Mechanisms contributing to angiotensin II regulation of body
weight
Lisa A.
Cassis,
Dana E.
Marshall,
Michael J.
Fettinger,
Brady
Rosenbluth, and
Robert A.
Lodder
Divisions of Pharmacology and Experimental Therapeutics and
Medicinal Chemistry and Pharmaceutics, College of Pharmacy, University
of Kentucky, Lexington, Kentucky 40536-0082
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ABSTRACT |
Previous studies in
our laboratory have implicated adipose tissue as a potential site for
local angiotensin II (ANG II) synthesis. However, functions of ANG II
in adipose tissue and the impact of ANG II on body weight regulation
are not well defined. To study the effect of ANG II on body weight, a
chronic ANG II infusion model was used. In study
1, a low dose of ANG II (175 ng · kg
1 · min
1)
was infused into rats for 14 days. Plasma ANG II levels were not
elevated after 14 days of infusion. ANG II-infused rats did not gain
weight over the 14-day protocol and exhibited a lower body weight than
controls on day 8. Food intake was not
altered, but water intake was increased in ANG II-infused rats. Blood
pressure gradually increased to significantly elevated levels by
day 14. Thermal infrared imaging
demonstrated an increase in abdominal surface temperature. Measurement
of organ mass demonstrated site-specific reductions in white adipose
tissue mass after ANG II infusion. In study
2, the dose-response relationship for ANG II infusion (200, 350, and 500 ng · kg
1 · min
1)
was determined. Body weight (decrease), blood pressure (increase), white adipose mass (decrease), plasma ANG II levels (increase), and
plasma leptin levels (decrease) were altered in a dose-related manner
after ANG II infusion. In study 3, the
effect of ANG II infusion (350 ng · kg
1 · min
1)
was examined in rats treated with the vasodilator hydralazine. Hydralazine treatment normalized blood pressure in ANG II-infused rats.
The effect of ANG II to decrease body weight was augmented in
hydralazine-treated rats. These results demonstrate that low levels of
ANG II infusion regulate body weight through mechanisms related to
increased peripheral metabolism and independent of elevations in blood
pressure.
weight gain; renin-angiotensin system; metabolism; infrared imagery
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INTRODUCTION |
ANGIOTENSIN II (ANG II), the primary peptide of the
renin-angiotensin system, is important in the regulation of blood
pressure and fluid and electrolyte balance (25). Alterations in the
synthesis of and responsiveness to ANG II have been implicated in the
disease states of hypertension (14) and congestive heart failure (2). On the basis of a variety of evidence, including demonstration of
components necessary for synthesis of ANG II, localization of ANG II
receptors, measurements of immunoreactive ANG II, and functional
responsiveness to ANG II, local tissue renin-angiotensin systems have
been proposed (13, 28). Many of the postulated tissue renin-angiotensin
systems have been implicated in the local control of cardiovascular
functions. For example, evidence supports the existence of tissue
renin-angiotensin systems in blood vessels (28), heart (12), kidney
(13), adrenal (24), and brain (9). Production of ANG II locally in
these tissues contributes to the regulation of vascular resistance and
structure, cardiac contractile state and hypertrophy, cell growth,
sodium and water retention, and activity of the sympathetic nervous
system.
Previous studies in our laboratory demonstrated a high level of
angiotensinogen mRNA expression (8), renin-like activity (32),
localization of high-affinity ANG II receptors (6), and ANG II
regulation of sympathetic neurotransmission (7) in rat adipose tissue.
These results suggest adipose tissue as a potential site for a local
tissue renin-angiotensin system. However, in contrast to the
well-defined role of ANG II in tissue sites of cardiovascular
relevance, the functional role of ANG II in adipose tissue metabolism
and associated alterations in body weight are not well defined.
Recent studies demonstrate that infusion of high pressor doses (500 ng · kg
1 · min
1)
of ANG II to rats resulted in a marked reduction (26%) in body weight
(3). The effect of high-dose ANG II infusion on body weight was
suggested to be independent of elevations in blood pressure (3). In
these studies, measurements of plasma ANG II levels in rats chronically
infused with ANG II were not performed; thus comparisons of plasma ANG
II levels in rats from this chronic high-dose ANG II infusion model
with levels observed previously in patients with cardiovascular
abnormalities such as congestive heart failure could not be made.
Interestingly, it was noted that in heart failure patients exhibiting
five- to eightfold elevations in plasma ANG II levels (26, 35),
anorexia, wasting, and cachexia are frequent problems, culminating in
the dysregulation of body weight (29). Alternatively, in the obese
population, expanded fluid volumes result in suppressed activity of the
systemic renin-angiotensin system (17, 19). We hypothesize that
conditions characterized by chronic alterations in the systemic or
tissue renin-angiotensin systems are associated with dysregulation of
body weight due to the metabolic effects of ANG II. The present study
utilized a previously established model for examination of ANG II
mechanisms in hypertension, the chronic ANG II infusion model, to
determine mechanisms for ANG II regulation of body weight.
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METHODS |
General experimental design.
Three different studies were performed. In study
1, the effect of chronic low-dose (175 ng · kg
1 · min
1)
ANG II infusion on body weight, food and water intake, and blood pressure was examined. ANG II or saline
(n = 5/group) was infused into rats
for 14 days. In study 2, the
dose-response relationship for ANG II infusion on body weight, blood
pressure, and food and water intake was examined. Four groups of rats
(n = 3/group) were examined for 7 days
and infused with saline or ANG II at doses of 200, 350, and 500 ng · kg
1 · min
1.
In study 3, the effect of ANG II on
body weight was examined in rats that were treated with the vasodilator
hydralazine. Four groups (n = 4/group)
of rats were examined for 7 days, saline infused with or without
hydralazine, and ANG II infused with or without hydralazine. In all
three studies, body weight and food and water intake were measured
daily, and measurements of blood pressure were taken every 3-5
days.
Angiotensin II infusion model.
Male Sprague-Dawley rats (Harlan Laboratories, Indianapolis, IN) were
used in all studies. Rats ranged in body weight from 350 to 450 g. All
rats were housed two per cage in an approved animal facility for 1 wk
before use under normal light-dark cycles and were given free access to
food and water. During each experimental protocol, rats were housed
individually for measurement of body weight and food and water intake
on a daily basis at 10:00 AM. Baseline measurements of food intake,
water intake, and blood pressure were performed on all rats in an
individual study for a minimum of 3 days preceding each experimental
protocol.
For ANG II infusion, rats were anesthetized with diethyl ether and
shaved in the interscapular region; then osmotic minipumps (model 2002 for 14-day infusion, model 2001 for 7-day infusion; Alza, Palo Alto,
CA) were implanted subcutaneously. Minipumps contained either ANG II
(Sigma Chemical, St. Louis, MO; 175-500 ng · kg
1 · min
1
infusion rate) or sterile saline and were primed according to the
manufacturer's instructions preceding implantation to assure immediate
subcutaneous delivery of ANG II. The skin overlaying the minipump was
closed with surgical staples, and rats were allowed to recover on
warmed heating pads.
Indirect systolic pressure by tail cuff plethysmography.
In study 1, systolic pressure was
measured in conscious restrained rats by use of a Narco system. In
studies 2 and
3, systolic pressure was measured on
ether-anesthetized rats using an inflatable tail cuff, a pressure and
pulse transducer, and a recording polygraph. Alterations in blood
pressure from anesthesia were controlled for across ANG II- and
saline-infused rats. The systolic pressure from three separate
measurements was averaged from each rat. Baseline systolic pressure was
recorded for 3 days preceding implantation of osmotic minipumps. After
implantation of ANG II-containing minipumps, blood pressure was
measured every 3-5 days.
Hydralazine treatment.
Hydralazine (15 mg/kg) was administered in the drinking water of
individual rats in study 3.
Hydralazine dosing was based on an average water consumption of
40-60 ml of water intake per day. The dose of hydralazine in the
drinking water was adjusted daily on the basis of the preceding 24-h
water intake and daily body weight measurements in individual rats.
Thermal infrared imaging.
Thermal infrared (IR) imaging was used in study
1 as an index of peripheral energy expenditure. Thermal
IR imaging was performed using a liquid nitrogen-cooled InSb focal
plane array camera (temperature precision = 0.03°C;
3,000-5,000 nm; Cincinnati Electronics, Mason, OH) with sound
annotation capability. No external light source was used in comparing
heat radiation in the different rats, so the intensity of features in
each rat image corresponded to the level of blackbody emission from the
skin and fur. Temperature calibration was accomplished using a
blackbody source closely coupled to a mercury thermometer. The IR
images were collected as 1-s segments of real-time video and saved on
computer disk. The IR video camera had a frame collection rate of 51.44 frames/s, making sample target immobilization unnecessary. Thermal IR
heat radiation was determined in joules per second (W) using standard software based on Stefan's Law.
Measurement of plasma ANG II.
Trunk blood was collected in heparanized vacuum test tubes containing
the following buffer: 0.15 mM pepstatin A, 20 mM phenanthroline, 125 mM
EDTA, 0.2% neomycin, 2% ethanol, 2% DMSO, and 0.1 M kallikrein, pH
7.4. The inhibitors in this buffer were added to eliminate breakdown of
angiotensin peptides as well as further production of peptides during
sample handling (4). Plasma was obtained by centrifugation (3,000 g) of blood at 4°C for 30 min.
Plasma samples were partially purified using Sep-Pak
C18 column chromatography (Waters,
Milford, MA), with the columns preequilibrated with 4 ml of methanol, 4 ml of water, and 10 ml of buffer. Angiotensin peptides were eluted from
the columns with 2 ml of methanol-water-trifluoracetic acid (70:29:1).
The eluate was evaporated overnight using a speed-vac (Savant). Plasma
ANG II was measured in preextracted samples, which were reconstituted
in 100 µl of ANG II RIA buffer (0.1 M K2HPO4,
3.0 mM EDTA, 0.15 mM 8-hydroxyquinoline, and 0.25% BSA, pH 7.2),
sonicated for 5 min, and stored at
20°C. Angiotensin content
in each sample was measured by ANG II RIA using a polyclonal ANG II
antibody (kindly supplied by Dr. A. Freedlender, University of
Virginia) exhibiting minimal cross-reactivity to ANG I (2%) and
angiotensin 5-8 (4%) but 100% cross-reactivity to ANG III, angiotensin 3-8, and angiotensin 4-8. The sensitivity of the
RIA was 2 pg/ml.
Measurement of plasma leptin.
Blood was obtained as described above, and an aliquot (500 µl) of
plasma was removed for measurement of plasma leptin levels by use of a
commercial RIA kit (Linco Research, St. Louis, MO) with a rat leptin
antibody. The sensitivity of the kit for rat leptin was 0.5 ng/ml and
required 100 µl of rat plasma for assay.
Statistical analysis.
For all studies, data are means ± SE. In study
1, data (blood pressure, body weight, food and water
intake) were analyzed using a two-way ANOVA, with ANG II as a
between-group factor and time of infusion as a within-group repeated
measure. In study 2, data were
analyzed using a two-way ANOVA, with ANG II dose as a
between-group factor and time as a within-group repeated measure. In
study 3, data were
analyzed using a three-way ANOVA, with ANG II dose and hydralazine
treatment as between-group factors and time as a within-group repeated
measure. Post hoc analysis was performed using Duncan's multiple range
test.
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RESULTS |
Study 1.
The purpose of this study was to determine the effect of chronic
low-dose ANG II infusion on the regulation of body weight. In
study 1, ANG II was administered at an
infusion dose of 175 ng · kg
1 · min
1
to rats for a period of 14 days. Plasma ANG II levels after 14 days of
infusion were not significantly different between ANG II- and
saline-infused rats (saline: 37.3 ± 1.5; ANG II: 34.3 ± 8.2 pg/ml). Measurements of systolic blood pressure demonstrated a
significant between-group effect of ANG II
[F(1,7) = 57.7, P < 0.001] and a significant
interaction between ANG II and time of infusion
[F(5,35) = 2.7, P < 0.05]. Systolic blood
pressure was significantly increased in ANG II-infused rats over
baseline (day 0) levels by
day 1 of ANG II infusion (Fig.
1). Moreover, systolic pressure in ANG
II-infused rats was increased compared with saline controls at
day 1. Initial increases in blood
pressure (days 1 and
3) in ANG II-infused rats were
followed by a return to levels not significantly different from
saline-infused controls or from baseline measurements
(day 0, ANG II-infused rats) on days 4 and
7. Despite three baseline measurements
before initiation of the experimental protocol, systolic pressures
measured in conscious, restrained, saline-infused rats decreased over
the time course of the study, suggesting that habituation to the
restraining apparatus occurred over the time course of study. For this
reason, systolic pressures were measured on ether-anesthetized rats in
studies 2 and
3. After 14 days of ANG II infusion,
systolic blood pressure was significantly increased in ANG II-infused
rats compared with saline controls and compared with baseline
measurements (day 0; Fig. 1).

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Fig. 1.
Effect of angiotensin (ANG) II infusion (175 ng · kg 1 · min 1)
on systolic blood pressure. In study
1, rats were infused with saline or ANG II
subcutaneously by osmotic minipump for 14 days, and systolic blood
pressure was measured. Baseline measurements of systolic pressure were
performed for 3 days before pump implantation. Systolic pressure
increased in ANG II-infused rats over controls at 1 and 3 days of
infusion, followed by a decline to levels not significantly different
from controls on days 4 and
7. At 14 days, systolic pressure was
significantly increased in ANG II-infused rats over saline-infused
controls and from baseline measurements in the ANG II group before pump
implantation (day 0). Values are
means ± SE of 5/group. * Significantly different
(P < 0.05) from sham (saline-infused) controls;
f significantly different
(P < 0.05) from day 0 within each group.
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Daily body weight measurements revealed a significant interaction
between ANG II and time of infusion
[F(15,105) = 3.1, P < 0.05]. The body weight of
ANG II-infused rats was significantly different from saline-infused
controls from day 8 of infusion through the remainder of the experimental protocol (Fig.
2A). Saline-infused rats gained 27 g in body weight over the 14-day experimental protocol; in contrast, ANG II-infused rats did not gain
weight over the 14-day protocol. Thus the primary effect of low-dose
ANG II infusion was to eliminate weight gain. The time course for the
effect of ANG II on body weight (Fig.
2A) and mean arterial pressure (Fig.
1) illustrates that these two variables did not change in parallel.

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Fig. 2.
Effect of ANG II infusion (175 ng · kg 1 · min 1)
on body weight (A), food intake
(B), and water intake
(C). In study
1, rats were infused with saline or ANG II for 14 days,
and body weight, food intake, and water intake were measured daily at
10:00 AM. Saline-infused rats gained weight over the 14-day protocol;
in contrast, ANG II-infused rats did not gain weight
(A). Body weight was significantly
different in ANG II-infused rats compared with saline controls from
day 8 onward. After an initial
transient drop in food intake at day 1 after pump implantation, food intake was not altered in ANG
II-infused rats (B). Water intake was increased in ANG II-infused
rats compared with controls from day 3 onward (C). Values are means ± SE of 5/group. * Significantly different
(P < 0.05) from saline control.
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The effect of ANG II infusion on body weight was not the result of
reductions in food intake (Fig. 2B).
After an initial transient drop in food intake at 1-2 days after
minipump implantation, ANG II infusion did not result in significant
alterations in food intake. In contrast, infusion of ANG II
significantly increased water intake (Fig.
2C). Increases in water intake were
significant by 3 days of ANG II infusion and were maintained throughout
the ANG II infusion protocol. Moreover, increases in water intake occurred before ANG II-induced reductions in body weight.
Thermal IR imaging demonstrated an increase in regional surface
temperature in the tail and abdomen/thorax area of ANG II-infused rats
compared with saline controls (Fig.
3A). An
IR image of an ANG II-infused rat is illustrated in Fig.
3B. The surface temperature in the
abdomen of the ANG II-infused rat was of higher intensity than that of
the saline-infused control rat (Fig.
3A).

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Fig. 3.
Thermal infrared (IR) imaging demonstrates an increase in tail and
abdomen surface temperature. In study
1, rats were infused with saline or ANG II (175 ng · kg 1 · min 1)
for 14 days. Thermal IR imaging was performed on day
14 using an InSb focal plane array camera. Temperature
calibration was accomplished using a blackbody source closely coupled
to a mercury thermometer. Surface temperature was increased in the tail
and abdomen from ANG II-infused rats compared with saline controls
(A). * Significantly different (P < 0.05) from saline control.
B: image of an ANG II-infused rat.
Note visible detection of temperature in abdomen and tail.
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A variety of organs were removed from ANG II-infused and saline-infused
rats at the end of the experimental protocol, and organ weight-to-body
weight ratios were constructed to determine sites contributing to ANG
II-induced decreases in body weight (Fig.
4). Of the tissues examined, ANG II-infused
rats exhibited significant decreases in the relative mass of
retroperitoneal white fat (RPF) (adipose tissue) and the diaphragm
(skeletal muscle). The other organs examined maintained their relative
mass after ANG II infusion.

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Fig. 4.
Effect of ANG II infusion (175 ng · kg 1 · min 1)
on organ mass. In study 1, rats were
infused with saline (open bars) or ANG II (hatched bars) for 14 days.
Organs were removed at 14 days and normalized as a percentage of body
weight. ISBAT, interscapular brown adipose tissue; EF, epididymal fat;
RPF, retroperitoneal fat; LV, left ventricle; Diaph, diaphragm. Mass of
RPF and diaphragm was decreased in ANG II-infused rats compared with
saline controls. Values are means ± SE of 5/group.
* Significantly different (P < 0.05) from saline
control.
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Study 2.
To determine whether the effect of ANG II on body weight was dose
dependent, rats were administered either saline or 200, 350, or 500 ng · kg
1 · min
1
ANG II via osmotic minipump for 7 days. Measurement of plasma ANG II
levels demonstrated a significant effect of ANG II
[F(3,11) = 5.9, P < 0.05; Table
1]. Measurement of blood pressure
demonstrated a significant effect of ANG II dose
[F(3,8) = 7.2, P < 0.05], a significant
effect of time of infusion
[F(3,24) = 32, P < 0.05], and a significant
interaction between ANG II dose and time of infusion
[F(9,24) = 2.5, P < 0.05]. Mean arterial
pressure was significantly increased in rats receiving 350 ng · kg
1 · min
1
of ANG II infusion over controls at day
3, with blood pressure increased over controls at all
three doses of ANG II by day 7 of ANG
II infusion (Fig. 5). Moreover, at
day 7 of ANG II infusion, blood
pressure increases in rats receiving 500 ng · kg
1 · min
1
ANG II were significantly greater than those observed in rats receiving
the ANG II dose of 200 ng · kg
1 · min
1.

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Fig. 5.
Dose-related effects of ANG II infusion on systolic blood pressure. In
study 2, rats were infused with saline
or 200, 350, or 500 ng · kg 1 · min 1
ANG II for 7 days. Systolic blood pressure was measured before minipump
implantation (day 0) and at 3, 5, and 7 days postimplantation. Systolic pressure increased in a dose- and
time-dependent manner with ANG II infusion. Values are means ± SE
of 3/dose. * Significantly different (P < 0.05)
from control; f significantly
different (P < 0.05) from 200 ng · kg 1 · min 1
ANG II-infused rats.
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Examination of body weight in ANG II-infused rats demonstrated a
significant interaction between ANG II dose and time of infusion [F(8,64) = 25, P < 0.01]. At the lowest dose
of ANG II infused (200 ng · kg
1 · min
1),
body weight was significantly decreased from saline-infused controls at
day 7 of ANG II infusion (Fig.
6A).
However, at ANG II infusion doses of 350 and 500 ng · kg
1 · min
1,
body weight was significantly decreased from saline-infused controls by
day 5 of ANG II infusion and remained
lower throughout the remainder of the experimental protocol. The time
course for the effect of ANG II infusion on body weight (Fig.
6A) and blood pressure (Fig. 5)
illustrates that these two variables did not change in parallel.

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Fig. 6.
Dose-related effects of ANG II infusion on body weight
(A), food intake
(B), and water intake
(C). In study
2, rats were infused with saline or 200, 350, or 500 ng · kg 1 · min 1
of ANG II for 7 days. ANG II infusion resulted in a time-dependent
decrease in body weight (A) compared
with saline controls (sham). All doses of ANG II infusion resulted in
an initial transient drop in food intake
(B), followed by a return to food
intake levels that were not significantly different from control. There
were no significant effects of ANG II infusion on water intake
(C). Values are means ± SE of
3/dose. * Significantly different (P < 0.05) from
control.
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There was no significant effect of ANG II dose on food intake; however,
there was a significant effect of the time of ANG II infusion on food
intake [F(2,56) = 12.3, P < 0.01]. Reductions in food
intake were evident at all three doses of ANG II infused 1 day after
minipump implantation and returned to control levels by 7 days of ANG
II infusion (Fig. 6B). The time
course for the return of food intake to normal levels was dependent on
the ANG II infusion dose. Water intake was not significantly altered by ANG II infusion (Fig. 6C).
Examination of the relative mass (%body weight) of organs removed from
ANG II- and saline-infused rats demonstrated a site-specific decrease
in the mass of RPF (Fig. 7) that was
dependent on ANG II dose. Interestingly, measurement of plasma leptin
levels at day 7 demonstrated a
significant between-group effect of ANG II
[F(3,11) = 4, P < 0.05] and a decrease in
plasma leptin levels at the ANG II dose of 500 ng · kg
1 · min
1
(Fig. 8).

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Fig. 7.
Dose-related effects of ANG II infusion on organ mass. In
study 2, rats were infused with saline
or 200, 350, or 500 ng · kg 1 · min 1
of ANG II for 7 days. Organs were removed on day
7, and organ weight was normalized as a percentage of
body weight. See Fig. 4 for abbreviations. ANG II infusion resulted in
a dose-related decrease in mass of retroperitoneal white fat. Values
are means ± SE of 3/dose. * Significantly different
(P < 0.05) from saline control.
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Fig. 8.
ANG II infusion results in a decrease in plasma leptin levels. In
study 2, rats were infused with saline
or 200, 350, or 500 ng · kg 1 · min 1
of ANG II for 7 days. Plasma was collected on day
7, and leptin levels were measured with a commercial
rat leptin RIA. Statistical analysis demonstrated a significant effect
of ANG II dose on plasma leptin levels. At a dose of 500 ng · kg 1 · min 1,
ANG II infusion resulted in a decrease in plasma leptin levels. Values
are means ± SE of 3/dose. * Significantly different
(P < 0.05) from saline control.
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Study 3.
The purpose of this study was to determine whether the effects of ANG
II on body weight were independent of ANG II-induced increases in blood
pressure. In study 3, ANG II was
infused at a dose of 350 ng · kg
1 · min
1
for a period of 7 days. This dose of ANG II was chosen on the basis of
results from study 2 demonstrating
maximal effects of ANG II on body weight at an infusion dose of 350 ng · kg
1 · min
1.
The vasodilator hydralazine was administered (10 mg/kg) in the drinking
water of ANG II-infused and saline-infused rats for 3 days before
minipump implantation and for the period corresponding to ANG II
infusion.
Measurement of systolic pressure demonstrated a significant effect of
ANG II infusion [F(1,12) = 14.7, P < 0.01] and hydralazine treatment [F(1,12) = 6.1, P < 0.05] and a significant
interaction between ANG II infusion and hydralazine treatment
[F(1,12) = 5.4, P < 0.05]. Blood pressure was
significantly increased in ANG II-infused rats compared with
saline-infused controls by day 2 and
throughout the remainder of the experimental protocol (Fig.
9). In contrast, blood pressure did not
increase in ANG II-infused rats treated with hydralazine.

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Fig. 9.
Hydralazine treatment normalizes blood pressure in ANG II-infused rats.
In study 3, rats were infused with ANG
II (350 ng · kg 1 · min 1)
or saline for 7 days. Separate groups of ANG II-infused rats and saline
controls were treated with the vasodilator hydralazine (10 mg/kg) in
drinking water for 3 days before minipump implantation and for
remainder of protocol. Measurement of systolic pressure demonstrated an
increase in pressure in ANG II-infused rats that was prevented by
treatment with hydralazine. Values are means ± SE of 4/group.
* Significantly different (P < 0.05) from saline
control.
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Examination of body weight demonstrated a significant effect of time of
infusion [F(7,84) = 5.3, P < 0.01] and a significant interaction between ANG II and time of infusion
[F(7,84) = 26.4, P < 0.01]. Both groups of
saline-infused rats (with or without hydralazine) increased their body
weight over the 7-day experimental protocol (Fig.
10A).
In contrast, ANG II-infused rats did not change in body weight over the
7-day protocol. Body weight of ANG II-infused rats was significantly
decreased from saline-infused controls beginning at
day 5 of ANG II infusion.
Interestingly, ANG II-infused rats receiving hydralazine exhibited
reductions in body weight over the 7-day experimental protocol.
Beginning at day 3 of ANG II infusion,
body weights of hydralazine-treated rats infused with ANG II were
significantly less than controls. Moreover, beginning at
day 4, the body weights of ANG
II-infused rats receiving hydralazine were significantly less than ANG
II-infused rats. Thus, despite elimination of ANG II-induced increases
in blood pressure with hydralazine treatment, the effect of ANG II on
body weight was maintained in hydralazine-treated rats.

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Fig. 10.
Effects of treating ANG II-infused rats with hydralazine on body weight
(A), food intake
(B), and water intake
(C). In study
3, rats were infused with saline (with or without
hydralazine) or ANG II (350 ng · kg 1 · min 1;
with or without hydralazine) for 7 days, and body weight was measured 4 days before pump implantation and throughout remainder of protocol.
Body weight decreased in ANG II-infused rats in a time-dependent
manner. Body weight decreases were augmented in ANG II-infused rats
treated with hydralazine. Food intake
(B) decreased in ANG II-infused rats
compared with saline controls. Decreases in food intake were not
prevented in hydralazine-treated rats. Water intake
(C) increased in ANG II-infused rats
treated with hydralazine. Values are means ± SE of 4/group.
* Significantly different (P < 0.05) from saline
control; f significantly different
(P < 0.05) from ANG II-infused rats (without
hydralazine).
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In study 3, there was a significant
effect of ANG II on food intake
[F(1,12) = 29.8, P < 0.01] and a significant
effect of time of ANG II infusion
[F(1,12) = 10.2, P < 0.01]. Beginning at
day 1 of ANG II infusion, food intake
was significantly decreased in ANG II-infused rats with or without
hydralazine compared with saline controls (with or without hydralazine)
(Fig. 10B). In contrast, at a dose
of 350 ng · kg
1 · min
1
of ANG II infusion, water intake did not significantly increase (Fig.
10C). In ANG II-infused rats treated
with hydralazine, water intake increased by day
1 of infusion and throughout the remainder of the
experimental protocol.
Infusion of ANG II at 350 ng · kg
1 · min
1
resulted in an increase in the relative mass of the left ventricle and
a decrease in the relative mass of RPF (Fig.
11). Alterations in the mass of each of
these organs were not reversed with normalization of blood pressure in
hydralazine-treated rats. Plasma leptin levels were significantly
decreased in ANG II-infused rats compared with saline controls (Fig.
12). However, in ANG II-infused rats
treated with hydralazine, plasma leptin levels were diminished but not significantly different from controls (with or without hydralazine) or
ANG II-infused rats. Thus treatment with hydralazine resulted in a
partial reversal of ANG II-induced decreases in plasma leptin levels.

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Fig. 11.
Effect of hydralazine (Hyd) treatment on ANG II-induced alterations in
organ mass. In study 3, rats were
infused with saline (with or without Hyd) or ANG II (350 ng · kg 1 · min 1;
with or without Hyd) for 7 days. ANG II infusion resulted in a decrease
in organ mass of RPF, which was augmented in hydralazine-treated rats.
ANG II infusion resulted in an increase in LV mass that was not
prevented by treatment with hydralazine. Values are means ± SE of
4/group. * Significantly different (P < 0.05) from
saline controls.
|
|

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Fig. 12.
Effect of hydralazine treatment on ANG II-induced decreases in plasma
leptin levels. In study 3, rats were
infused with saline (with or without Hyd) or ANG II (350 ng · kg 1 · min 1;
with or without Hyd) for 7 days. ANG II infusion resulted in a decrease
in plasma leptin levels. In Hyd-treated rats, plasma leptin levels were
not different from ANG II-infused rats or saline controls (with or
without Hyd). Values are means ± SE of 4/group.
* Significantly different (P < 0.05) from saline
controls.
|
|
 |
DISCUSSION |
This study clearly demonstrates that ANG II infusion dose dependently
alters the rate of weight gain and decreases body weight through
pressor-independent mechanisms. Mechanisms defined in the present study
that contribute to the effect of ANG II on body weight include an
increase in surface body temperature (energy expenditure), transient
alterations in food intake, and alterations in plasma leptin levels. In
hydralazine-treated rats with normalized blood pressure, infusion of
ANG II resulted in marked reductions in body weight, demonstrating that
the effect of ANG II on body weight was independent of blood pressure.
With the use of infusion doses of ANG II that gradually increased blood
pressure and did not elevate plasma ANG II levels, ANG II infusion was
associated with a total elimination of weight gain.
Infusion of ANG II to rats has been studied extensively as a model for
human renovascular and high-renin hypertension (1, 23, 34). Models of
ANG II infusion have been classified as "pressor" and
"subpressor," referring to the direct vasoconstrictor effects of
ANG II to elicit immediate increases in blood pressure vs. slower
mediated effects of ANG II at doses that do not directly influence
blood pressure (34). Results from the present dose-response studies for
ANG II infusion demonstrate that, at doses of ANG II infusion
classified previously in the literature as pressor (>200
ng · kg
1 · min
1)
and subpressor (<200
ng · kg
1 · min
1)
(34), ANG II infusion resulted in a total elimination of weight gain
and a decrease in body weight compared with saline-infused controls.
Results from study 1 demonstrate that,
after infusion of ANG II at a dose of 175 ng · kg
1 · min
1
for 14 days, plasma ANG II levels were not elevated. Plasma ANG II
levels measured in control rats in the present study are in agreement
with literature values for rat plasma ANG II, ranging from 8 to 30 pg/ml (31). Previous studies suggest that at infusion doses of 200 ng · kg
1 · min
1,
plasma ANG II levels increased threefold; however, elevations in plasma
ANG II levels were not statistically significant because of a large
variability (61% coefficient of variation) in measurements (15). In
the present study, measurements of ANG II levels in rat plasma were not
associated with marked variability (controls: 4-10%; ANG II
infused: 10-20% coefficient of variation). The threshold dose of
ANG II infusion resulting in an increase in plasma ANG II levels in the
present study was 350 ng · kg
1 · min
1.
At doses of ANG II infusion <200
ng · kg
1 · min
1,
alterations in systemic ANG II levels were not evident and are suggested to represent the high end of physiological ANG II levels.
Measurement of plasma ANG II levels in ANG II-infused rats in the
present study demonstrated a three- and sixfold increase at the two
highest doses of ANG II infusion (350 and 500 ng · kg
1 · min
1,
respectively). Previous investigators have demonstrated a sevenfold increase in plasma ANG II levels in patients with human heart failure
(26). Increases in plasma ANG II levels in the rat model used in the
present study (0, 3-, and 6-fold) are below the reported multiples of
increase (7-fold) in systemic renin-angiotensin system activation in
human heart failure (26, 35). Thus alterations in body weight were
evident in ANG II-infused rats at doses resulting in minimal elevations
in plasma ANG II levels. The significance of the observed effects of
low-dose ANG II infusion on body weight may also relate to human
obesity, in which plasma volume expansion is typical with suppressed
activity of the renin-angiotensin system (17, 19).
Previous investigators have demonstrated that subcutaneous ANG II
infusion at a dose of 200 ng · kg
1 · min
1
resulted in an increase in blood pressure within 1 day postinfusion (15). At a subcutaneous ANG II infusion dose of 76 ng · kg
1 · min
1,
systolic blood pressure increased by day
2 of ANG II infusion (21). In agreement with previous
studies, results from this study demonstrate that infusion of ANG II at
a dose of 175 ng · kg
1 · min
1
resulted in an initial transient increase in systolic pressure at
days 1 and
3 of infusion. In contrast to previous
reports and results from the present study, at doses of 280 (11) and
200 ng · kg
1 · min
1
(18), administered intraperitoneally, systolic blood pressure did not
increase after 7 days of ANG II infusion, suggesting that these ANG II
doses were subpressor. Results from the present study demonstrate
dose-dependent effects of ANG II infusion on systolic blood pressure
that were influenced by the time of infusion. However, all doses of ANG
II infusion used in the present study resulted in an early increase in
systolic pressure. Thus distinctions between pressor and subpressor
doses of ANG II infusion in the present study were not readily
apparent.
At low-dose ANG II infusion in study
1, water intake increased. In agreement with these
results, previous studies demonstrate dipsogenic effects of
systemically administered ANG II (37). Interestingly, in the present
study, high ANG II infusion doses (>175
ng · kg
1 · min
1)
that significantly elevated plasma ANG II levels did not result in an
increase in water intake. In contrast to results from the present
study, previous investigators have suggested that the threshold for the
dipsogenic effect of acutely administered ANG II is
200 pg of ANG II
per milliliter of plasma (22). Interestingly, previous investigators
have shown that, after repeated intracerebroventricular administration
of ANG II, tachyphylaxis to the dipsogenic effect of ANG II developed
(30). On the basis of results from previous studies, a lack of
dipsogenic response to chronic high-dose ANG II infusion in the present
study may have resulted from tachyphylaxis or desensitization of the
ANG II receptor involved in the dipsogenic effects of ANG II (30, 38).
Previous investigators demonstrated that at a dose of ANG II infusion
approximately threefold greater than that used in
study 1 (175 ng · kg
1 · min
1),
body weight was decreased from baseline starting values after 14 days
of infusion (3). Results from study 1 extend previous findings by demonstrating that low doses of ANG II
infusion classified at the threshold level for direct pressor effects
markedly affected the rate of weight gain and body weight. In agreement
with previous studies (3), results from this study demonstrate that
higher pressor doses of ANG II result in a loss of body weight from
baseline starting values. Throughout the present studies, the time
course for increases in blood pressure in ANG II-infused rats did not parallel that for the effects of ANG II on body weight. Typically, increases in blood pressure were manifested 3-5 days before
alterations in body weight. The time delay between blood pressure
increases and elimination of weight gain after ANG II infusion suggests that these two variables are independent. Alternatively, the effect of
ANG II to regulate body weight may be indirectly related to elevations
in blood pressure with a time-lag delay.
Further studies using the vasodilator hydralazine demonstrated that the
effect of ANG II on body weight was independent of blood pressure.
These results are in agreement with previous studies demonstrating that
decreases in body weight in high-dose ANG II-infused rats (500 ng · kg
1 · min
1)
were independent of elevations in blood pressure (3). Interestingly, the effect of ANG II to decrease body weight was augmented in hydralazine-treated rats. Potential mechanisms for augmentation of ANG
II regulation of body weight include reflex increases in sympathetic
neurotransmission in hydralazine-treated rats in response to decreased
peripheral vascular resistance. Hydralazine-mediated increases in
sympathetic neurotransmission would potentially increase peripheral
metabolism and elevate systemic ANG II production (increased kidney-derived renin release).
The present study utilized the noninvasive method of thermal IR imaging
for the regional determination of surface temperature as an index of
energy expenditure. Previous investigators have demonstrated the
ability of IR thermography to detect changes in mean body surface
temperature in postsurgical patients receiving total parenteral
nutrition or in healthy subjects in the fasting state or after meal
ingestion (33). Results from the present study demonstrate that after
chronic low-dose ANG II infusion, tail surface temperature increases,
suggesting that heat dissipation mechanisms were activated. In
agreement with these results, previous investigators have demonstrated
that acute high-dose ANG II injection resulted in an increase in tail
skin temperature (36). In addition to alterations in tail temperature,
results from this study demonstrate an increase in abdominal/thorax
surface temperature after chronic ANG II infusion.
A variety of evidence demonstrates that ANG II facilitates the
sympathetic nervous system (40). Moreover, the sympathetic nervous
system is important in the control of peripheral lipid metabolism.
Previous investigators chronically measured plasma norepinephrine (NE)
levels in rats infused with ANG II (150 ng · kg
1 · min
1)
and demonstrated that plasma NE levels increased by
days 4-6 of infusion (16). Plasma
catecholamine measurements were not performed in the present study.
Thus it is unclear whether the effect of ANG II to decrease body weight
and increase surface temperature was mediated indirectly through
activation of the sympathetic nervous system. Future studies will
determine the role of the sympathetic nervous system in the metabolic
effects of ANG II.
Results from this study do not support a role for alterations in food
intake as the primary mechanism for the effect of ANG II on body
weight. Previous investigators demonstrated that pair feeding control
rats to food intake levels of ANG II-infused rats (500 ng · kg
1 · min
1)
resulted in similar levels of body weight reduction (3), suggesting
that decreased food intake contributes to ANG II regulation of body
weight. In the present study, high-dose ANG II infusion (>350
ng · kg
1 · min
1)
resulted in a time-dependent reduction of food intake. Initial reductions in food intake in the present study and in previous studies
(3) may represent effects of ANG II related to initial pressor-mediated
increases in blood pressure and general animal malaise. However, at low
doses of ANG II infusion, as in study 1, the effect of ANG II on weight gain and body weight
occurred in the absence of significant reductions in food intake.
Assessment of relative organ mass in the present study demonstrated a
preferential effect of ANG II infusion to reduce white adipose tissue
mass. Retroperitoneal white adipose tissue was significantly reduced in
all of the studies performed. In contrast, other organs examined
maintained their relative mass after ANG II infusion, with the
exception of the diaphragm (decreased) and left ventricle (increased).
The relatively specific effects of ANG II to decrease the mass of
retroperitoneal white adipose tissue suggest that effects of ANG II on
weight gain and body weight may arise from augmented lipid metabolism.
However, in the present study, the epididymal white fat pad did not
exhibit reductions in mass after ANG II infusion. These results suggest
site-specific alterations in adipose lipid metabolism and mass from ANG
II infusion.
The cloning of the ob/ob gene and the
identification of leptin have greatly expanded the field of obesity
research (39). Biological effects of adipose-derived leptin include a
decrease in food intake and an increase in energy expenditure (5, 27). Increases in energy expenditure after leptin administration are associated with elevations in NE turnover and enhanced brown adipose thermogenesis (10). In a feedback endocrine regulatory loop, the
sympathetic nervous system has been demonstrated to negatively modulate
leptin gene expression in white adipose tissue (20). In the present
study, measurement of plasma leptin levels after chronic ANG II
infusion demonstrated that high-dose ANG II resulted in a decrease in
plasma leptin. A limitation of the present study is that chronic
measurements of plasma leptin were not obtained during ANG II infusion;
thus it is unclear whether suppressed leptin levels may represent a
compensatory response to chronic ANG II infusion, potentially mediated
through sympathetic nervous system negative feedback. Alternatively,
decreases in plasma leptin levels after chronic ANG II infusion may
arise from semifasted states of rats (decreased food intake) or
reductions in the mass of white adipose tissue. Future studies will
determine the role of leptin in ANG II regulation of body weight.
Regardless, these studies are the first to demonstrate that ANG II
influences plasma leptin secretion.
In summary, results from this study demonstrate that ANG II regulates
body weight through pressor-independent mechanisms in a dose-dependent
manner. Furthermore, mechanisms contributing to ANG II regulation of
body weight include alterations in plasma leptin, mobilization of fat
mass, and increased energy expenditure. These findings are relevant to
disease states associated with heightened (congestive heart failure) or
diminished (obesity) activity of the renin-angiotensin system.
Moreover, results from this study support a functional role for ANG II
production in adipose tissue and strengthen the physiological
significance of an adipose renin-angiotensin system.
 |
ACKNOWLEDGEMENTS |
The authors acknowledge Dr. Allen Hacker for the use of the Narco
blood pressure equipment.
 |
FOOTNOTES |
This work was supported by National Heart, Lung, and Blood Institute
Grant HL-52934.
Address for reprint requests: L. A. Cassis, Rm. 417, College of
Pharmacy, Rose St., Univ. of Kentucky, Lexington, KY 40536-0082.
Received 20 October 1997; accepted in final form 5 February 1998.
 |
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