developmental aspects of the adipose tissue renin-angiotensin system: therapeutic implications

DRUG DEVELOPMENT RESEARCH 32~117-125 (1994)

Research Article

Developmental Aspects of the Adipose Tissue Renin-Angiotensin System: Therapeutic Implications

David 1. Crandall, Helen E. Herzlinger, Brian D. Saunders, and John G. Kral Department of Cardiovascular Research, Medical Research Division, American Cyanamid Company, Pearl River (D.L.C., H.E.H., B.D.S.), and Department of Surgery, State University of New York Health

Science Center, Brooklyn (/.C. K.), New York

I Strategy, Management and Health Policy I

ABSTRACT Adipose tissue possesses considerable growth potential, and excess adipose tissue mass is associated with noninsulin dependent diabetes mellitus (NIDDM). Adipose tissue is a significant source of angiotensinogen, the precursor of angiotensin I1 (All), a vasoactive peptide with in vitro mitogenic effects. The renin-angiotensin system remains only partially identified in adipose tissue, however, and knowledge of whether Al l has a physiologic role in this tissue is dependent in part upon other components of the system being characterized. We have chosen to further investigate aspects of the renin-angiotensin system in separate adipose tissue depots of rats during growth and development of the tissue. Either epididymal or retroperitoneal adipose tissue was examined in young lean, or adult obese male Sprague-Dawley rats. Adipose tissue was subjected to collagenase digestion, yielding separate vascular and fat cell fractions. Each fraction was analyzed for angiotensin converting enzyme (ACE) activity and Al I receptor binding capacity. ACE activity was significantly greater when expressed per gram of adipose tissue in the young lean rats. Vascular and fat cell fractions exhibited All receptors of high affinity, with a greater density of receptors observed in the epididymal adipose tissue fractions. Incubation of adipocytes with exogenous All was associated with the release of prostaglandin into the medium, and analyses of adipocyte cytosol indicated the presence of angiotensinogen. Growing rats given oral losartan, an All receptor antagonist, once daily (15 mg/kg) for 2 weeks exhibited reductions in body weight gain, fat mass, fat cell volume, and Al l receptor number. These data further document the presence of a peripheral renin-angiotensin system in anatomically distinct adipose tissue depots at different stages of growth. While the characterization of the effects of All upon adipose tissue development remains incomplete, future investigations with agents affecting the aclipocyte renin-angiotensin system could provide additional information on the role of this system in adipose tissue growth.

Key Words: adipose tissue, growth and development, renin-angiotensin system, rat

o 1994 WiIey-Liss, Inc.

INTRODUCTION

Among the numerous peptide growth factor ac- tivities described Over the last decade, the dual roles of angiotensin I1 (AII) in cardiovascular homeostasis and mitogenesis have attracted considerable atten- tion. Data implying that A11 may be involved in growth have ken generated from a Variety Of experimental Sources. Perhaps the first investigations suggesting that A11 might have functions

0 1994 Wiley-Liss, Inc.

other than fluid and electrolyte balance were performed by Campbell and Habener in 1986. In these studies, molecular probes revealed a distribution of

Accepted for publication January 21, 1994.

Address reprint requests to David L. Crandall, Department of Cardiovascular Research, Medical Research Division, American Cyanamid Company, ~ ~ i l d i ~ ~ 200, R~~~ 4603, N. Middletown Road, Pearl River, NY 10965.

118 CRANDALL ET AL.

the angiotensinogen gene in tissues not classically ex- pected to be involved in cardiovascular homeostasis [Campbell and Habener, 19861. Other investigations followed, and the term “peripheral” renin-angiotensin system (RAS) was used to describe tissue sites possessing the message, substrate, or enzymatic coin- ponents of the system outside of the classic hepatic- renal axis.

As A11 became recognized as an in vitro initogen in cell culture studies [Naftilan et al., 19891, investigations attempting to link the function of the peripheral RAS to growth and development were undertaken by a number of laboratories. The initial experiments successfully demonstrated components of the system in developing fetal tissue. In 1989, Millan et al. used autoradiographic analyses in determining that A11 receptors were expressed differentially in the developing rat fetus [Millan et al., 19891. These studies were compleinented by similar experiments describing the transient expression of the AT, subtype of the receptor in developing rat mesen- chyme [Grady et al., 19911, a correlation between A11 receptor subtype and development in rat brain [Tsu- tumi arid Saavedra, 19911, the relative expression of A11 receptor subtypes dependent upon the age ofcul- tured fetal fibroblasts [Johnson and Aguilera, 19911, and finally, the differential regulation of the AT, subtype of the receptor during kidney development in rats [Tufro-McReddie et al., 19933. While the volume of literature detailing the expression of components of the RAS during development continues to increase, it can be summarized to date h y stating that the angiotensinogen gene and the A11 receptor are expressed differentially in specific tissues during mammalian development, and that A11 has mitogenic effects in vitro.

During the course of investigations on the peripheral U S , several groups identified components of the system in adipose tissue. This series of studies began with the observation that the angiotensinogen gene was expressed in brown adipose tissue of rats [Campbell and Habener, 19871, and similar observations were subsequently extended to white adipose tissue [Cassis et al., 19881. These initial observations were complicated by the fact that adipose tissue slices were used, making the identification of the cellular source of the angiotensinogen gene difficult. How- ever, very recently, Frederich et al. [1992] identified the angiotensinogen gene in isolated adipocytes of rats and, through estimates of the number of adipocytes in mammals, correctly concluded that fkit cells are the largest potential source of angiotensinogen in the mammalian body. Studies from our laboratory which identified angiotensin converting enzyme in rat

and human adipose tissue fractions for the first time [Crandall et al., 1992a,b] as well as receptors for A11 on rat adipocyte membranes [Crandall et al., 19931, complemented the studies of Frederich et al. [1992] by identifying other components of the peripheral RAS in adipose tissue.

While adipose tissue possesses important components of the peripheral RAS, little is known con- cerning its physiologic role, especially with respect to growth and development. However, adipose tissue is one of the few tissues capable of continued growth throughout adult life which, if left unchecked, can cause obesity and contribute to NIDDM and hypertension. Therefore, the present research was de- signed to identify additional components of the RAS in adipose tissue and to investigate changes in some of these components during adipose tissue development. Finally, specific effects of an orally active A11 receptor antagonist, losartan, upon adipose tissue development have been studied.

MATERIALS AND METHODS Experimental Design

The purpose of these experiments was, in part, to observe components of the RAS during development. Therefore, rats at two different ages were studied. Male Sprague-Dawley rats (Charles River Labo- ratories, Wilmington, MA) were maintained on ad libitum food (Ralston Purina Rat Chow 5001, 4.5% fit., Purina, St. Louis, MO) and water in a controlled en- vironment. At either 40 or 125 days of age, rats were weighed, killed by carbon dioxide inhalation, and adipose tissue depots excised and weighed. These two groups were designated as either young lea11 or adult obese because of their age and adipose tissue mass. The epididymal depot adjacent to each testis and the retroperitoneal depot surrounding each kidney were specifically studied because of the well-documented differences in the pattern of growth [Newby et al., 19901. The experimental design therefore provided for the analyses of adipose tissue at different ages and from anatomic sites classically exhibiting different growth patterns.

Cell Separation and Membrane Preparation

After weighing, the process of dividing the tissue into specific cellular fractions was begun (Rodbell, 1964). Each depot was minced into sinall pieces and approximately 10 g transferred to a flask containing 25 in1 of Krebs-Ringer-bicarbonate buffer (KRB), 6 mM glucose, and 50 mg of collagenase, pH 7.4. The flask containing the minced adipose tissue was shaken vig- orously (150 strokeslmin) at 37°C for 30 min, fo1lowt:d

ANGIOTENSIN II IN RAT ADIPOSE TISSUE 119

by passing of the contents through 150 p nylon mesh. Intact adipocytes which had become dissociated from the tissue passed freely into waiting tubes, while vascular tissue was trapped on the screen. The tubes containing the adipocytes were washed an additional 4 times with 20 ml of cold Tris buffer (10 mM Tris HCI, pH 8.0) by first allowing the adipocytes to separate from the digestion medium by flotation, remov- ing the infranatant through polyethylene tubing at- tached to a syringe, resuspending the cells in the Tris buffer, and repeating this procedure. The vascular tissue was removed from the nylon screen with a stainless steel spatula, and placed into cold Tris buffer. Immediately after the final wash, an appropri- ate aliquot was taken from each adipocyte preparation for determination of lipid content, microscopic sizing of cell diameter, and visual verification of the purity of the preparation [ Goldrick, 1967; DiGirolamo et al., 19711. All enzymes and reagents used in these proce- dures were purchased froin Sigma Chemical, St. Louis, MO.

Adipocyte membranes were prepared essen- tially according to the method of Mauriege et al. [1987]. Using a Brinkmann polytron with a small, blunt probe, adipocytes were homogenized for 20 sec at a medium speed, and the resulting homogenate centrifuged at 40,OOOg for 35 minutes and 4°C. Fol- lowing centrifugation, the fat cake and supernatant were carefully removed, and the pellet resuspended in 1 ml of Tris buffer. Crude vascular membranes were prepared by homogenization and centrifugation as described for the adipocytes. Protein content of each pellet was determined by the method of Lowry et al. [1951]. The resuspended protein was either assayed immediately or stored at -75°C for no longer than 1 week before binding assays were performed.

Receptor Binding Studies

The receptor binding studies were undertaken for two purposes: (1) to further characterize developmental aspects of the adipocyte A11 receptor which we initially described [Crandall et al., 19931, and (2) to investigate for the first time, A11 receptors in the stromal-vascular fraction of adipose tissue. Mem- branes were diluted with 0.25% BSA buffer (bovine serum albumin, 50 mM Tris EICl, 5 mM MgCl,, pH 7.4) to a final concentration of 5 pg proteinA0 pl buffer. For Scatchard analysis, membranes were incubated with 6-12 different concentrations of 12'1- [Sar',Ile'] A11 (Dupont-NEN, Boston, MA) ranging from 0.15 to 5.0 nM. A typical incubation tube contained 160 pl (80 pg) fat cell or vascular membrane protein, 20 pl of radiolabeled angiotensin 11 and 20 p1 of 1 p M angiotensin I1 as appropriately required for

determination of nonspecific binding. The assay was initiated by the addition of membrane protein, followed by incubation at 22°C for 30 min in a slowly shaking water bath. Separation of bound from free radioactivity was performed on a Brandel filtration apparatus containing a Whatman GF/B filter, followed by 6 additional rinses with 5 ml each of cold 0.9% saline. The filters were placed in tubes, and radioactivity measured in a gamma scintillation counter programmed to correct for the half-life of the isotope (Packard Cobra 5010; 80% counting effi- ciency). Data was processed by RS232 interface di- rectly into a VAX mainframe computer containing software for determination of equilibrium dissociation rates (Kd) and maximal binding sites (B,,,,) (Lundon, Inc., Chagrin Falls, OH).

Metabolic Effects of All on Adipocytes

In separate experiments, isolated adipocytes from the epididymal and retroperitoneal depots were isolated with a collagenase buffer as described earlier. Cells were then resuspended in KRB without collagenase at a final concentration of 250,000 cells/ml, and incubated at 37°C for 1 h in the presence of 10 p M AII. At the end of the incubation, tubes were placed on ice and the medium was decanted and stored over- night at -20°C. The next morning, the medium was thawed and assayed for PGF,,, a stable metabolite of prostacyclin, using a commercially available assay system (DuPont-NEN). Data were corrected for cell number, and were expressed as prostacyclin release per adipocyte.

Adipocyte Angiotensinogen Assay

In order to assess adipocyte angiotensinogen content, the production of angiotensin I was determined in the presence of renin [Frederich et al., 19921. Ad- ipocytes were isolated by collagenase digestion and filtration as described earlier, following by homogenization in a buffer composed of 0.25 M Tris acetate (pH 7.0), 3% albumin, 5 mM Na,EDTA, and 0.1 mM cap- topril (Sigma). A1 was generated for 1 h in the presence of excess (0.05 U/ml) porcine renin as described by Frederich et al. [1992], followed by quantitation of A1 in triplicate using a commercially available radioiin- munoassay system (Dupont-NEN). A standard curve was generated using different quantities of AI, and the amount of A1 generated in each adipocyte homogenate was calculated by comparison with that in samples incubated in the absence of renin.

Angiotensin Converting Enzyme (ACE) Activity

The ACE Microvial Radioassay System (Ven- trex, Portland, ME) provided a quantitative deter-

120 CRANDALL El AL.

~~ ~ ~

TABLE 1. Experimental Design to Determine Angiotensin II Binding in Crowing Rat Adipose Tissue'

Total depot weight (g)" Body weight Age No. rats/

Group (g) (days) preparation Epididymal Retroperitoneal

Young, lean 149 2 6 40 12 9.75 k 0.94 6.78 I 0 . 2 0 * " Adult. obese 620 ? 17' 125 1 12.55 k 1.62 13.72 t 3.07

Values are the means t s.e.m. of n = 6 preparations for each age examined. "Depot weight per animal is equivalent to total weight divided by the number of ratdprepara tion. *Significantly different from other group body weight at P i 0.001. **Significantly different from young epididvnial weight at P < 0.05.

mination of ACE. This assay served the purpose of verifying the separation of vascular tissue from adipocytes and determining ACE levels in adipose tissue during developinent.

Effect of Oral Losartan on Adipose Tissue Development

To determine the effects of an orally active A11 receptor antagonist on adipose tissue, an in vivo study was performed. Male Sprague-Dawley rats, 100 days of age, were divided into two groups of 6 rats each. Rats were gavaged with either the orally active A11 receptor antagonist, losartan (gift of Dr. Ronald 1). Smith, DuPont Merck, Wilmington, DE), once daily for 2 weeks (1s mg/kg body weight; 15 mg/ml distilled water) or distilled water (control group). Body weight and food intake were inonitored daily. At the end of the %week period, rats were killed approximately 15-18 h following the final dosing of the antagonist. Epididymal adipose tissue depots were quickly excised and weighed, fat cells harvested and sized, membranes isolated, and binding studies performed.

Statistical Analysis

Statistical analysis was performed using Statis- ticalMac (Statsoft, Tulsa, OK). Group means were considered significantly different at P < 0.05.

RESULTS

The experimental design for determining A11 binding characteristics in adipocyte memhrane~ from the adipose tissue of growing rats is shown in Table 1. Two different groups of rats with differing ages, body weights, and fat depot weights were used. Body weights were significantly different between groups. When cornparing individual paired depot weights between young lean and adult obese rats, epididyrnal weight increased from 0.82 g to 12.5Ei g ( 1 5 ~ ) , and

(25 x ). Table 2 contains the cellular composition of the two adipose tissue depots, indicating that at each stage of growth, retroperitoneal fat cells were significantly larger in volume than the epididyrnal cells, although the quantitative mean difference in the young rats was less than 20 pl. As the adipocytes enlarged, the nuinher of cells per mg of membrane protein necessarily decreased.

Data analysis for identification of the number and affinity of A11 binding sites on fat cell membranes is shown in Table 3, and indicates similar affinities for all 4 groups. Significant differences were observed in B,,,L,X, however, and the pattern of hinding varied between depots. When expressed as fmol/mg protein, epididyrnal adipocytes exhibited significantly fewer binding sites in the adult animals. A similar pattern was observed in retroperitoneal depots, suggesting an age-associated decrease in receptor concentration in both depots. When comparing interdepot values in rats of the same age, significant differences in B,,,,, ~7ere observed between retroperitoneal and epididy- ma1 adipocytes, with retroperitoiieal cells exhibiting consistently fewer receptor sites. Because the retroperitoneal fat cells were larger than the epididyinal in the adult rats, it is important to note that estimates of correction of hinding for cell size would not normalize the interdepot differences observed.

While A11 receptors have been well characterized in vascular tissue from a variety of anatomic sites, specific analysis in adipose tissue microvasculature has not been described. Since the methodology we employed for the isolation of adipocytes necessarily involved the removal of the stromal-vascular fraction, vascular tissue was readily available for binding studies. Scatchard analysis of binding data obtained from the vascular fraction of adipose tissue was examined only in adult rats, and indicated the presence of an A11 receptor with high affinity and a density that varied between depots (Fig. 1). BlllilX for epididymal microvascular membranes was significantly greater than

retroperitoneal weight increased from 0.57 to 13.72 g retroperitoneal, 48.3 (12.5) compared. to 25.9 (7.7)

ANGIOTENSIN II IN RAT ADIPOSE TISSUE 121

TABLE 2. Effects of Age and Fat Mass on lnterdepot Differences in Adipocyte Morphology*

Epididymal Retroperitoneal

diameter volume membrane diameter volume membrane Cells/mg Cell Cell Cellshng Cell Cell

Rat size (wn) (PI) protein (pm) (PI) protein

Young, lean 50.0 ? 0.8 a2 -c 4 7.78 !z 0.44 51.9 ? 0.2 98 ? 4** 6.84 ? 0.48 Adult, obese 95.1 ? 5.2 508 ? 80 2.76 ? 0.50 98.3 t 4.0 603 +- 68** 2.63 ? 0.26

*Values represent cells x 10'. Cell volume, diameter, and membrane protein values are always significantly different ( P < 0.01) from the other group within each depot. No significant differences between cells/mg protein for epididymal vs. retroperitoneal. **Significantly greater than epididymal value at P < 0.01,

TABLE 3. Analysis of Angiotensin I1 Receptor Binding to Rat Fat Cell Membranest

Epididymal Retroperitoneal

~ m a x

Rat size (fmol/mg protein) K, (nM)

Young, lean 46.4 1.44 (6.8) (0.20) n = 8

Adult, obese 26.0*** 1.68 13.9) (0.26) n = 3

Bmax (fmol/mg protein) K, (nM)

30.0' 1.84 (4.6) (0.31) n = 8

1 1.2*,** 1.76 (3.2) (0.51) n = 3

'All values are mean with s.e.m. in Parentheses of n experiments. *Significantly different from corresponding epididymal value at P < 0.05. **Significantly different from young retroperitoneal membranes at P < 0.05 ***Significantly different from young epididymal membranes at P < 0.05.

fmol/mg protein (P < .05; s.e.m. in parentheses). However, the affinity (Kd) of the receptors was similar (0.48 * 0.08 nM for epipidyinal vessels and 0.41 * 0.76 nM for retroperitoneal vessels).

Following the characterization of adipocyte A11 receptors, other aspects of the RAS in adipose tissiie were examined only in adult rats. In Figure 2, tlie effect of 10 pM A11 upon PGF,, release b y isolated adipocytes is shown. Adipocytes from both depots exhibited a basal PG release which was elevated upon the addition of AIL Angiotensin converting enzyme activity was also quantitated in whole adipose tissue depots of growing rats. When expressed as ACE activity/g wet weight, young lean rats had 426.3 (55.1) U/g and adult obese rats had 84.0 (13.7) U/g in the epididymal fat (s.e.m. in parentheses; P < .01). In retroperitoneal adipose tissue, ACE activity was 443.9 U/g in young lean rats and 91.4 (55.5) U/g in adult obese rats ( P < .01). No significant dif€erences were observed between depots at each age examined. ACE activity was never observed in adipocyte fractions after separation from the whole tissue, suggesting that the source of ACE was of vascular origin. Fat cell angiotensinogen content was also estimated in epididymal and retroperitoneal adipocytes. Epididymal

adipocytes contained 6.53 (1.12) ng/107 fat cells ofan- giotensinogen compared to 4.85 (. 74) ng/107 fat cells in the retroperitoneal depot (s.e.m. in parentheses). Although there was a trend for reduced angiotensinogen in retroperitoneal adipocytes, differences between depots were not statistically significant.

Effects of oral losartan on body weight and rat adipose tissue during a %week in vivo study are shown in Table 4. While rats were initially of an equivalent body weight, 14 consecutive days of 15 mglkg body weight of oral losartan resulted in a significant reduc- tion in growth as estimated by mean body weight. A quantitatively small, yet statistically significant reduc- tion in daily food intake was observed for losartan- treated animals. Additional analysis of epididyinal adipocyte physiology is shown in Table 4, indicating reduced adipose tissue weight, adipocyte volume, a i d A11 receptor binding in losartan-treated rats.

DISCUSSION Adipose tissue possesses the potential to grow

throughout most of adulthood, yet the basis for continued adipose tissue growth remains largely un- known. Because of the putative role of A11 in devel-

122 CRANDALL ET AL.

5 0

4 0

3 0

2 0

1 0

0

5 0

4 0

3 0

2 0

1 0

0 0

0 .5 i 1 .5 2

T

b

0 . 5 1 1 . 5

51-ANGIOTENSIN I I

2

Figure 1. Saturation binding and Scatchard analysis (insets) of 1251-All to crude membrane preparations isolated from adipose tissue microvessels of the (a) epididymal or (b) retroperitoneal depots of 600 g rats (n = 5). Lines represent best-fit to actual

means of raw data indicated by solid blocks. s.e.m. bars are shown for each data point, and where not visible, lie within the data point. See text for additional details.

ANGIOTENSIN I I IN RAT ADIPOSE TISSUE 123

Epldidymal Ret roper l tonea l

FAT DEPOT

Figure 2. Release of PGF,,, a stable metabolite of prostacyclin, in isolated adipocytes from 600 g rats (n = 3). Prostaglandin was measured in the medium after a 1 h incubation of 250,000 adipocytes in the presence or absence of 10 pM angiotensin II (All) and saralasin. Values are expressed as ng/106 cellsih.

TABLE 4. In Vivo Effect of All Receotor Blockade on EDididvmal AdiDose Tissue in Rats'

Epididymal adipose tissue

Initial body Final body Bodv weight Daily food Cell B*ax

weight weight gain intake Weight volume (fmol/mg Kd (g) ( g) (g) (g) (g) (PI1 protein) (nM1

Control 399 455 5 6 32.8 5.2 256 55.6 0.97 (9.2) (1 1.31 (4.11 (0.82) (0.391 (30.3) (1 3.5) 10.241

Losartan 396 432 36* 30.1 * 4.1 * 163* 20.5 0.64 (1 5 mgikg) (7.0) (12.21 16.2) (0.781 (0.46) (4.91 (4.6) (0.07)

'Values are mean (2 s.e.m.] of n = 6 rats in each treatment. "Significantly different from control group value at P < 0.05.

opment, we recently began investigations identifying components of the RAS in adipose tissue. To d, $1 t e, we h v e successfully identified ACE in rat and human adipose tissue of obese individuals [Crandall et al., 1992a,b]. An A11 receptor was also recently identified and characterized in fat cells from aged rats [Crandall et al., 19931. These data complement identifica.tioi1 in adipose tissue slices of the angiotensinogen gene [Cassis et al., 19883, which is now known to be locally

regulated within the adipocyte by the nutritional state of the aniinal [Frederich et al., 19921. To date, no attempts have been made to characterize aspects of the adipose tissue RAS during different stages of development, even though a number of investigations in other fetal tissues suggest a general role in this process [Millan et al., 1989; Grady et al., 1991; Tufro- McReddie et al., 19931.

Our original studies on A11 receptor identifica-

124 CRANDALL ET AL.

tion were in fat cell membranes from a single anatomic site at an end-stage of growth, but because adipose tissue growth varies with anatomic location in both rodents and humans [Crandall et al., 1984; Ohl- son et al., 19851, the present experiments were performed to include fat cell membranes obtained from different anatomic locations at different ages. The receptor previously identified in epididymal adipocyte membranes had additionally been observed to vary in number with age and stage of development of the animal. An A11 receptor has also been identified in retroperitoneal adipocytes, an anatomic site which classically has greater growth potential than the epididymal [ Lemonnier, 1972; Newby et al., 19901. When expressed per unit of membrane protein, there was a significant decrease in retroperitoneal binding when compared to epididyinal values from the same animal, and the retroperitoneal B,,,2,X decreased as the adipocytes enlarged. The same pattern was observed for microvasculature harvested from the adipose tissue: retroperitoneal vessels exhibited significantly fewer binding sites than epididyrnal vessels in adult rats. It is therefore apparent from A11 hinding data in cellular fractions of rat adipose tissue that the pattern of receptor expression varies with the anatomic site of the depot, the age of the animal, and the potential for growth of thc tissue.

Other components of the adipose tissue RAS were also oliserved in these studies. Total adipose tissue ACE activity was determined in both depots in young lean and adult obese rats. When expressed as activitylg wet weight of the depot, ACE was observed to decrease in tmth sites with age. However, no differences were observed between sites for total ACE activity when comparing rats of the same age. Adipo- cyte angiotensinogen content and AII-stimulated prostacyclin release were similar between depots. The successful identification of various cornponents of the U S prompted an in vivo study of the effects of losartan, an orally active inhibitor of A11 receptor binding, on body weight and adipose tissue growth in rats. During a limited study at a modest concentration of compound, normotensive rats were observed to continue to grow, hut at a reduced rate cornpared to control animals. Epididymal adipose tissue was further examined because it had exhibited the greatest B,,,,, in the receptor binding experiments. Drug- treated animals had significantly less epididymal fat and smaller adipocytes. The number of A11 receptor binding sites was also reduced in losartan-treated rats.

While the effects of angiotensin I1 on adipose tissue are in the early stages of laboratory investiga- tion, the putative importance of A11 in growth is sug- gested by experiments conducted at different levels of

physiological organization. At the molecular level, the gene encoding angiotensinogen is under tissue-specific hormonal and developmental regulation. An increase in expression of this gene occurs during the differentiation of fibroblasts to preadipocytes [ Mc- Gehee et al., 19931, indicating that this hormone is in fact involved in adipose tissue development. At the cellular level, prostacyc!in has been proposed to be a potent effector in adipocyte differentiation [Negrel et al., 19891. At the metabolic level, studies have shown that A11 stimulates release of prostacyclin by adipocytes, and this release has been postulated to promote local vasodilation, maintenance of luminal patency, and supply of substrate to the cell required for enlargement [Axelrod et al., 1985; Richelson, 19871. Fi- nally, at the symptomatic level, local angiotensin I1 production by adipose tissue could contribute to the hypertension associated with obesity [Cassis et al., 19881, and, in fact, specific patterns of fat deposition are associated with a greater incidence of hypertension [Larsson et al., 19841. The potential therefore exists for A11 to affect cell growth and differentiation at several physiological levels. In this context, it is important to note that our adipocyte binding data to- gether with microvascular receptor identification, fat cell angiotensinogen content, and metabolic re- sponses to A11 firmly establish adipose tissue as an important effector Yite for the peripheral RAS.

While receptor binding studies in rats have revealed an adipocyte A11 receptor, the expression of which varies with the growth potentid of the adipose tissue depot, the relevance of this finding to normal growth and development requires further investiga- tion. Hypothetically, if signal transduction mecha- nisms following A11 receptor binding are invo1t.d in the regulation of adipose tissue growth, drugs block-. ing the receptor or affecting other components of the local renin-angiotensin system could impact upon this process. Relative enlargement of visceral fat depots is associated with most metabolic abnormalities of obesity resulting in hypertension, NIDDM, and dyslipi- demia. Thus clarification of the relationship between the local RAS and adipose tissue growth and metabolism could lead to significant therapeutic advances based on pharinacologic modulation of visceral adipose tissue growth. The availability and comparative ease of detailed analyses of adipose tissue make it an ideal tissue for studies of this nature.

ACKNOWLEDGMENT

We appreciate the assistance of Dr. Joyce Harp, Emory University, in development of the adipocyte angiotensinogen assay.

ANGlOTENSlN II IN RAT ADIPOSE TISSUE 125

REFERENCES Axelrod L, Minnich A, Ryan C (1985): Stimulation of prostacyclin

production in isolated rat adipocytes by angiotensin 11, vaso- pressin, and hradykinin: Evidence for two separate mechmisms of prostaglandin synthesis. Endocrinology 1162548-2553.

Campbell DJ, Hahener J F (1986): Angiotensinogen gene is expressed and differentially regulated in multiple tissues of the rat. J Clin Invest 78:31-39.

Campbell DJ, Hahener J F (1987): Cellular localization of angioteit- sinogen gene expression in brown adipose tissue and mesentery: Quantification of messenger ribonucleic acid abundance using hybridization in situ. Endocrinology 121:1616-1626.

Cassis LA, Saye J , Peach MJ (1988): Location and regulation of rat angiotensinogen messenger RNA. Hypertension 11:59-596.

Crandall DL, Goldstein B, Huggins F, Cervoni P (1984): Adipo- cyte blood flow: Influence of age, anatomic location, and dietary manipulation. Am J Physiol 247:R46-R51.

Crandall DL, Herzlinger HE, Cervoni P, Scalea TM, Kral JC (1992a): Vasoactive substances in human adipose tissue biopsies. In Ailhaud G, et al. (eds): Obesity in Europe. London: John Lihbey & Company Ltd, pp 405-409.

Crandall DL, Gordon G , Herzlinger HE, Saunders AD, Zolotor HC, Cervoni P, Kral JG (19921~): Transforming growth factor alpha and atrial natriuretic peptide in white adipose tissue depots in rats. Eur J Clin Invest 22676-680.

Crandall DL, Herzlinger HE, Saunders BD. Zolotor KC, Feli- ciano L, Cervoni P (1993): Identification and characterization of angiotensin 11 receptors in rat epididymal adipocyte men- hranes. Metaldisin 42:511-515.

DiGirolamo M, Mendlinger S, Fertig JW (1971): A simple method to determine fat cell size and number in four mammalian spe- cies. Am J Physiol 221:850-858.

Frederich RC, Kahn BB, Peach MJ, Flier JS (1992): Tissue-specific nutritional regulation of angiotensinogen in adipose tissue. Hypertension 19(4):339-344.

Goldrick R (1967): Morphological changes i n the adipocyte during fat deposition and mobilization. A m J Physiol 212:777-'782.

Crady EF, Sechi LA, Griffin CA, Schamhelan M, Kalinysk JE (1991): Expression of AT, receptors in the developing rat fetus. J Clin Invest 88:921-933.

Johnson MC, Agiiilera C (1991): Angiotensin-I1 receptor suhtypes and coupling to signaling systems in cultured fetal fibroblasts. Endocrinology 129(3): 1266-1274.

Larsson B, Svardsudd K, Welin L, Wilhelmsen L, Bjorntorp P, Tibl~lin G (1984): Abdominal adipose tissue distril)ution, ol~e-

sity, and risk of cardiovascular disease and death: 13 year follow-up of participants in the study of men horn in 1913. Br Med J 288:1401-1404.

Lemonnier D (1972): Effect of age, sex and site on the celliilarity of the adipose tissue in inice and rats rendered obese by il high- fat diet. J Clin Invest 51:2907-2915.

Lowry OH, Rosebrough NJ, Fdrr AL, Randall KJ (1951): Protein measurement with the Folin phenol reagent. J Riol Chem 1

Mauriege P, Galitsky J , Berlan M, Lafontan M (1987): Heteroge- neous distrihution of beta and alpha-2 adrenoceptor binding sites in human fiat cells from various fat depots: Functional con- sequences. Eur J Clin Invest 17:156-165.

McGehee R, Ron D, Brasier A, Habener J (1993): Differentiation- specific element: a cis-acting developmental switch required for the sustained transcriptional expression of the angiotensinogen gene during hormonal-induced differentiation of 3T3-Ll fihro- hlasts to adipocytes. Mol Endocrinol 7:551-560.

Millan MA, Carvallo P, lzumi SI, Zemel S, Catt K, Aguilera G (1989): Novel sites of expression of functional angiotensin I1 receptors in the late gestation fetus. Science 244: 1340-1342.

Naftilan AJ, Pratt RE, Dzau VJ (1989): Induction of platelet-de- rived growth factor A-chain and c-myc gene expression b y an- gioteiisin I1 in cultured rat vascular smooth muscle cells. J Clin Invest 83: 1419-1424.

Negrel R, Gaillard D, Ailhaud C (1989): Prostacyclin as a potent effector of adipose-cell differentiation. Biochern J 257:399-405.

Newby FD, DiGirolamo M, Cotsonis G , Kutner M (1990): Model of spontaneous obesity in aging male Wistar rats. Am J Physiol

Ohlson LO, Larsson B, Svardsudd K, Welin I,, Erikssori H, Wilhelinsen L, Bjorntorp P, Tibblin G (1985): The influence of body fat distrihution on the incidence of diabetes inellitus: 13.5 years of follow-up of the participants in the study of inen born in 1913. Diabetes 34: 1055-1058.

Richelsen B (1987): Factors regulating the production of prostaglandin E, and prostacyclin in rat and human adipocytes. Bio- chein J 247:389-394.

Hodhell M (1964): Metabolism of isolated fat cells. J B i d Chem

Tsutsuni K, Saavedra J M (1991): Characterization and development of angiotensin I1 receptor subtypes (AT, arid AT,) in rat brain. Am J Physiol 26:R209-H216.

Tufro-McHeddie A, Harrison JK, Everett AD, Gomez HA (1993): Ontogeny of type 1 angiotensin I1 receptor gene expression in the rat. J Clin Invest 91:530-537.

265 -275.

259:Kll17-1125.

239:375-380.

developmental aspects of the adipose tissue renin-angiotensin system: therapeutic implications

Documents