Effect of central angiotensin II on body weight gain in young rats

Effect of central angiotensin II on body weight gain in young rats

Brain Research 959 (2003) 20–28 www.elsevier.com / locate / brainres Research report Effect of central angiotensin II on body weight gain in young r...

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Brain Research 959 (2003) 20–28 www.elsevier.com / locate / brainres

Research report

Effect of central angiotensin II on body weight gain in young rats James P. Porter*, Jared M. Anderson, Reid J. Robison, Adam C. Phillips Department of Physiology and Developmental Biology and the Neuroscience Center, Brigham Young University, Provo, UT 84602, USA Accepted 15 August 2002

Abstract Systemic infusion of ANG II decreases body weight and food intake and increases energy expenditure. We recently reported that young rats receiving a 1-week intracerebroventricular (i.c.v.) infusion of angiotensin II (ANG II) exhibited decreased body weight compared to control. The aim of the present investigation was to determine if chronic i.c.v. infusion of ANG II also decreases food intake and increases energy expenditure. Young rats were infused with i.c.v. 0.9% saline or ANG II (16.7 or 4.2 ng / min) for at least 10 days and body weight and food intake were monitored daily. Pair-fed rats had the same daily food intake as the ANG II-infused rats. The i.c.v. ANG II decreased body weight gain and food intake. The decrease in weight gain was greater than in the pair-fed groups. The expression of mRNA for uncoupling protein-1 (UCP-1) in BAT was increased significantly in the ANG II-infused rats compared to the pair-fed animals. Subcutaneous infusion of ANG II at the same doses used for i.c.v. infusion had no effect on body weight or food intake. The expression of CRH mRNA in the paraventricular nucleus was not increased in the ANG II-infused rats. These data are consistent with the idea that i.c.v. ANG II decreases body weight gain in young rats, in part, by decreasing food intake and, in part, by increasing thermogenesis (although via a CRH-independent mechanism). This central effect of ANG II may contribute to or complement the effect of peripheral ANG II on body weight.  2002 Elsevier Science B.V. All rights reserved. Theme: Neural basis of behavior Topic: Ingestive behaviors Keywords: Arterial blood pressure; Brown adipose tissue; RT-PCR; Water intake

1. Introduction Over the years, there have been isolated reports that chronic infusion of angiotensin II (ANG II) in rats not only affects drinking behavior and cardiovascular function, but also decreases body weight [10,13,15]. For example, in 1978, it was reported that following surgery to implant intracerebroventricular (i.c.v.) cannulae and osmotic minipumps for chronic infusion, rats receiving i.c.v. ANG II regained weight slower than rats receiving i.c.v. saline [13]. Rats receiving chronic intraperitoneal ANG II via minipumps for 2 weeks showed no increase in body weight during the infusion period (while control rats continued to gain weight) and had a significant decrease in cumulative food intake [10]. More recently, the effects of chronic *Corresponding author. Tel.: 11-801-422-9160; fax: 11-801-4220700. E-mail address: james [email protected] (J.P. Porter). ]

subcutaneous infusions of ANG II on body weight have been more closely studied. Depending on the dose administered, peripheral ANG II has been reported to produce its effect on body weight by decreasing food intake [3], by increasing energy expenditure but not by decreasing food intake [8], or by decreasing food intake early with later increases in oxygen consumption [7]. Chronic systemic infusions of ANG II also have long-term effects on plasma levels of IGF-I [3] and on muscle protein degradation [4]. We recently reported that young, 3-week-old rats, receiving chronic i.c.v. infusion of ANG II for 1 week exhibited an increase in the expression of brain AT1 receptor mRNA and protein [17]. Interestingly, the ANG II-treated rats had a significantly smaller body weight at the end of the week than the saline controls. Given conflicting proposed mechanisms for weight reduction by peripheral ANG II, it was not apparent how the i.c.v. ANG II mediated this decrease in weight gain in our young rats.

0006-8993 / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 02 )03676-4

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Some of the confusion with systemic infusion is likely due to the fact that the ANG II has both peripheral and central effects since circulating ANG II has access to the brain via circumventricular organs that lack a blood–brain barrier. Depending on the dose used, the peripheral ANG II could have more or less central effect. By studying the central effects of ANG II using i.c.v. infusion without evoking peripheral responses, it may be possible to more clearly identify the overall role of this peptide in control of body weight. The initial aim of the present investigation was to determine if the decrease in weight gain observed with chronic i.c.v. infusion of ANG II in young rats was accompanied by a decrease in food intake, and to determine if decreased food intake could account for all of the decrease in body weight gain. Once it was determined that food intake was indeed decreased in the ANG IIinfused rats, uncoupling protein-1 (UCP-1) mRNA expression in brown adipose tissue (BAT) was assessed to estimate the potential contribution of increased thermogenesis to the decreased weight gain. Finally, the potential for ANG II-induced increases in CRH expression in the paraventricular nucleus (PVN) as a mechanism for the decreased food consumption was determined using in situ hybridization. Establishing a clear central mechanism for control of body weight by ANG II will provide a more complete understanding of how this peptide affects energy balance. Furthermore, since research concerning the role of brain ANG II in other areas, such as cardiovascular control, has largely ignored potential changes in body weight or energy expenditure, these studies will raise the awareness of an important confounding factor that should be considered when making interpretations. Portions of this work have been published in abstract form [18].

2. Materials and methods Young (50–70 g) male Sprague–Dawley rats (Harlan Sprague–Dawley, Indianapolis, IN, USA) were individually housed in hanging metal cages and were fed standard rodent diet (Harlan Teklad, 8604). The animal room was maintained at 24 8C with a 12:12 h light–dark cycle. The rats were prepared for chronic i.c.v. infusions as follows. Anesthesia was induced using a mixture of ketamine (125 mg / kg) and acepromazine (1.5 mg / kg) i.p. An i.c.v. infusion cannula (Alzet) connected to a primed osmotic minipump (Alzet, Model 2002) was placed into the right lateral cerebroventricle and cemented in place using two small skull screws and cranioplastic cement. The minipump was inserted under the skin between the scapulae and the incision was closed.

2.1. Effect of high-dose i.c.v. ANG II infusion In this initial experiment, rats were infused with the

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same dose used in our earlier studies looking at the effect of central ANG II on hypothalamic expression of AT1 receptors. Experimental rats (n510) received i.c.v. infusion of ANG II dissolved in sterile saline at a rate of 16.7 ng / min. Control rats (n59) received i.c.v. infusion of sterile 0.9% saline delivered at the same rate (0.5 ml / h). Pair-fed rats (n56) were given the same daily intake measured for the ANG II-infused rats, adjusted to include estimated loss as crumbs. For all rats, body weight and food intake were measured every morning (between 8:00 and 9:00 a.m.) for 10 days, beginning with the first postoperative day. In some rats (control, n54; experimental, n55), blood pressure was determined at the end of the experiment as follows. On the tenth day, the rats were anesthetized and catheters (PE 50, heat-fused to PE 10) were inserted into the femoral artery. The catheters were tunneled subcutaneously to the nape, exteriorized, and tied off so that the rats could move freely during the subsequent 24 h. On the eleventh day, mean arterial pressure was determined in each rat while conscious and freely moving. At the end of the experiment, all rats were again anesthetized. The tubing connecting the minipump to the i.c.v. cannula was cut and 10 ml of fast green dye was infused to verify patency of the infusion system. Nose– anus length was measured and epididymal fat pad wetweight was determined. Brains were removed and the presence of dye in the ventricular system was verified.

2.2. Effect of low-dose i.c.v. ANG II infusion Since i.c.v. infusion of ANG II at a rate of 16.7 ng / min produced a pronounced hypertension, subsequent studies used a dose that was arbitrarily chosen to be one fourth the original dose (4.2 ng / min, n510). Control rats (n59) and pair-fed rats (n56) were included as described above. Food intake and body weight were monitored daily as above for eleven days. In some rats (control, n55; experimental, n55), water intake was also measured daily. Blood pressure was determined in all rats (except the pair-fed group) on the twelfth day as described above. At the time of sacrifice, the rats were perfused with ice-cold 0.9% saline through the left cardiac ventricle. Nose–anus length and epididymal fat pad wet-weight were determined. Brown adipose tissue (BAT) was harvested from between the scapulae and immediately frozen in liquid nitrogen. Patency of the infusion system was verified as described above. A final group of rats were treated as above (i.c.v. ANG II, n54; i.c.v. saline, n53; and pair-fed, n53) and were sacrificed for subsequent in situ hybridization for CRH mRNA in PVN. These rats did not have blood pressure measured. On the morning of the 12th day of infusion, these rats were anesthetized with the ketamine–acepromazine cocktail and were killed by decapitation. Brains were rapidly removed and frozen in plastic molds (con-

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taining OCT compound) that were placed in 100% ethanol cooled with dry ice.

2.3. Effect of systemic infusion of ANG II In a final experiment, rats were given subcutaneous infusions of ANG II at the two rates utilized for the i.c.v. infusion studies. Primed minipumps filled to deliver ANG II at 16.7 ng / min (n54) or 4.2 ng / min (n54) were inserted into a subcutaneous pouch at the nape of the neck. Control rats (n54) received pumps containing sterile saline. Body weight, food intake, and water intake were measured daily for 11 days. In these rats, MAP was determined just before sacrifice while the rats were anesthetized with the ketamine–acepromazine.

2.3.1. Expression of UCP-1 mRNA using relative RTPCR Total RNA was isolated from BAT after homogenization in Trizol (GibcoBRL) reagent. RNA (2 mg) was reverse transcribed to cDNA using 200 U of Superscript II (GibcoBRL) in the presence of 1 ml of dNTP mix (10.0 mM each), 0.4 ml of random decamers (250 mM, Ambion), 2 ml of 0.1 M DTT, 40 U of RNAse inhibitor (GibcoBRL), and 2 ml of 103 buffer (100 mM Tris–HCl, pH 8.3, 500 mM KCl, and 15 mM MgCl 2 ). Each tube had a final volume of 20 ml and was incubated at 42 8C for 60 min and then cooled immediately to 4 8C. PCR was performed on the cDNA using 2 ml of the RT reaction product combined with 1 mM of the UCP-1 primer pairs, 0.625 ml of the dNTP mix, 1 mCi 32 P-dCTP, 5 ml of a 103 buffer (200 mM Tris–HCl, pH 8.4, 500 mM KCl), 1.4 mM MgCl 2 , and 0.4 ml of JumpStart Taq DNA polymerase (Sigma). Each tube had a final volume of 50 ml and was subjected to the following optimized temperature profile for amplification, 94 8C (20 s), 60 8C (30 s), 72 8C (45 s) repeated 20 times followed by a final elongation period (72 8C) for 10 min. The primer pairs (197-bp fragment) used were, sense: 59-GTGAAGGTCAGAATGCAAGC and antisense: 59-AGGGCCCCCTTCATGAGGTC [9]. For each RT sample, a second 2-ml aliquot was used in a separate tube to amplify a fragment (324 bp) of 18S RNA as an external control. To increase the number of cycles where the 18S fragment was amplified in the linear range, 18S competimers (Ambion) were included with the 18S primers at a ratio of 3:7. Each PCR reaction was subjected to nondenaturing PAGE and gels were exposed to autoradiographic film for 8–24 h. In an initial protocol, a cycle titration was performed and 20 cycles was selected because it was clearly in the linear range for amplification of both products (UCP-1 and 18S). The amplified fragment for UCP-1 was submitted to the BYU DNA Sequencing Center to confirm that the DNA sequence was an identical match to that expected. 2.3.2. In situ hybridization Coronal sections (20 mm) through the PVN were cut

with a cryostat and thaw-mounted onto slides (Superfrost Plus, Fisher). The sections were subsequently fixed (4% buffered paraformaldehyde) and acetylated. Hybridization, in situ, was carried out overnight at 57 8C using a 33 PUTP-labeled (1310 6 cpm per section) antisense riboprobe to CRH mRNA (Dr. Kelly Mayo, Northwestern University, Evanston, IL, USA). Unincorporated probe was removed by incubating the slides in RNAse (14 mg / ml, Sigma) for 30 min followed by washes in buffer without RNAse, 13 SSC (room temperature) and 0.53 SSC (60 8C). Initial visualization of the hybridized probe utilized an overnight exposure to autoradiographic film (Hyperfilm MP, Amersham). The slides were subsequently dipped in NTB-2 emulsion and exposed in light-tight boxes for 24 h. The emulsion was then developed and fixed and the sections were lightly stained with cresyl violet, dehydrated in ethanol, placed in xylene, and coverslipped using permount.

2.3.3. Data analysis For each dose of ANG II, the effect of treatment on daily food intake and body weight was compared to the corresponding saline-infused and pair-fed control using two-way ANOVA for repeated measures ( SIGMASTAT, Jandel). Posthoc pair-wise analysis was performed using the Duncan’s method. For each dose of ANG II, differences in nose–anus length and epididymal fat pad weight were compared using one-way ANOVA. Mean arterial pressure of saline- and ANG II-infused rats was compared using the Student t-test. For the relative RT-PCR data, the optical density (O.D.) of the UCP-1 band was determined using SIGMAGEL (Jandel). The O.D. value was then standardized by dividing it by the O.D. value of its corresponding 18S band. This O.D. ratio was then set at 1 for saline-control animals and the O.D. ratio of the experimental and pair-fed rats was adjusted accordingly. Differences in these averaged values were determined using one-way ANOVA. For all analyses, P,0.05 was considered to be significant. Semiquantitative analysis of the emulsion-dipped slides was carried out under dark field using MICROSUITE (Olympus) image analysis. For each brain, a comparable level of the PVN (approximate midrostrocaudal level) was imaged. One side of the PVN was outlined to define the region of interest and mean optical density was determined. A region of similar area just ventral to the PVN was used to determine the background. The final optical density values (region of interest minus background) for each of the saline-control rats were averaged and set at 1. The values in the ANG II- and pair-fed groups where then adjusted accordingly.

3. Results High-dose (16.7 ng / min) i.c.v. infusion of ANG II produced a significant decrease in body weight gain (Fig. 1, upper panel) and food intake (Fig. 1, lower panel)

J.P. Porter et al. / Brain Research 959 (2003) 20–28

Fig. 1. Effect of chronic i.c.v. infusion of ANG II (16.7 ng / min) on body weight and food intake in young rats. *, P,0.05, i.c.v. ANG II versus i.c.v. Saline; **, P,0.05, Pair-fed group versus i.c.v. Saline; ***, P,0.05, i.c.v. ANG II versus Pair-fed group.

compared to saline-infused control rats. The difference in body weight between the control and experimental rats reached significance by day 8. Pair-fed rats also exhibited a decrease in weight gain that was significant at day 9. By the final day (10), body weight in the ANG II-infused rats was less than the pair-fed animals. Ten-day cumulative food intake was significantly decreased by 22.3% (148.867.0 vs. 115.766.0 g). The high dose also produced a significant increase in MAP (Table 1). Nose–anus length and epididymal fat pad weight were both decreased significantly in the ANG II-infused rats compared to the saline control rats. Pair feeding produced a similar decrease in epididymal fat pad weight, but nose–anus length was not decreased. The lower dose (4.2 ng / ml) also produced a decrease in body weight gain (Fig. 2, upper panel) and food consumption (Fig. 2, lower panel) compared to the saline control rats. Body weight was significantly less in the ANG

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Fig. 2. Effect of chronic i.c.v. infusion of ANG II (4.2 ng / min) on body weight and food intake in young rats. *, P,0.05, i.c.v. ANG II versus i.c.v. Saline; **, P,0.05, Pair-fed group versus i.c.v. Saline; ***, P,0.05, i.c.v. ANG II versus Pair-fed group.

II-infused rats than the saline controls from day 7 on. On days 10 and 11, the body weight of the ANG II-infused rats was significantly less than the pair-fed animals. The decrease in food intake was less pronounced and biphasic. Ten-day cumulative food consumption was 13.3% less in the ANG II-infused rats compared to the saline controls (119.965.7 g vs. 103.964.7 g). This effect was significantly less than with the high dose (two-way ANOVA). MAP was not significantly increased in the low-dose group (Table 1). Nose–anus length and epididymal fat pad weight were both decreased in the ANG II-infused rats compared to saline-infused controls. As with the higher dose, epididymal fat pad weight was also decreased in the pair-fed rats, but nose–anus length was not. The effect of the low-dose of i.c.v. ANG II on water intake was variable. Average daily water intake tended to be higher in the low-dose ANG II-infused rats, but the effect was not significant (Table 1). There was no correlation between daily water intake (for every ANG II-infused

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Table 1 Effect of high- and low-dose intracerebroventricular (i.c.v.) or subcutaneous (s.c.) infusion of ANG II on nose–anus length, epididymal fat pad weight, mean arterial pressure (MAP), and daily water intake n i.c.v. Saline (0.5 ml / h) i.c.v. ANG II (16.7 ng / min) Pair-fed i.c.v. Saline (0.5 ml / h) i.c.v. ANG II (4.2 ng / min) Pair-fed s.c. Saline s.c. ANG II (4.2 ng / min) s.c. ANG II (16.7 ng / min)

Nose–anus length (cm)

Epididymal fat pad (g)

MAP (mmHg)

9

16.260.2

0.6260.03

10863 (5)

10

15.260.3*

0.4960.02*

174614 (5)*

6

15.860.18

0.4260.02*

9

15.760.4

0.4860.03

11164

17.461.0 (5)

10

14.360.4*

0.3460.03*

11867

37.1611.4 (5)

6

15.560.10

0.3760.03*

4 3

16.160.3 16.260.2

0.6060.05 0.6760.05

4

15.860.2

0.5460.06

9661 9362 11266 (3)

Daily water intake (ml)

19.461.4 20.260.9 22.361.1

*, P,0.05 compared to i.c.v. saline.

rat, for every day except the first two postoperative days) and daily food intake (slope520.01, r50.14) (Fig. 3). UCP-1 mRNA expression in BAT of pair fed rats was significantly less than saline control rats (Fig. 4). ANG II-infused rats had a relative expression of UCP-1 mRNA that was not different from saline control animals, but was significantly greater than pair-fed rats. The expression of CRH mRNA in the PVN was significantly decreased in the ANG II-infused rats compared to saline-infused and pair-fed rats (Fig. 5). One of the brains from the pair-fed group did not cut well and no usable sections were obtained.

Subcutaneous infusion of ANG II at either dose had no effect on food intake or body weight gain (Fig. 6). It should be noted that one rat receiving the low dose of ANG II died before the end of the experiment. However, even with only three rats in that group, the absence of effect on body weight and food consumption is clearly observed. Daily water intake (P50.2) and mean arterial pressure (P50.06) tended to be higher in the rats receiving the higher dose of ANG II, but the effect was not significant due to the limited number of animals (Table 1).

4. Discussion The aim of the initial high-dose i.c.v. infusion-study was to determine if the decrease in weight gain observed

Fig. 3. Relationship between water intake and food intake for every rat (n55; saline-infused rats, n55; ANG II-infused rats) on each infusion day except the first 2 postoperative days. Filled circles; saline-infused rats, open circles; ANG II-infused rats. The line depicts the correlation for the ANG II-infused rats.

Fig. 4. Relative expression of UCP-1 mRNA from brown adipose tissue determined by relative RT-PCR in rats receiving i.c.v. saline, pair feeding, or i.c.v. ANG II (4.2 ng / min). *, P,0.05.

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Fig. 5. Example of expression of CRH mRNA in the PVN of i.c.v. saline-infused (A), i.c.v. ANGII-infused (B), and pair-fed (C) rats. *, P,0.05.

previously in young rats [17] was accompanied by decreased food intake, and whether the decrease in food consumption could account for the decreased weight gain. The data clearly show that i.c.v. infusion of ANG II at a rate of 16.7 ng / min for 10 days produced a significant decrease in the rate of weight gain in 3-week-old rats. This dose of ANG II also produced a significant decrease in food consumption. However, decreased food consumption cannot account for the entire decrease in weight gain. Pair-fed rats did not exhibit as great a decrease in the rate of weight gain as the i.c.v. ANG II-infused rats. The effect of the ANG II appeared to be mediated within the central nervous system since subcutaneous administration of the peptide at the same dose had no effect on either weight gain or food intake. In support of this notion, others have infused ANG II intracerebroventricularly in rats for 5 days at a rate 24 times (100 ng / min) that used in the present investigation and reported no increase in circulating ANG II levels [5,6]. Thus, the present study extends the well-known effect of peripheral

ANG II on body weight and energy expenditure to include a significant central neural effect. Since the high-dose i.c.v. infusion of ANG II also produced a significant increase in MAP, a lower dose of ANG II was used in subsequent studies that was selected arbitrarily to be one fourth the original dose in an attempt to minimize blood pressure changes. With the lower dose of ANG II there was a slight tendency for increased MAP, but the effect was not significant. Furthermore, no significant correlation existed between arterial pressure and food intake on the final day of the study (r50.4) when all rats for both doses were considered (data not shown). Hence, any effect of the low-dose i.c.v. ANG II on body weight gain or food intake could not be explained by the ‘stress’ of high blood pressure. Two other studies have reported that chronic peripheral infusion of ANG II decreased body weight in a blood-pressure-independent mechanism [3,8]. In both instances, concomitant infusion of the vasodilator, hydralazine, prevented the increase in arterial pressure with ANG II, but not the decrease in body weight.

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Fig. 6. Effect of chronic subcutaneous infusion of ANG II (4.2 or 16.7 ng / min) on daily body weight and food intake.

The lower dose of i.c.v. ANG II also produced a significant decrease in the rate of weight gain. The effect was not dose related since both doses produced comparable decreases in weight gain. Food consumption was also decreased by the low-dose infusion, but in this case the effect was biphasic and less pronounced. As with the high dose, the decreased food intake with the lower i.c.v. dose did not account for the entire decrease in weight gain since pair fed rats did not exhibit as great a decrease in weight gain. Peripheral infusion of ANG II at the low dose had no effect on body weight gain or food consumption, again suggesting a central effect of the peptide. Thus, both doses of i.c.v. ANG II decreased food consumption through a central mechanism. However, additional factors, not related to decreased food intake also contributed to the lower body weights in the treated rats. The decreased food intake with i.c.v. ANG II was not likely due to increased water intake and the accompanying over-distended stomach. Although somewhat variable, the low dose of ANG II tended to increase water consumption.

Rats were included in the analysis if injection of fast green dye into the i.c.v. cannulae at the end of the experiment confirmed the patency and correct placement of the injection system. The lack of correlation between daily water intake and daily food intake (Fig. 3) makes it unlikely that the decreased food consumption can be explained by increased drinking. Chronic peripheral infusion of ANG II is known to increase heat production in the scapular region [8].This could be due to a direct presynaptic effect of ANG II on sympathetic nerves to increase norepinephrine release at adipose tissue [11]. The present study raises the possibility that activation of centrally driven sympathetic pathways may also contribute to the increased energy expenditure. Chronic i.c.v. infusion of ANG II in adult rats has been reported to increase sympathetic outflow, at least, with respect to cardiovascular function [5]. We found that the i.c.v. ANG II-infused rats exhibited an increased expression of UCP-1 mRNA in BAT when compared to the pair-fed control rats. UCP-1 mRNA expression in BAT is controlled by sympathetic innervation [9]. The additional decrease in weight gain beyond that produced by decreased food consumption could have been due to increased heat production. A recent report showed that oxygen consumption is affected by chronic peripheral ANG II infusion [7]. With a 28-day infusion of ANG II, oxygen consumption was decreased during the first 10 days even though body weight was decreased. At day 16, oxygen consumption was greater in ANG II-treated rats. It was concluded that much of the early decrease in body weight was due to the anorexic effect of ANG II, but later reductions included effects on energy expenditure. This could explain why the differences in body weight gain took so long to become significant in the present investigation. The lack of a dose relationship between i.c.v. ANG II and decreased body weight gain is puzzling, especially since the decrease in food consumption was dose related as was the blood pressure effect. The effects on nose–anus length and epididymal fat pad weight were also not dose related. With the low dose of ANG II, 60% of the decreased weight gain (from day 8 on) was due to factors other than anorexia. With the high dose of ANG II, this was reduced to 46%. Perhaps the higher dose caused additional effects such as decreased oxygen consumption [7] that were responsible for the smaller contribution. It is not known to what degree the results in the present study are dependent on the rats being young and in a growth phase. The decrease in body weight gain with the high dose of ANG II appeared to be due to decreased lean body mass accumulation (nose–anus length), rather than decreased fat deposition since both ANG II-infused and pair-fed rats had a similar decrease in epididymal fat pad weight. This is consistent with reports that peripheral ANG II produced muscle protein degradation rather than decreased epididymal fat [4]. I.c.v. injection of ANG II has been reported to decrease GH release [20], and i.c.v.

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injection of ANG II antiserum was shown to increase plasma growth hormone levels [12]. Could the decrease in rate of weight gain in the young rats used in the present study be in part due to decreased GH and, hence, decreased circulating IGF-1? Brink and co-workers showed that chronic peripheral infusion of ANG II in adult rats [3,4] or 6-week-old young rats [4] decreased plasma levels of IGF-1. However, replacement infusion of IGF-1 failed to prevent the decrease in weight gain [4]. These authors provided evidence that local IGF-1 mechanisms may be diminished by the systemic ANG II infusion. CRH-containing neurons in the paraventricular nucleus (PVN) are known to co-express the AT1 ANG II receptor [1], and acute i.c.v. injection of ANG II increases CRH levels in hypophyseal portal blood [16] and increases CRH mRNA expression in the PVN [1] in adult rats. Furthermore, CRH is known to produce anorexia [14,19] and increased energy expenditure [2]. We hypothesized that the decreased food consumption evoked by chronic i.c.v. infusion of ANG II may have been due to increased expression of CRH. However, the in situ hybridization data show that CRH mRNA is, if anything, decreased in the PVN by chronic i.c.v. ANG II. These data suggest that the acute effects of i.c.v. ANG II on CRH expression are not maintained during chronic infusion and that the decrease in food consumption was not due to increased CRH, at least at the end of the infusion period. The decrease in food intake with the low dose of ANG II was biphasic. Perhaps the early effect involved increased hypothalamic CRH mRNA expression that normalized over time. The significant late decrease in CRH mRNA expression in the ANG II-infused rats may have been compensation in response to decreased weight gain evoked by some other non-CRH mechanism. A decrease in CRH mRNA expression in the pair-fed rats was also expected. It is not known why this effect was not observed. It may have been due to the limited number of rats in the group. In summary, chronic i.c.v. infusion of ANG II produced a decrease in weight gain in young rats that was accompanied by decreased food intake and increased UCP-1 mRNA expression in BAT. The decrease in weight gain appeared to be partly due to the anorexia, and partly due to increased energy expenditure. These results show that ANG II has the ability to decrease body weight gain in young rats through central mechanisms. It is not know if these mechanisms come into play only when circulating ANG II is increased (via circumventricular organs that lack a blood–brain barrier and are rich with ANG II receptors) or if a separate intrinsic brain angotensinergic pathway exists that functions to regulate body weight.

Acknowledgements This work was supported by a Grant-in-Aid from the Western States Affiliate of the American Heart Associa-

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tion, and by Brigham Young University. The in situ hybridization technique was learned in the laboratory of Dr. Mary Dallman, University of California, San Francisco under the auspices of a Career Enhancement Award (to J.P. Porter) provided by the American Physiological Society.

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