 7.4). Protein concentration was determined spectrophotometrically using coomassie blue dye with bovine serum albumin as the standard (Bradford, 1976).

Saturation binding isotherms were performed by incubating duplicate aliquots of membrane (70 g) with an increasing concentration of [n-methyl-³H]NIS (5.9 Ci/mmol specific activity; New England Nuclear, Boston, MA) (0.1 - 20 nM, 8 points, 50 l) and binding buffer (250 l) for 30 min at 22C. Non-specific binding was determined at each concentration of [³H]NIS by the addition of mazindol (2 M; RBI Natick, MA). Binding was terminated by filtration over pre-soaked (0.3% polyethleneimine) glass microfiber filters (#32; Schleicher & Schuell Keene, NH) using a Brandel cell harvester. Radioactivity retained on the filters was measured by liquid scintillation spectrometry. Saturation isotherms for specific [³H]NIS binding were constructed using GraphPad Prism 2 software. For the determination of Kd and Bmax, data was analyzed by nonlinear regression analysis using LIGAND.

Measurement of NE turnover. Measurement of NE turnover in tissues was according to previously published methods (Brodie et al., 1966; Cassis et al, 1988). Rats designated to control (saline-infused) or AngII groups (400 ng/kg/min) were treated with either vehicle or the tyrosine hydroxylase inhibitor, -methyl-p-tyrosine methyl ester (AMPT, Sigma, St. Louis, MO; 300 mg/kg, i.p.) either once (rats designated to 2 - 6 hr time points) or twice (second injection at 6 hours for rats designated to 6 - 24 hr time points) on the final day of the study. At designated times (0 - 24 hrs) after AMPT injection, rats were killed and tissues [ISBAT, LV, EF, kidney) were removed and frozen for measurement of tissue NE content using HPLC with electrochemical detection by previously published methods (King et al., 1999). Under conditions of synthesis inhibition, the decline of tissue NE over time is mono-exponential (Brodie et al, 1966). Tissue NE content (log) was regressed against time following AMPT injection, and the slope of the line was used to calculate the rate of decline (k, rate constant of NE efflux) from the following equation: k = slope/0.434. The turnover rate of NE (K, rate of NE synthesis) was calculated as the product of the endogenous NE content present in untreated tissue and k, the rate of decline of NE after synthesis inhibition. The turnover time (total time required for dissipation of NE content) was calculated as 1/k, the rate of NE decline).

Measurement of catecholamines.

(i) Description of the high performance liquid chromatography (HPLC) system.

The HPLC system consisted of a System Gold Model 116 pump (Beckman, Fullerton, CA), a Model 7725 injection valve fitted with a 50 µl sample loop (Rheodyne, Cotati, CA), a Coulochem Model 5100A electrochemical detector (ECD) (ESA, Bedford, MA), an ESA Model 5011 analytical cell, and a catecholamine ESA HR-80 reverse phase column packed with 3 µm spherical silica bonded with octadecylsilane with a graphite guard filter. The mobile phase consisted of (in mM): citric acid anhydrous, 70; EDTA , 0.16; 1-octane sulfonic acetate trihydrate, 100; NaCl, 11; and 1.5% (v/v) methanol, pH 4.0. The HPLC column was equilibrated with mobile phase for 12 hours before use and separation was achieved at a flow rate of 1.0 ml/min. HPLC chromatograms were displayed on an Omniscribe chart recorder (Houston Instruments, Houston,TX).

(ii). Extraction and quantification of catecholamines. Methods for measurement of tissue NE content were previously established (King et al., 1999). Tissues were homogenized on ice in 1 ml of 0.4 N perchloric acid containing 300 pg of the internal standard DHBA and 200 µl 0.5% EDTA solution containing 283 mM metabisulfate. Homogenized samples were centrifuged at 12,365 X g for 10 min at 4C and the supernatant decanted for analysis of catecholamines. Free catecholamines were extracted from an aliquot of the supernatant (representing approximately 1-2 mg of tissue) by activated alumina (Bioanalytical Systems, West Lafayette, IN). Upon addition of alumina (25 mg), the pH of the sample was adjusted to 8.6 by the addition of 3 M Tris HCl buffer (pH 10.9). The samples were vortexed for 10 min, interrupting every 2 min to allow the alumina to settle, followed by centrifugation at 5,000 rpm for 3 min (4C). Supernatant was decanted and the alumina pellet was washed three times with 3 ml of a 1:1 dilution of Tris HCl buffer (1.5 M Tris HCl, 0.5 mM EDTA and 0.4 mM sodium metabisulfate, pH 8.7) and water. The supernatant was removed and the alumina-slurry transferred to microfilter tubes (0.45 µm nylon; Alltech, Deerfield, IL) and centrifuged at 5,000 rpm for 1 min. The supernatant from this centrifugation was discarded. Catecholamines were eluted from the alumina utilizing a two step procedure consisting of the addition of 100 µl of 0.15 N perchloric acid, vortexing for 10 sec and centrifugation at 5,000 rpm for 1 min (total elution volume of 200 µl). The eluent (50 µl) was injected onto the HPLC for catecholamine analysis using the following parameters: electrode potentials set at (E1) -50 mV and (E2) +320 mV. A set of catecholamine standards [10 - 300 pg, NE and epinephrine (EPI), 250 pg DHBA] were prepared on a daily basis. The peak height of the standards were determined and analyzed by linear regression analysis. The retention time of the standards were used to identify catecholamines. Peak heights in sample unknowns were extrapolated from standard curves to quantify catecholamines. Amount and response (peak height) were linearly related (correlation coefficient > 0.95) up to 200 pg of NE. The peak height of DHBA in sample unknowns was used to correct for recovery of the extraction procedure. Recovery was typically 86% with a limit of detection of 5 pg of NE.

Statistical analysis. For the dose-response data for AngII infusion (water intake, body weight, food intake), a one-way ANOVA with time as a repeated measure was performed followed by Tukey-Kramer multiple comparisons test for post-hoc analysis. For the data with AngII infusion +/- propranolol (water intake, body weight, food intake), a two-way ANOVA with repeated measures with AngII, propranolol as between group factors and time as a within group repeated measure was performed. Post-hoc analysis was by Tukey-Kramer multiple comparisons test. For the [³H]NIS binding parameters (Bmax, Kd), a one-way ANOVA was performed. For the NE turnover studies, NE tissue content was converted to log, plotted against time, and linear regression performed. The slope of the line was used to calculate the rate constant for decline and turnover. The error of the parameters for NE turnover were calculated assuming a Gaussian distribution. An unpaired t-test was used to determine statistical difference between groups.

Results

The dose-dependent effect of chronic AngII infusion on the regulation of the NE uptake transporter. The purpose of this study was to determine the dose-dependent effect of chronic AngII infusion on the regulation of the peripheral NE uptake transporter. AngII (200, 400, 600 ng/kg/min; n = 8/group) or saline were infused for 14 days. Food intake, water intake and body weight were measured daily. Systolic blood pressure was measured on anesthetized rats by tail cuff at baseline (day 0, pre-pump implantation) and on the final day of the study. Analysis of variance demonstrated a significant effect of AngII on systolic blood pressure [F_3,16 = 4, P < 0.05]. At 200 ng/kg/min of AngII, blood pressure was increased compared to baseline (day 0; data not shown) and compared to saline control (Table 1). Heart rate was significantly increased in rats treated with all doses of AngII compared to control (Table 1). Plasma NE concentration was significantly increased in rats treated with all doses of AngII compared to control (Table 1).

Analysis of variance demonstrated a significant effect of AngII on organ mass (tissue weight normalized to final body weight) (Table 2). In each tissue examined, the highest dose (600 ng/kg/min) of AngII resulted in a significant decrease in organ mass. Chronic infusion of AngII (600 ng/kg/min) resulted in a marked decreased in organ mass of white adipose tissue deposits including retroperitoneal fat (RPF, 83% decrease) and epididymal fat (EF, 52% decrease). All other organs examined decreased in mass by approximately 25 - 35% following chronic AngII infusion.

Analysis of variance of daily body weight measurements demonstrated a significant effect of AngII dose [F_3,16 = 42, P < 0.05], a significant effect of time of infusion [F_16,48 = 51, P < 0.05] and a significant interaction between AngII dose and time of infusion [F_48,256 = 34, P < 0.05]. At 200 ng/kg/min of AngII, body weight was significantly decreased compared to control on days 5 - 14 (Fig. 1A). At higher doses (400, 600 ng/kg/min) of AngII, body weight was significantly decreased compared to control on days 2 - 14 (Fig 1A). Moreover, reductions in body weight in response to 400 and 600 ng/kg/min of AngII were significantly greater than those observed at 200 ng/kg/min. The magnitude of the reduction in body weight was similar at doses of 400 and 600 ng/kg/min of AngII, demonstrating that maximal responses had occurred.

Analysis of variance of daily food intake measurements demonstrated a significant effect of AngII dose [F_3,16 = 53, P < 0.05], a significant effect of time of AngII infusion [F_15,45 = 18, P < 0.05], and a significant interaction between AngII dose and time of infusion [F_45,250 = 4, P < 0.05]. Food intake was significantly decreased compared to control at all doses of AngII on days 1 - 3 (Fig. 1B). Moreover, reductions in food intake were significantly greater in rats infused with 400 and 600 ng/kg/min of AngII compared to 200 ng/kg/min. The magnitude of the reduction in food intake was similar at doses of 400 and 600 ng/kg/min of AngII, demonstrating that maximal responses had occurred. In all rats infused with AngII, food intake gradually returned towards control in a dose- and time-dependent manner.

Analysis of variance of daily measurement of water intake demonstrated a significant effect of AngII dose [F_3,16 = 10, P < 0.05], a significant effect of time of infusion [F_15,45 = 15, P < 0.05], and a significant interaction between AngII dose and time of infusion [F_45,250 = 2, P < 0.05]. At 200 ng/kg/min of AngII, water intake was significantly increased compared to control on days 8 - 14 (Fig. 1C). At 400 and 600 ng/kg/min of AngII, water intake increased significantly compared to control on days 3 - 14.

Saturation isotherms for [³H]NIS binding were performed in ISBAT and LV from AngII-infused rats. In ISBAT membranes from all rats, [³H]NIS binding was saturable, of high affinity and to a single class of sites (Fig. 2A,B). There was no effect of AngII infusion on the affinity of [³H]NIS binding (Table 3). Analysis of variance of results for [³H]NIS binding density demonstrated a significant [F_3,13 = 10, P < 0.05] effect of AngII on the density of [³H]NIS binding sites in ISBAT membranes (Fig. 2C,D; Table 3). At 400 ng/kg/min of AngII, [³H]NIS binding density in ISBAT was increased by 77% (Table 3). At 600 ng/kg/min of AngII infusion, [³H]NIS binding density in ISBAT was significantly increased (by 63%) compared to control, but was not increased compared to 400 ng/kg/min.

In LV, [³H]NIS bound to a single class of sites with slightly lower affinity than observed in ISBAT membranes (Table 3). Moreover, in tissues from control rats, the density of [³H]NIS binding sites was 9.5-fold lower in LV than ISBAT. Chronic infusion of AngII did not significantly alter [³H]NIS binding density or affinity in LV compared to control.

The effect of chronic AngII infusion on NE turnover: The purpose of this study was to determine the effect of chronic AngII infusion on the turnover of NE in tissues with metabolic and cardiovascular relevance to the effects of AngII. AngII (400 ng/kg/min) or saline were infused for either 7 (n = 5 rats/group/time point after AMPT injection) or 14 days (n = 5 rats/group/time point after AMPT injection). Body weight was measured preceding AngII infusion, and on the final day of the study. On the final day of each study, rats from each group (saline, AngII) were injected with AMPT and killed at different time points (2 - 24 hours) after injection for measurement of tissue NE decline.

Following 7 days of AngII infusion, blood pressure and heart rate were significantly increased (Table 1), and body weight was significantly decreased (control: 367 + 6; AngII: 302 + 9 g, P < 0.05) compared to controls. Endogenous NE content in control rats was greatest in ISBAT, followed by LV and then EF. The endogenous NE content in EF and ISBAT was not significantly different between AngII and control rats (Table 4). Infusion of AngII for 7 days resulted in a significant decrease in NE content in LV (Table 4). The decline of NE following synthesis inhibition with AMPT in each tissue examined was a mono-exponential process (data not shown). The rate of NE decline (k) was not significantly influenced in any organ following 7 days of AngII infusion; however, the turnover rate (k X NE₀), equal to the rate of NE synthesis, was significantly decreased in LV from AngII-infused rats compared to control (Table 4). The turnover time of NE, or total time required for the dissipation of tissue NE, was significantly increased in EF following AngII infusion; however, in ISBAT the NE turnover time was significantly decreased compared to control.

Following 14 days of AngII infusion, blood pressure and heart rate were significantly increased (Table 1), and body weight was decreased (control: 440 + 10; AngII: 356 + 8 g, P < 0.05) compared to control and compared to starting body weight (data not shown). The endogenous NE content in kidney and LV from AngII-infused rats was significantly decreased compared to control (Fig. 3 and 4A). In contrast, AngII infusion resulted in a significant increase in ISBAT NE content. The decline of NE in tissues from rats in each group following synthesis inhibition was a mono-exponential process (Fig. 3; correlation coefficient > 0.85). The slope of this relationship (Fig. 3), indicating the rate of NE decline, was significantly greater in LV and kidney from AngII-infused rats compared to control (Fig. 4B). The turnover rate of NE was significantly increased in ISBAT from AngII-infused rats compared to control (Fig. 4C). The turnover time was significantly decreased in all tissues from AngII-infused rats compared to control (Fig. 4D).

The effect of propranolol administration on AngII regulation of body weight. The purpose of this study was to determine the effect of -adrenergic receptor blockade on AngII regulation of body weight. Four groups of rats (n = 6 rats/group) were examined: saline control, saline + propranolol, AngII, AngII + propranolol. AngII was infused at dose of 350 ng/kg/min for 7 days. The non-selective -adrenergic receptor antagonist, propranolol was administered in the drinking water (50 µg/kg/day) for 3 days prior to mini-pump implantation, followed by co-infusion of propranolol (7 µg/kg/min) with AngII in the osmotic mini-pump for the remainder of the experimental protocol. Food intake, water intake, and body weight were measured daily. Systolic blood pressure was measured by tail cuff in anesthetized rats on day 0 (pre-pump implantation) and day 7.

Analysis of variance demonstrated a significant effect of AngII on systolic blood pressure [F_1,12 = 6, P < 0.05) (Table 1). Infusion of AngII increased systolic blood pressure compared to baseline values (data not shown) and compared to control (Table 1). When rats were co-infused with propranolol, AngII-mediated increases in blood pressure were eliminated.

Analysis of variance of daily body weight measurements demonstrated a significant effect of propranolol [F_1,12 = 17, P < 0.05], a significant effect of AngII [F_1,12 = 10, P < 0.05], and a significant effect of time of infusion [F_10,120 = 7, P < 0.05]. Administration of propranolol to control rats resulted in an increase in body weight on days 1 - 7 (Fig. 5A). Infusion of AngII resulted in a reduction in body weight compared to control on days 2 - 7. In rats treated with AngII and propranolol, AngII-mediated reductions in body weight were totally abolished.

Analysis of variance of daily measurements of food intake demonstrated a significant effect of propranolol [F_1,12 = 12, P < 0.05], a significant effect of AngII [F_1,12 = 9, P < 0.05], and a significant effect of time of infusion [F_8,8 = 15, P < 0.05]. There was no effect of propranolol administration on food intake in control rats (Fig. 5B). Infusion of AngII resulted in a decrease in food intake on days 1 - 7. Co-infusion of AngII and propranolol eliminated AngII-mediated reductions in food intake from days 2 - 7.

Analysis of variance of daily measurements of water intake demonstrated a significant effect of propranolol [F_1,12 = 4, P < 0.05], a significant effect of AngII [F_1,12 = 5, P < 0.05], and a significant effect of time of infusion [F_8,8 = 10, P < 0.05]. Propranolol administration had no effect on water intake in control rats (Fig. 5C). Infusion of AngII resulted in an increase in water intake on days 1 - 7. Co-infusion of AngII and propranolol totally eliminated AngII-mediated increases in water intake.

Discussion

Results from this study demonstrate that AngII regulation of body weight is associated with tissue-specific increases in sympathetic activity. Increases in the turnover time of NE occurred in tissues with cardiovascular (LV, kidney) and metabolic (EF, ISBAT) relevance to the effects of AngII. In ISBAT, a tissue involved in peripheral energy expenditure through non-shivering thermogenesis, chronic AngII infusion resulted in an increase in tissue NE content and the rate of NE synthesis. Coincident with these observations, the density of NE uptake transporter sites was dose-dependently increased in ISBAT following chronic AngII infusion, indicative of a greater density of sympathetic innervation. Finally, blockade of -adrenergic receptors in AngII-infused rats by co-administration of propranolol reversed AngII-mediated reductions in body weight. Collectively, these results support the hypothesis that AngII regulates body weight by activating the sympathetic nervous system.

Previous results demonstrate that AngII facilitates sympathetic neurotransmission through mechanisms including enhanced release of NE (Storgaard et al., 1997; English and Cassis, 1999), regulation of the uptake and removal of NE (Lu et al, 1996), and regulation of tyrosine hydroxylase activity and the synthesis of NE (Yang and Raizada, 1998). These effects of AngII, either formed systemically or through local tissue renin-angiotensin systems, have been implicated in the pathology of cardiovascular diseases such as hypertension and congestive heart failure. Previous results from our laboratory suggest adipose tissue as a site for a local renin-angiotensin system, with adipose AngII production involved in the regulation of sympathetic neurotransmission and the control of lipid metabolism (Cassis and Dwoskin, 1992; Cassis, 1994; English and Cassis, 1999). Based on these observations, we hypothesized that AngII, formed either systemically or through an adipose renin-angiotensin system, would stimulate sympathetic neurotransmission to adipose tissue for the control of lipid metabolism, and thereby regulate body weight.

Previous results from this laboratory demonstrated that chronic infusion of AngII decreased body weight in a dose-dependent manner independent of elevations in blood pressure (Cassis et al., 1998). In this study, AngII regulation of body weight occurred in a dose-dependent manner, and was associated with dose-dependent reductions in food intake and elevations in water intake. In addition, chronic AngII infusion decreased organ mass in a tissue-specific manner with the most pronounced reductions in white adipose tissue mass. Results from this study confirm and extend previous findings by demonstrating parallel dose-dependent effects of AngII to reduce food intake, adipose tissue mass and body weight.

We examined several indices of sympathetic function in rats chronically infused with AngII, including plasma and tissue NE concentration, NE uptake transporter density and the tissue-specific turnover of NE. Results from this study demonstrate that chronic infusion of AngII elevated plasma NE concentration, indicative of a generalized increase in sympathetic activity. These results are in agreement with previous findings demonstrating that the chronic infusion of similar doses of AngII increased plasma NE concentration in a time-dependent manner (Henegar et al., 1995). The measurement of plasma NE concentration, while indicative of systemic sympathetic activity, represents the balance between spillover and clearance of NE in plasma, and thus does not provide tissue-specific information. Therefore, we determined the effect of chronic AngII infusion on tissue-specific indexes of sympathetic activity, including NE turnover and NE uptake transporter binding density.

Previous studies demonstrated that acute and chronic AngII exposure stimulated NE uptake in neuronal cell cultures (Lu et al., 1996). To examine the effect of AngII infusion on the NE uptake transporter, we used [³H]nisoxetine as a highly selective ligand for the NE uptake transporter binding site (Tejani-Butt, 1992). Both the affinity and the density for [³H]NIS binding in ISBAT were similar to previously reported values (King et al., 1999). Moreover, in tissues from control rats, the density of NE uptake transporter sites in ISBAT was approximately 10-fold greater than in LV, demonstrating the dense sympathetic innervation of ISBAT. Results from this study demonstrate that chronic infusion of AngII dose-dependently increased ISBAT NET binding density. These findings are in agreement with previous results demonstrating that chronic AngII exposure increases the gene transcription and translation of the NET (Yu et al., 1996). Moreover, results from this study extend previous findings by demonstrating regulation of the NET in a peripheral tissue following chronic in vivo AngII exposure. Interestingly, the ability of AngII to regulate the NET was specific to ISBAT, with no changes in NET binding density in LV. Differential tissue-specific effects of AngII to regulate the NET may be related to the density of sympathetic innervation (10-fold higher NE content in ISBAT than LV) or the relative density of AngII receptors (2-fold greater in ISBAT than LV, Cassis et al., 1998a). Interestingly, maximal regulation of the ISBAT NET by AngII occurred at doses that maximally regulate body weight, suggesting inter-dependence of these two measured variables.

In addition to examining the NE uptake transporter, we measured tissue NE turnover following chronic AngII infusion. NE turnover is a useful indicator of sympathetic function in specific tissues. The method we chose for assessment of NE turnover is based on that originally described by Brodie et al. (1966), which examines the decline of NE in specific tissues following synthesis inhibition. In all of the tissues examined in this study the rate of NE decline after synthesis inhibition followed a mono-exponential process with correlation coefficients of the slope of decline greater than 0.85. Interestingly, the calculated rate of NE decline and the turnover time of NE were similar in all tissues examined from control rats. The original report by Brodie et al., (1966) suggested that similarities in NE turnover time across tissues with varying densities of innervation implies that individual neurons within a tissue synthesize NE at the same rate. In contrast, the endogenous NE content and the rate of NE synthesis (K) differed markedly across these same tissues. Thus, differences in the rate of NE synthesis, despite the similarity in NE turnover time, arise from regional differences in neuron density and are reflected by the endogenous NE content. In agreement with previous observations, the rate of NE synthesis (K) in the present study across the various tissues examined from control rats paralleled differences in the endogenous NE content.

We examined the effect of chronic AngII infusion on NE turnover in tissues (LV, kidney) of cardiovascular relevance (hypertension) to the effects of AngII. In the LV from rats treated with AngII for 7 days, endogenous NE content was decreased, as was the rate of NE synthesis. However, the rate of NE decline and the turnover time for NE were not influenced by 7 days of AngII exposure. These results suggest that chronic infusion of AngII for 7 days decreases primarily the synthesis of NE, reducing the endogenous NE store. Alternatively, reductions in the endogenous NE content and rate of NE synthesis may result from a reduction in the density of sympathetic innervation following 7 days of AngII infusion. The observed decrease in NE content in LV is in agreement with previous findings demonstrating reductions in cardiac NE content following 10 days of i.v. AngII infusion (Kline et al., 1990). However, in contrast to results from the present study, the rate constant for NE decline was not significantly altered by 10 days of AngII infusion (Kline et al., 1990). Differences in results between these studies may arise from the dose, route, and duration of AngII administration. In this study following 14 days of AngII infusion, endogenous NE content remained reduced compared to controls and was accompanied by an increase in the rate of NE decline and a decrease in the NE turnover time. Taken together, results from this study suggest that chronic AngII infusion initially decreases NE synthesis in the heart, followed by a marked increase in NE turnover (and presumably NE release).

In the kidney, chronic AngII infusion for 14 days increased the rate of NE decline, reduced the endogenous NE content, but did not influence NE synthesis rate. The observed decrease in NE content in kidney is in agreement with previous findings demonstrating reductions in kidney NE content following 10 days of i.v. AngII infusion (Kline et al., 1990). Reductions in endogenous NE content in the kidney following chronic AngII exposure are suggested to result from enhanced NE release, without accompanying compensatory increases in NE synthesis, ultimately reducing endogenous NE tissue stores. In summary, results from this study demonstrate that chronic AngII infusion increases NE turnover in tissues of cardiovascular relevance to the effects of AngII.

In the EF, a white adipose tissue of metabolic relevance, the effects of AngII on NE turnover were influenced by the time of exposure. Following 7 days of AngII infusion, the turnover time of NE was increased, suggesting that AngII initially reduced sympathetic activity to white adipose tissue. However, more prolonged exposure to AngII (14 days) resulted in a reduction in turnover time of NE in EF, demonstrating that sympathetic activity had increased with longer durations of AngII exposure.

In ISBAT, an tissue important in non-shivering thermogenesis, chronic AngII exposure resulted in a progressive increase in NE turnover. In contrast to the other tissues examined, endogenous NE content and the rate of NE synthesis increased in ISBAT following 14 days of AngII infusion, despite a faster turnover time of tissue NE. Of note, the turnover time of NE was similar across all tissues from AngII infused rats, suggesting that the individual neurons within each tissue synthesized NE at the same rate. Taken together, the findings of an increase in NE content and synthesis rate in ISBAT with similar turnover times of NE across tissues suggest that chronic AngII infusion may have increased the density of sympathetic neurons in ISBAT. In support, results from this study demonstrate an increase in NE uptake transporter density following the same dose and duration of AngII infusion, suggesting an increased density of sympathetic innervation. Collectively, these results demonstrate that chronic AngII infusion increases sympathetic activity in brown adipose tissue. This effect of AngII is suggested to increase non-shivering thermogenesis of brown adipose tissue and increase peripheral energy expenditure, potentially contributing to AngII regulation of body weight.

We examined the effect of co-administration of the -receptor antagonist, propranolol, on AngII regulation of body weight. The hypothesis of these studies was that AngII facilitation of sympathetic neurotransmission would regulate body weight through -adrenergic receptor effects in organs of metabolic relevance. Our results demonstrate that the administration of propranolol totally eliminated AngII regulation of body weight, food and water intake, and organ mass. The ability of propranolol to eliminate the effect of AngII on food intake suggests interactions between the renin-angiotensin and the sympathetic nervous system in the control of food intake. Previous studies demonstrated that a portion of the drinking response to acute AngII injection was blocked by the administration of propranolol (Katovich and Barney, 1985). Our results extend these findings by demonstrating that the non-selective -receptor antagonist propranolol completely inhibits the chronic dipsogenic response to AngII. In summary, results from this study demonstrate that the primary target of AngII regulation of body weight involves -adrenergic receptor stimulation.

In conclusion, reductions in body weight following chronic AngII infusion are dose-dependent, involve the regulation of food and water intake, and are eliminated by the administration of -receptor antagonists. Moreover, chronic AngII infusion resulted in increased turnover of NE, elevations in plasma NE, and a tissue-specific increase in the density of the NE uptake transporter in brown adipose tissue. Collectively, these results support the hypothesis that AngII regulates body weight by stimulating the sympathetic nervous system, and suggest an important physiological role for an adipose renin-angiotensin system.

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Footnotes

This research was supported by NIH HL58927.

Reprint requests:

Lisa A. Cassis, Ph.D.

Room 434, College of Pharmacy

Division of Pharmaceutical Sciences

University of Kentucky

Lexington, KY 40536-0082

Table 1. Characteristics of Rats.

Data are mean + SEM.

*, denotes significantly different from saline group (P < 0.05).

Table 2. Organ weights normalized to % body weight.

Data are mean + SEM of individual organ wet weight normalized as a percentage of body weight.

ISBAT, interscapular brown adipose tissue; RPF, retroperitoneal fat; EF, epididymal fat; diaphrgm, diaphragm.

*, denotes significantly different from saline group (P < 0.05).

, denotes significantly different from saline/prop group (P < 0.05).

Table 3. [³H]Nisoxetine Binding Kinetics in Left Ventricle and ISBAT Membrane Preparations.

Data are mean + SEM from n = 8/group.

*, denotes significantly different from saline group (P < 0.05).

Table 4. Tissue NE turnover following 7 days of AngII infusion.

Data are mean + SEM. *, denotes significantly different from saline, P < 0.05.

k, the fractional turnover, rate of NE decline (the slope of the decay/0.434).

K, the rate of NE synthesis, the turnover (k x tissue NE content at time 0).

Turnover time, (1/k), the time required for dissipation of tissue NE.

Figure 1. Chronic AngII infusion dose-dependently regulates body weight (A), food intake (B) and water intake (C). Baseline measurements of body weight, food and water intake were taken for 3 days prior to osmotic mini-pump implantation (arrow). Rats were administered either saline (Con), or AngII (200 - 600 ng/kg/min) for 14 days by osmotic mini-pump. Measurements were taken daily at 10:00am. A, Body weight. Chronic infusion of AngII resulted in a significant decrease in body weight. At 200 ng/kg/min of AngII, body weight was decreased compared to control from days 6 - 14. At higher doses of AngII (400, 600 ng/kg/min), AngII infusion resulted in a reduction in body weight on days 2 - 14. The magnitude of the decrease in body weight was dose-dependent at AngII doses of 200 and 400 ng/kg/min. B, Food intake. Chronic infusion of AngII decreased food intake. Reductions in food intake were dose-dependent at AngII doses of 200 and 400 ng/kg/min. C, Water intake. Chronic infusion of AngII increased water intake. Increases in water intake were dose-dependent at AngII doses of 200 and 400 ng/kg/min. Data are mean + SEM from n = 8 rats/group. *, denotes significantly different from control.

Figure 2. Saturation isotherm (top) and Scatchard plot (bottom) for [³H]NIS binding in ISBAT membranes from saline and AngII-infused rats. Rats received either saline or AngII (200 - 600 ng/kg/min) by osmotic mini-pump for 14 days. ISBAT was removed and membranes prepared for radioligand binding analysis of the NE uptake transporter binding site. Top, Saturation binding isotherm for [³H]NIS binding to ISBAT membranes. Aliquots of ISBAT membrane were incubated with increasing concentrations of NIS for 30 minutes. [³H]NIS bound to a single, high affinity site in ISBAT membranes. The density of [³H]NIS binding sites was increased in ISBAT membranes from AngII-infused rats compared to control. Bottom, Scatchard transformation of [³H]NIS binding to ISBAT membranes. The affinity of [³H]NIS binding was not influenced by AngII infusion; however, the density of [³H]NIS binding sites was increased following AngII infusion. Data are mean + SEM from n = 8 rats/group.

Figure 3. Chronic AngII infusion increases the turnover of NE. Rats were administered saline or AngII (400 ng/kg/min) by osmotic mini-pump for 14 days. On the final day of the study, rats were injected with either saline or AMPT and tissues (LV, ISBAT, kidney and EF) were removed for determination of NE content using HPLC with electrochemical detection. The LogNE was plotted against the time after AMPT injection. The decline in tissue NE content was a mono-exponential process in each tissue examined. In LV and kidney, endogenous NE content at time 0 was significantly reduced following 14 days of AngII infusion; in ISBAT, endogenous NE content was increased in AngII-infused rats compared to control. Significant differences in the rate of NE decline were observed in kidney and LV from AngII-infused rats compared to control. Data are mean from n = 5 rats/group/time point.

Figure 4. NE turnover parameters from AngII- and saline-infused rats. Rats were infused with saline or AngII (400 ng/kg/min) by osmotic mini-pump for 14 days. On the final day of the study, rats were injected with either saline or AMPT and tissues (LV, ISBAT, kidney and EF) were removed for determination of NE content using HPLC with electrochemical detection. A, Endogenous NE content following 14 days of AngII or saline infusion. Tissue NE content was increased in ISBAT, and decreased in LV and kidney following 14 days of AngII infusion. B, k, the rate of NE decline. The rate of NE decline was calculated from the slope of the line in Figure 3 for individual tissues. In LV and kidney, chronic infusion of AngII increased the rate of NE decline. C. K, the Turnover rate or rate of NE synthesis. The turnover rate was calculated as k x NE₀. Chronic infusion of AngII increased the turnover rate in ISBAT. D, Turnover time, or time required to dissipate total tissue NE content. The turnover time was calculated as 1/k. The rate of NE dissipation or turnover time was significantly decreased in all tissues examined from AngII-infused rats compared to control. Data are mean + SEM from n = 5 rats/group/time point.

Figure 5. Propranolol administration prevents AngII regulation of body weight (A), food intake (B) and water intake (C). Rats were administered either propranolol (7 µg/kg/min) alone, or propranolol with AngII (350 ng/kg/min) for 7 days. Measurements of body weight, food and water intake were taken daily at 10:00am. Arrow denotes implantation of osmotic mini-pump and beginning of drug infusion. A, Body weight. Chronic infusion of AngII decreased body weight. Reductions in body weight by AngII were totally eliminated by co-infusion with propranolol. B, Food intake. Chronic infusion of AngII decreased food intake. Except for the initial drop in food intake at day 1, chronic administration of propranolol to AngII-infused rats eliminated reductions in food intake. C, Water intake. Chronic infusion of AngII increased water intake. Increases in water intake by AngII were totally eliminated by co-infusion with propranolol. Data are mean + SEM from n = 6 rats/group. *, denotes significantly different from saline. f, denotes significant difference between saline vs saline+ propranolol group.

Index Terms: Angiotensin, body weight, sympathetic nervous system, norepinephrine, turnover.