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Endocrinology, doi:10.1210/en.2006-0754
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Endocrinology Vol. 147, No. 11 5078-5086
Copyright © 2006 by The Endocrine Society

Prevention of Adipose Tissue Depletion during Food Deprivation in Angiotensin Type 2 Receptor-Deficient Mice

Laurent Yvan-Charvet, Patrick Even, Noël Lamandé, Pascal Ferré and Annie Quignard-Boulangé

Institut National de la Santé et de la Recherche Médicale (INSERM) (L.Y.-C., P.F., A.Q.-B.), Unité 671, and Université Pierre et Marie Curie (L.Y.-C., P.F., A.Q.-B.), Centre Biomédical des Cordeliers, Paris F-75006, France; and Institut National de la Recherche Agronomique (P.E.), Unité Mixte de Recherche 914, Institut National Agronomique Paris-Grignon, and INSERM (N.L.), Unité 36, College de France, Paris F-75005, France

Address all correspondence and requests for reprints to: Annie Quignard-Boulangé, Institut National de la Santé et de la Recherche Médicale, Unité 671, 15 rue de l’Ecole de Médecine, 75270 Paris Cedex 06, France. E-mail: quignard{at}bhdc.jussieu.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Angiotensin (Ang) II is produced locally in various tissues, but its role in the regulation of tissue metabolism is still unclear. Recent studies have revealed the role of type 2 Ang II receptor (AT2R) in the control of energy homeostasis and lipid metabolism. The contribution of the AT2R to adaptation to starvation was tested using AT2R-deficient (AT2R y/–) mice. Fasted AT2R y/– mice exhibited a lower loss of adipose tissue weight associated to a decreased free fatty acid (FFA) release from stored lipids than the controls. In vitro studies show that Ang II causes an AT1R-mediated antilipolytic effect in isolated adipocytes. AT1R expression is up-regulated by fasting in both genotypes, but the increase is more pronounced in AT2R y/– mice. In addition, the increased muscle ß-oxidation displayed in AT2R y/– mice on a fed state, persists after fasting compared with wild-type mice. In liver from fed mice, AT2R deficiency did not modify the expression of genes involved in fatty acid oxidation. However, in response to fasting, the large increase of the expression of this subset of genes exhibited by wild-type mice, was impaired in AT2R y/– mice. Taken together, decreased lipolytic capacity and increased muscle fatty acid oxidation participate in the decreased plasma FFA observed in fasted AT2R y/– mice and could account for the lower FFA metabolism in the liver. These data reveal an important physiological role of AT2R in metabolic adaptations to fasting.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SEVERAL REPORTS HAVE demonstrated the existence of a functional renin angiotensin system (RAS) in adipose tissue (1, 2). The adipose RAS has been implicated in modulation of adiposity through its ability to stimulate lipogenesis in adipocytes (3, 4). The biological effects of angiotensin II (Ang II) are mediated by two subtype receptors (AT1R and AT2R) that belong to the large family of G protein-associated receptors (5). The importance of these receptors in the control of fat mass has been recently demonstrated using transgenic mice (6, 7). We reported that AT2R-deficient mice (AT2R y/–) exhibited a striking hypotrophy of adipose cell associated with decreased fat storage (6). Besides its effect on fatty acid storage, Ang II could also modulate the lipolysis pathway to control adiposity. Different studies reported contrasting results on Ang II effect on lipolysis (8, 9, 10). It could be suggested that hypotrophied adipocyte displayed by AT2R y/– mice is also linked to an excessive lipolytic activity. Whether Ang II through AT2R is involved in the control of adipocyte free fatty acid (FFA) release remains to be elucidated.

The transition through the fasting cycle is an important metabolic adaptation in response to food availability. Adipose tissue plays a major role in this adaptation because of the release of large quantities of FFAs into the circulation from stored lipids. Increased FFA into the circulation provide an important alternative energy source to glucose for many tissues allowing survival during times of food deprivation. In muscle, FFAs are primarily used as a source of energy for ATP synthesis through ß-oxidation (11, 12). In the liver, FFA can either be used for ß-oxidation and ketogenesis, or re-esterified into triglycerides (13, 14). Recently we have demonstrated that AT2R deletion led to an increase in muscle ß-oxidation (6) pointing out a role for AT2R in the balance between FFA storage and oxidation.

We conducted the present study to investigate the role of AT2R in response to fasting. Our data show that fasted AT2R y/– mice exhibited a resistance to fat loss together with a smaller increase in plasma FFA than in wild-type (WT) mice. This could be explained by a down-regulation of lipolytic activity of the adipocyte compared with WT mice. In vitro studies demonstrate that Ang II exerts an antilipolytic effect, which is mediated by AT1R. Furthermore, the present study attempted to determine whether valsartan, an AT1R antagonist, prevents the down-regulation of lipolysis in AT2R y/– mice. Our findings provide strong evidence for the participation of AT1R in FFA release from adipose tissue, and the activation of which might represent an additional mechanism for the control of adipose tissue mass. This study suggests an important role for AngII-dependent mechanisms in the control of adipose tissue fat stores.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
AT2R-deficient mouse strains
The generation of AT2R gene null mice has been previously described (15). Male C57BL/6 AT2R y/– and WT littermates were obtained by crossing male WT and female AT2R +/– mice (a gift from Dr. Ichiki, Vanderbilt University, Nashville, TN). Animals had free access to food and water and were housed in a controlled environment with a 12-h light, 12-h dark cycle and constant temperature (22 C). All mice were maintained on standard chow (Usine d’Alimentation Rationnelle, Meylan, France) from weaning to 13 wk of age. All animal protocols were undertaken according to the Guidelines for Care and Use of Experimental Animals, France.

Energy expenditure
In vivo indirect open circuit calorimetry was performed in metabolic chambers. Before food deprivation, 12-wk-old animals were allowed a 2-h period to become accustomed in experimental chambers at 25 C ± 1 with free access to food and water. Constant airflow (0.5 liter/min) was drawn through the chamber and monitored by a mass-sensitive flowmeter. Gas concentrations were monitored at the inlet and outlet of the scaled chambers allowing calculation of oxygen consumption (VO2), carbon dioxide production (VCO2), and the respiratory quotient (RQ: ratio of VCO2 to VO2). Total metabolic rate (energy expenditure) was calculated from oxygen consumption and carbon dioxide production using Lusk’s equation and expressed as Watts per kilogram to the 0.75 power of body weight. Glucose and lipid oxidation were calculated as previously described (16).

Metabolite assays
After 24 h fasting, mice were killed and blood was collected by cardiac puncture. Plasma FFAs, triglycerides, and glycerol were determined using an enzymatic colorimetric method (Wako Chemical, Neuss, Germany; Sigma Chemical, St. Louis, MO; and Enzytec, Diffchamb, France, respectively). ß-Hydroxybutyrate was assayed using standard laboratory procedures (17). Plasma leptin levels were determined using a commercial kit (Clinisciences, Montrouge, France). Plasma corticosterone was determined by RIA using a commercial kit (MP Biomedicals, Orangeburg, NY). Plasma angiotensinogen was cleaved by mouse renin into Ang I. RIA of Ang I was carried out as previously described (18). Tissue triglyceride content was measured from a sample of adipose tissue, muscle, or liver using the commercial kit described above.

Adipose tissue cellularity
Cell size and number of epididymal adipose tissue was determined as previously described (19). Briefly, images of isolated adipocytes were acquired from a light microscope fitted with a camera and approximately 400 cell diameters were measured using Perfect Image software (Numeris, Paris, France) and the mean fat cell volume calculated. Fat cell number was estimated by dividing the tissue lipid content by fat cell weight (fat cell volume x triolein density).

Measurement of rates of lipolytic activity
To estimate the in vivo lipolytic activity, we measured the rise in plasma FFA and glycerol during the first 4 h of food deprivation. Isolated adipocytes from epididymal adipose tissue of fed mice were used to measure in vitro lipolysis. Briefly, adipocytes were incubated in Krebs-Ringer bicarbonate buffer (pH 7.4), containing 3% serum albumin for 1 h at 37 C in an atmosphere of 5% CO2-95% O2 as previously described (20). The amount of glycerol released into the incubation medium in basal or stimulated conditions (100 nM isoproterenol) was determined. Ang II (10 nM) was added in the presence or absence of 1 µM Losartan (AT1R antagonist) or 1 µM of PD 123319 (AT2R antagonist). The AT2 nonpeptide antagonist PD123319 competes for 80–90% of Ang binding and Losartan is the first orally active AT1R antagonist available. In vitro Losartan competes with the binding of Ang II to AT1R (IC50 of 20 nmol/liter). Lipolytic activity was expressed as micrograms of glycerol released by 106 cells/h.

Chronic treatment with valsartan
To test the role of ATR1 on fasting lipolysis in vivo, we used an AT1R-antagonist, valsartan (a kind gift from Novartis Pharma S.A.S., Rueil-Malmaison, France). Twelve-week-old male AT2R y/– and WT mice were randomly assigned to the following four groups: a WT control group, a valsartan-treated (5 mg/kg·d in drinking water) WT group, an AT2R y/– control group, and a valsartan-treated AT2R y/– group. This low dose was comparable with that of Losartan, which was found to have no significant hypotensive effects (21). After 7 d of treatment, mice were fasted for 24 h, killed, and their blood collected. Adipose tissue was removed and adipocyte size was determined as described above. In all groups, the number of animals was four to five per group.

Enzyme activities
Carnitine palmitoyl transferases (CPT-1 and -2) activities in the mitochondrial fraction of liver and hind limb skeletal muscle were determined by spectrophotometric assay according to Bieber et al. (22). Data are expressed as millimoles per minute (mU) per milligram of mitochondrial protein.

RNA preparation and analysis
Total RNA was extracted from tissues, as previously described (23) and cDNA was synthesized from 1 µg of total RNA with superscript reverse transcriptase (Invitrogen, Cergy, France). Real-time PCR was performed using a Bio-Rad Thermocycler (Bio-Rad, Richmond, CA) and the PCR was carried out as described elsewhere (6). Sequences of the primers are listed in Table 1Go. The expression of all genes reported was normalized to ribosomal 18S RNA expression.


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  		TABLE 1. Sequences of PCR primers

 
Statistical analysis
Data are given as means ± SEM. Comparison of mean values between groups was evaluated by Student’s two-tailed unpaired t test or by one-way ANOVA with a Tukey multiple comparison posttest (GraphPad Software, San Diego, CA). Differences were considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of fasting on adipose tissue in AT2R-deficient mice
In response to 24-h fasting, we observed a slight but significant body weight loss in WT mice. In AT2R-deficient mice (AT2R y/–), body weight tended to be lower in fasted mice although this decrease did not reach the level of significance (Table 2Go). Similarly, epididymal adipose tissue weight from WT mice was decreased by 60% after 24 h-fasting, whereas AT2R y/– mice exhibited no significant change in fat mass (Table 2Go). Similar results were obtained in adipose triglyceride contents, where triglyceride contents were significantly reduced by 40% and 20% in WT and AT2R y/– mice respectively, reflecting the resistance to triglyceride breakdown in the adipose tissue of AT2R y/– mice (Table 2Go). This decreased adiposity was related to the 80% and 30% decrease in fat cell weight observed in WT and AT2R y/– mice, respectively (Table 2Go). Thus, when compared with WT mice, the hypotrophied adipocyte observed in AT2R y/– mice in fed state was abolished after fasting mainly due to the drastic change in cell weight exhibited by WT mice. Because fasting did not modify adipocyte number whatever the genotype, the increase in fat cell number observed in fed AT2R y/– mice persisted after fasting and accounted for the higher epididymal fat weight than controls (Table 2Go). Only in WT mice, we observed a significant decrease in plasma leptin concomitant with the fat mass loss (Table 3Go). Table 3Go shows that plasma corticosterone levels were increased in both genotypes by fasting but no genotype effect was evident either in fed or fasted mice. By contrast, neither genotype nor the nutritional state affected the plasma angiotensinogen levels (Table 3Go). Thus, AT2R deletion allows protection against adipose tissue weight loss during fasting, suggesting that Ang through the AT2R contribute to the maintenance of lipid stores in adipose tissue.


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  		TABLE 2. Effects of AT2R deficiency on body weight, blood pressure, and fat composition

 

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  		TABLE 3. Effects of AT2R deficiency on parameters in the plasma

 
Effect of AT2R deletion on adipocyte lipolysis
The lower effect of food deprivation on adipose tissue weight in AT2R-deficient mice prompted us to examine the lipolytic capacity of their adipocytes. In adipocytes from WT mice, isoproterenol-stimulated lipolysis was decreased in the presence of Ang II (Fig. 1AGo). This inhibitory effect was counteracted by the addition of the AT1R antagonist, Losartan, suggesting that this effect was mediated by AT1R. The inability of the AT2R-antagonist (PD 123319) to affect lipolysis confirmed the implication of AT1R in the antilipolytic effect of Ang. In basal conditions, the rate of glycerol release was slightly but significantly lowered in the adipocytes from AT2R y/– than WT mice (compare Fig. 1Go, A and B). This difference was drastically augmented after stimulation by isoproterenol due to the lower sensitivity of adipocytes from AT2R y/– mice. In addition, adipocytes from AT2R y/– mice exhibited similar antilipolytic effect in response to Ang II that was abolished by Losartan treatment. As expected, PD 123319, an AT2R-antagonist, did not modify the Ang II effect in AT2R y/– adipocytes (Fig. 1GoB). The depressed lipolytic activity demonstrated in isolated adipocytes fits in with the moderate increase in plasma FFA and glycerol levels of AT2R-deficient mice compared with WT mice, after 24 h of fasting (Table 3Go). These observations indicate that Ang II acts as a modulator of lipid stores in adipose tissue not only by its well-known stimulatory effect on lipogenesis, but also by inhibiting the lipolytic pathway.


Figure 1
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  		FIG. 1. Influence of AT1R on adipose tissue lipolysis. A, Effect of Ang II and Ang receptor blockers on glycerol release by isolated adipocytes from WT mice. The lipolytic activity of adipocytes was determined in the absence (basal) or in the presence of isoproterenol (Iso) without treatment (control) or with different treatments as described in the figure. B, Effect of Ang II and Ang receptor blockers on glycerol release by isolated adipocytes from AT2R y/– mice. Results are means ± SEM (n = 5 each conditions). {dagger}, P < 0.01 isoproterenol vs. basal conditions; {ddagger}, P < 0.01 Ang II and/or receptor antagonists vs. isoproterenol condition. C, Expression of AT1R mRNA in epididymal adipose tissue from WT and AT2R y/– mice under fed or fasted states (n = 5 per group). Open and filled bars indicate WT and AT2R y/– mice, respectively. Values were normalized to ribosomal 18S quantity and expressed as percent over fed WT mice. *, P < 0.01 fasted vs. fed state within genotype; #, P < 0.01 AT2R y/– vs. WT

 
Effect of valsartan on fasting-related lipolytic activity and fat mass loss in AT2R-deficient mice
To determine whether AT1R could be involved in the lower lipolysis seen in AT2R y/– mice in vivo, we investigated the effect of fasting on Ang II receptors expression. In response to fasting, no significant change in adipose AT2R expression in WT mice was observed (data not shown). By contrast, AT1R expression was increased 2- and 3-fold in epididymal adipose tissue from WT and AT2R y/– mice, respectively (Fig. 1CGo). Thus, AT2R deficiency promotes a higher AT1R expression in the fasting state. This effect on AT1R expression between genotypes occurs without a change in plasma angiotensinogen (Table 3Go). To find out whether an increase in AT1R expression could be involved in the prevention of fat loss in AT2R y/– mice during fasting, we treated fasted mice with an AT1R antagonist. Treatment with valsartan for 7 d did not modify the body weight of the mice, whatever the genotype (data not shown). After 24 h fasting, valsartan treatment led to 2-fold decrease in adipocyte weight of AT2R-deficient mice (Fig. 2AGo). This was also observed in control mice in which there was a significant drop in fat cell weight due to fasting (Table 2Go and Fig. 2AGo). Similarly, fasting in valsartan-treated mice, provoked in AT2R-deficient mice a higher increase in plasma FFA than in control mice (Fig. 2BGo). These results demonstrate that the presence of AT1R antagonist increases in vivo the lipolytic activity in control mice leading to heightened fat loss. In AT2R-deficient mice, the protection from fat mass loss during fasting was abolished by valsartan treatment showing that the in vivo antilipolytic effect of Ang II was dependant on AT1R activation. These findings were consistent with a resistance to fat mass loss during fasting exhibited by AT2R-deficient mice in which AT1R was overexpressed (Table 2Go and Fig. 1CGo).


Figure 2
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  		FIG. 2. Effect of fasting on epididymal adipose cellularity (A) and plasma FFA (B) from control and valsartan-treated WT and AT2R y/– mice. Results are expressed as means ± SEM (*, P < 0.05 or **, P < 0.01 fasted vs. fed state within genotype; #, P < 0.01 AT2R y/– vs. WT).

 
In vivo FFA oxidation in fasted AT2R y/– mice
Because plasma FFA level results from the balance between adipose tissue release and peripheral utilization, we also investigated whole body lipid oxidation in AT2R-deficient mice in response to fasting. Indirect calorimetry revealed that RQ was decreased in the first 4 h after food deprivation in both genotypes (Fig. 3AGo). During this step, AT2R y/– mice displayed a more rapid drop in RQ indicating a faster switch to lipid oxidation than in control mice (Fig. 3Go, A and B). This increased lipid oxidation exhibited by AT2R y/– mice occurred concomitantly with lower plasma FFA levels than in controls, from the second hour of the postabsorptive period (Fig. 4AGo). On the contrary, the plasma glycerol accumulation in AT2R y/– mice was unchanged during the first 4 h of fasting compared with controls (Fig. 4BGo). After 4 h of fasting, RQ and lipid oxidation patterns became identical in both genotypes (Fig. 3AGo). Nevertheless, the decreases in both plasma FFA and glycerol levels exhibited by AT2R y/– mice persisted after 24 h of fasting, suggesting that the rate of lipolysis in the adipose tissue could largely account for the control of plasma FFA (Table 3Go). Throughout the experimental period, glucose oxidation was unchanged between genotypes (Fig. 3CGo). Taken together, these data demonstrate that both impaired FFA release and up-regulated lipid oxidation could contribute to the diminution in plasma FFA observed in AT2R y/– mice upon fasting.


Figure 3
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  		FIG. 3. Effect of fasting on whole-body oxidation. RQ (A), lipid oxidation (B), and glucose oxidation (C) in WT ({circ}) and AT2R y/– ({blacksquare}) mice were measured by indirect calorimetry. Values are expressed as means ± SEM (n = 5 per group). Comparisons show a significant genotype effect (P < 0.05) on RQ and lipid oxidation values during the first 4 h after food deprivation.

 

Figure 4
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  		FIG. 4. Early effects of fasting on plasma FFA and glycerol. Plasma FFA (A) and glycerol (B) were measured over the first 5 h of fasting in WT ({circ}) and AT2R y/– ({blacksquare}) mice and expressed as percent increase over fed values. Values are expressed as means ± SEM (n = 5 per group). Differences were significant between genotypes (*, P < 0.05).

 
Higher muscle ß-oxidation persists in fasted AT2R y/– mice
In response to fasting, WT mice exhibited a significant 60% increase in expression of the ß-oxidation marker, CPT-1 along with peroxisome proliferator-activated receptor (PPAR) {delta}, whereas no change in their expression occurred in AT2R y/– mice (Fig. 5Go). PPAR{alpha} expression was down-regulated by a similar extent in both genotypes (Fig. 5AGo). As previously shown, CPT-1, PPAR{alpha} and PPAR{delta} mRNA levels were increased by approximately 2-fold in fed AT2R y/– compared with WT mice and, despite this up-regulation, the expression of these genes remained slightly but significantly higher in fasted AT2R y/– mice (Fig. 5Go, A and B). Accordingly, the higher muscle CPT activity exhibited by fed AT2R y/–, remained evident in fasted state compared with controls (Table 4Go). Pertaining to an increase in fatty acid oxidation capacity, our data show that whether the mice were fed or fasted, the expression of succinate dehydrogenase, a marker of mitochondrial complex II, was increased by 2-fold in AT2R y/– compared with WT mice (Fig. 5BGo). Despite the difference in fatty acid oxidation, there was no significant change in muscle triglyceride content between the genotypes irrespective of the nutritional state (Table 4Go). Thus, after 24 h-fasting, AT2R y/– mice maintained a higher muscle lipid oxidation capacity than WT mice. Compared with adipose tissue, we did not observe any change in muscle AT1R expression during fed/fasting transition suggesting that AT1R was not implicated in the regulatory mechanisms of ß-oxidation (Fig. 5CGo).


Figure 5
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  		FIG. 5. Effect of fasting on gene expression in skeletal muscle. PPARs (A), mitochondrial CPT-1 and succinate dehydrogenase (SDH) (B) expression in WT and AT2R y/– mice under fed or fasted states (n = 5 per group). C, Expression of AT1R mRNA in skeletal muscle from WT and AT2R y/– mice under fed and fasted states (n = 5 per group) Values were normalized to ribosomal 18S quantity and expressed as percent over values from fed WT mice. *, P < 0.01 fasted vs. fed state within genotype; #, P < 0.01 AT2R y/– vs. WT mice.

 

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  		TABLE 4. Effects of AT2R deficiency on indicators of skeletal muscle and liver metabolism A

 
AT2R deletion has no effect on hepatic FFA metabolism
It is well known that liver plays a crucial role in fatty acid uptake and oxidation, especially during fasting. This prompted us to study whether the deletion of AT2R could alter lipid metabolism in liver. In the fed state, AT2R deletion did not significantly modify the expression of the genes related to fatty acid oxidation such as CPT-1, acyl coenzyme A oxidase (AOX), and PPARs compared with WT mice (Fig. 6Go, A and B). This is in agreement with barely detectable AT2R mRNA levels in liver from WT mice (data not shown). In contrast to the muscle, the liver from fed AT2R y/– mice exhibited similar CPT activity when compared with the WT mice (Table 4Go). However, starvation provoked an increase in liver triglyceride content and CPT activity in WT mice (Table 4Go). Conversely, the response to fasting was blunted in AT2R y/– mice leading to a significant decrease in both liver triglyceride and CPT activity, compared with fasted control mice (Table 4Go). This decrease in liver fatty acid oxidation capacity was also confirmed by the lower plasma ß-hydroxybutyrate in fasted AT2R y/– mice than WT mice (Table 3Go). In addition, mRNA analysis revealed that fasting induced a blunted rise in CPT-1 and AOX expression in AT2R y/– when compared with WT mice along with PPAR{alpha} expression (Fig. 6Go, A and B). No significant change on PPAR{delta} expression during fasting was observed between genotypes (Fig. 6AGo). The decreased hepatic FFA metabolism in fasted AT2R y/– mice was independent of the change in AT1R expression whatever the genotype or the nutritional status (Fig. 6CGo), suggesting that this hepatic metabolic phenotype could be the consequence of the lower plasma FFA found in these mice.


Figure 6
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  		FIG. 6. Effect of fasting on gene expression in the liver. PPARs (A), CPT-1 and AOX (B) expression in WT mice and AT2R y/– mice under fed and fasted states (n = 5 per group). C, Expression of AT1R mRNA in liver from WT and AT2R y/– mice under fed and fasted states (n = 5 per group). Values are means ± SEM and expressed as percent over values from fed WT mice. *, P < 0.01 fasted vs. fed state within genotype; #, P < 0.01 AT2R y/– vs. WT mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study reveals an inability of AT2R y/– mice to lose weight after fasting. This defect is partly due to Ang II as a regulator of the lipolytic pathway mediated by an up-regulation of AT1R. Thus, the fact that fasting leads to a higher AT1R mRNA expression in AT2R y/– mice is consistent with the moderate increase seen in their plasma FFAs in response to fasting. This observation is further supported by the in vivo treatment by an AT1R antagonist, which abrogates the protection against fat mass loss in AT2R y/– mice during starvation and improves the increase in their plasma FFA. Our findings also indicate that AT2R deficiency is associated with a high fatty acid oxidation capacity in a muscle-specific manner.

The physiological importance of angiotensinogen, the Ang II precursor, and AT2R in the metabolism of adipose tissue has been documented in recent studies using transgenic models (4, 6). Ang II was not only known to increase lipogenesis (3, 24) but also to decrease plasma FFA and glycerol in part due to an inhibition of adipocyte lipolysis (8, 9, 25). We recently showed in AT2R y/– mice, that adipocyte hypotrophy is related to defective lipogenesis (6). However, an excessive lipolytic activity might also contribute to the smaller size of adipocytes observed in AT2R y/– mice. This observation prompted us to explore their adaptation to starvation, to determine whether AT2R y/– mice developed an excessive lipolytic activity during fasting. Surprisingly, AT2R y/– mice exhibited a resistance to fasting-induced fat mass loss together with a preservation of adipose triglyceride stores suggesting that adipocytes from AT2R y/– mice hydrolyzed less triglycerides than WT mice. To support this observation, both plasma FFA and glycerol levels were moderately increased in AT2R y/– mice compared with the WT.

In the present study, by using isolated adipocytes from WT mice, we demonstrated that Ang II could act as an antilipolytic hormone through AT1R. This inhibitory effect could involve different signaling pathways including that of adenylyl cyclase (5). The ability of Ang II to inhibit adenylyl cyclase activity through its coupling to an inhibitory G protein has been demonstrated in hepatocytes and vascular smooth muscle cells (26, 27). In adipocytes, such an inhibition of adenylate cyclase results in a down-regulation of lipolysis and could account for the antilipolytic effect of Ang II in adipose tissue. Besides, Ang II is well known to increase intracellular calcium concentration, which leads to decreased lipolytic activity (28). Furthermore, Ang II has been reported to stimulate adipose prostaglandin E2 production, an antilipolytic factor (29). This new role of AT1R-mediated antilipolytic action of Ang II could account for the protection offered against fat mass loss during starvation observed in AT2R y/– mice.

Using an AT1R antagonist in vivo, we were able to demonstrate that the protection against fat mass loss en AT2R y/– mice during starvation was linked to the overexpression of AT1R. These in vivo findings also suggest that, in addition to the role of AT2R in lipogenic effect of Ang II, AT1R signaling is involved in an independent metabolic pathway leading to increase lipid stores in adipocytes. Moreover, when mice were subjected to valsartan treatment, lipolytic activity was enhanced during starvation in WT mice showing a physiological significance of the AT1R receptor. Although our data allow us to presume that AT1R signaling mediates the antilipolytic effect of Ang II, we cannot exclude the possibility of AT2R signaling being involved in the control of adipose tissue lipolysis. Indeed, it is known that the activation of this receptor results in the stimulation of the cGMP pathway, which could increase lipolysis by inducing phosphorylation of hormone-sensitive lipase (HSL) and perilipin A (30). Further investigations are required to elucidate the contribution of each receptor in the lipolytic activity of adipose tissue and know the underlying mechanisms. This agrees with the difference in adipose tissue weight observed between AT2R and angiotensinogen-deficient mice. Indeed, compared with angiotensinogen deficiency, AT2R y/– mice provoke a lower decrease in adiposity in accordance with the contribution of AT1R in antilipolytic effect of Ang II. Thus, whatever the mechanisms by which Ang II regulates lipolysis, the role of AT1R in mediating the antilipolytic effect of Ang II has physiological implications in the control of adiposity.

Despite a defective lipolytic activity in the adipose tissue of AT2R y/– mice, there was an increased lipid oxidation during the first hours after food deprivation in these animals. These findings confirm our previous report where a similar feature in the postprandial period of AT2R y/– mice was shown to be associated with an increased muscle ß-oxidation capacity (6). Similarly, the muscle ß-oxidation capacity remained higher in AT2R y/– mice after 24 h fasting, as suggested by their higher CPT activities (31). Hence, an increased peripheral fatty acid utilization in AT2R y/– mice could also contribute with the defective lipolysis to the lower increase in plasma FFA observed during the first hours of food deprivation. This is concomitant with an increased expression of PPAR{delta}, which plays a role in the up-regulation of lipid oxidation in the skeletal muscle in vivo (32). As previously described in mice overexpressing muscle PPAR{delta}, this phenotype could be related, in part, to an increase in oxidative muscle fibers as suggested by the higher succinate deshydrogenase expression observed in AT2R y/– mice (32). Despite a higher muscle ß-oxidation capacity exhibited by AT2R y/– mice, the whole-body lipid oxidation was similar in both genotypes after 4 h of fasting. This occurs concomitantly with a lower increase in plasma glycerol, suggesting that the decreased FFA release from adipose tissue is the limiting step for in vivo lipid oxidation in this animal model.

In the liver, it must be noted that AT2R deficiency does not affect FFA metabolism in fed mice, as shown by similar levels of expression of genes related to FFA oxidation. A possible explanation for this observation is the relatively low level of the expression of this receptor in the liver from WT mice. However, in response to fasting, we have shown that AT2R y/– mice display a lower liver CPT activity and plasma ß-hydroxybutyrate, reflecting a decreased liver ß-oxidation and ketogenesis. These changes in fatty acid utilization were in accordance with the minor effect of fasting on the mRNA quantity of PPAR{alpha} and its targets genes involved in FFA metabolism (33, 34). Therefore, it is surprising that the diminished ß-oxidation capacity did not provoke an increase in liver lipid stores in AT2R y/– mice. We can postulate that the lower increase in liver triglyceride content observed in AT2R y/– mice in response to fasting, resulted from the impaired adipose tissue release of FFA into the circulation, limiting the flux of FFA reaching the liver. Such a decreased hepatic triglyceride accumulation as a result of a defective lipolysis in adipose tissue has been previously described in mice lacking HSL (35). As a consequence, we observed a decreased plasma triglyceride levels in fasted AT2R y/– mice that could result in decreased hepatic very low-density lipoprotein synthesis as has been previously demonstrated in HSL-knockout mice (35).

In summary, mice lacking AT2R exhibit a resistance to fasting-induced fat mass loss, which is in agreement with the participation of Ang II in the control of adipose tissue metabolism. Our observations provide the first evidence that Ang II directly inhibits adipocyte lipolysis through AT1R and could hence participate in the regulation of FFA metabolism in AT2R y/– mice. This study also reveals the involvement of AT2R in the regulation of whole body FFA metabolism during fasting via a muscle-specific effect on ß-oxidation capacity. Such a phenotype displayed by AT2R y/– mice confirms the physiological importance of this receptor in the control of adiposity and lipid homeostasis.


   Acknowledgments
 
We would like to acknowledge Dr. Meneton for providing us with AT2R y/– mice originally supplied by Dr. Ichiki. We are indebted to Novartis Institutes for BioMedical Research, Inc. for providing valsartan.


   Footnotes
 
This work was partially supported by a grant from The Fondation de France.

Disclosure statement: the authors have nothing to declare.

First Published Online August 3, 2006

Abbreviations: Ang, Angiotensin; AOX, acyl coenzyme A oxidase; AT1R or AT2R, angiotensin type 1 or 2 receptor; CPT, carnitine palmitoyl transferase; FAT/CD36, fatty acid translocase; FFA, free fatty acid; HSL, hormone-sensitive lipase; L-FABP, liver fatty acid binding protein; PPAR, peroxisome proliferator-activated receptor; RAS, renin angiotensin system; RQ, respiratory quotient; WT, wild type.

Received June 6, 2006.

Accepted for publication July 26, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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