The effect of anti-hypertensive drug treatment on brown adipocyte diameter and locule distribution in rats

Camp. Biochem. Phvsiol. Vol. 19C, No. 2, pp. 317-320. 1984 Printedin Great Bhtain

0306-4492/84 S3.00 + 0.00 6~: 1984 PergamonPress Ltd

THE EFFECT OF ANTI-HYPERTENSIVE DRUG TREATMENT ON BROWN ADIPOCYTE DIAMETER AND LOCULE DISTRIBUTION IN RATS ORIEN

L.

TULP,*

DANIEL RooTt

and

RUTH FRINK~

*Department of Nutrition and Food Science, Nesbitt College, Drexel University, Philadelphia, PA, USA. Telephone: (215) 895-2419 and tDepartment of Anatomy and Neurobiology, The University of Vermont, Burlington, Vermont, USA (Received 8 February 1984)

Abstract-l. To determine the effects of sympathoplegic anti-hypertensive drug treatment on brown adipose tissue morphology, groups of adult male Wistar-Kyoto (WKY) and spontaneous hypertensive (SHR) rats were orally administered a solution containing 2.3 PM reserpine, 0.5 M hydralazine and 1.68mM hydrochlorothiazide ad libitutn or tap water and brown adipocyte diameter and extent of fat loculization were determined 3 weeks later. 2. Pulse rates of rats were significantly greater in SHR than WKY and were unaffected by treatment, while drug treatment resulted in significant decreases in mean arterial pressure of both groups at the end of the study. 3. Mean adipocyte diameters were smaller in untreated SHR than WKY and drug treatment of both groups was associated with increases in adipocyte diameter and cytological change from physiologically more active to less active cells. 4. These drug-induced alterations in BAT morphology area consistent with decreased sympathetic

activity and suggest that thermogenic capacity of brown adipose tissue may be pharmacologically modified, thereby altering an animal’s capacity for energy expenditure.

INTRODUCTION

Brown adipose tissue is the only tissue of homeotherms whose primary function is thermogenesis and is important, therefore, in the regulation of energy expenditure (Hahn and Novak, 1975; Tulp, 1981). Brown adipocytes differ from white adipocytes both on the basis of their principal physiologic function of heat production rather than energy storage, and morphologically by the presence of multiple small locules of fat and a centrally-positioned nucleus, rather than a single, larger fat droplet and a flattened, peripherally-located nucleus (Tulp, 1981). Recent studies have shown that the quantity of interscapular brown adipose tissue became markedly increased during experimental overnutrition in the rat, but that mature brown adipocyte diameters were similar to those of normally-fed animals (Tulp, 1981; Rothwell and Stock, 1979). The increased mass of brown adipose tissue of young rats would appear to be a consequence of proliferation and/or differentiation of adipocytes similar to that which follows chronic cold exposure rather than hypertrophy of existing cells (Tulp, 1981; Cameron and Smith, 1964). During adrenergic stimulation, brown adipocyte metabolism is stimulated resulting in generation of heat, a diminution of adipocyte lipid content, and a decrease in locule size (Brooks et al., 1980; Mrosovsky, 1962, 1964; Mrosovsky and Rowlatt, 1968). In contrast, when the thermogenic activity of brown adipose tissue is decreased, lipid accumulates and the locules may enlarge to the point of coalescing into a single, combined microdroplet of lipid nearly as large as the intact cell in which it is contained (Mrosovky 317

and Rowlatt, 1968; Himms-Hagen and Desaultels, 1978; Himms-Hagen, 1980; Tanuma ef al., 1975). Morphologic analysis during which the contained lipid stores are subjected to a qualitative grading of the diameter and frequency of their contained lipid locules, accompanied by measurements of adipocyte diameter may provide a valid indicator of the recent thermogenic activity of this tissue (Tanuma et al., 1975). The purpose of the present study was to determine if brown adipocyte morphology differed in strains of hypertensive and normotensive rats, and if a sympathoplegic antihypertensive regimen would result in morphologic evidence of decreased thermogenic activity and which might be characterized by the intracellular accumulation of lipid. The spontaneously hypertensive rat (SHR) and its normal Wistar-Kyoto (WKY) control were selected for these studies because of the ability to assess the effectiveness of the sympathoplegic effects via measurements of blood pressure, and because of the prominent interscapular brown adipose tissue depots normally present in these strains.

MATERIALS AND METHODS

Adult, male Okamoto-derived WKY (Wistar-Kyoto) and SHR (spontaneously hypertenive) rats averaging 27.8 weeks of age and at stable body wt (382 k 8 and 361 + 4g, respectively) were obtained from the University of Vermont colony, maintained in groups of 34 rats each and housed in plastic wire topped cages fitted with filter-barrier covers. They were maintained at 22%23°C with an automatic (12: 12) light-dark cycle, and free access to Purina laboratory chow No. 5000 (Ralston Purina Co., St. Louis, MO.). Each strain

ORIEN L. TULP et al.

318

Brown

was divided into two equal treatment groups (N = 6 rats/group), and given free access to either tap water or a solution containing the sympathoplegic agents reserpine and hydralazine and the diuretic agent hydrochlorothiazide (Ciba-Geigy Corp., Summit, NJ) at final concentrations of 2.4 FM, 0.5 M and 1.68 mM, respectively, for a period of 21 days. Measurements of body wt were taken initially and at the end of the study. Pulse measurements and systolic arterial blood pressure measurements were obtained with a tail cuff and an electroplesmograph initially and after 21 days of treatment when hypotensive effects appeared to be maximal. Following sacrifice, the interscapular brown adipose tissue (BAT) depots were rapidly excised in their entirety, weighed and fixed in 10% buffered formalin. Semi-thin (6nm) histologic sections were stained with hematoxylin and eosin. Cell diameters were measured with the aid of a stage micrometer. and the morphologic character of the adipocyte locularity evaluated using the criteria outlined by Tanuma et al. (I 975). Briefly. with these criteria. type I cells were devoid of fat locules; type II contained several small locules; type III contained abundant small locules; type IV contained several larger fat locules and type V contained a single, large locule of fat. Cells were distinguished from white adipocytes on the basis of lipid locularity, a more abundant, granular cytoplasm and a more centrally located nucleus than is observed in white adipocytes. In additional groups of untreated animals the IBAT and epididymal fat pads were removed and weighed in their entirety. Statistical treatment of data was accomplished with an HP4lCV calculator (Hewlett-Packard Corp, Corvalhs, OR) using standard, statistical programs for two-way analysis of variance (ANOVA), x2 analysis and unpaired Student’s t-test. In some cases more than one statistical test was applied to ensure the most vigorous treatment of data.

Dim&r,

A. Experiment

WKY-T

Loculority

Type

control animals of both strains maintained weight during the 3 weeks of study. Drug treatment resulted in an average loss of 15 g per rat in the WKY animals, but SHR rats gained 46 g per rat during the 3 weeks of observation. Visual observations of drinking activity and of liquid wastes during the study indicated that drug treatment resulted in relatively greater deficits in water balance in the WKY than in SHR rats. Behavior of animals during handling was consistent with decreased activity levels in treated animals of both strains. 4nalysis of variance indicated that both the factors of treatment and rat strain were significant with respect to final body wt and mean arterial pressure, but that only rat strain was a significant factor with respect to pulse rate. The weights of the interscapular brown adipose tissue and the epididymal fat pads of control WKY and SHR rats are shown in Table 1 and indicate that initial IBAT weight was similar in both strains, but that the weights of the epididymal fat depot were significantly less in the SHR than in WKY animals in untreated rats. Visual inspection of the carcasses indicated that other white fat depots also were much

pressure Within strain P

of WKY and SHR rats SHR-C

SHR-T

364 k 7 369 i-8 NS NS

358+21 357 i_ 19 NS 448+23

203 i IO 5

114*7 5

Within strain P

Between strain P

I

BWk)t Initial Final Treatment P Pulse (beats/min)$ SAP (mmHg)f Final N B. Experiment 2 IBAT(mg)$ IBAT (BW x IO-‘) Epididymal fat pads (g) Epididymal W (BW x 10e2) N

392 i 24 391 i 22 NS

98 i 8 6

129k6 5 388 9.4 6.3 I .4s

+ f k * 4

382fl8 369 + 14 NS 311 k43

36 I.1 0.3 0.02

*

Treated

contml

pulse and blood

WKY-C

u

Distribution

Fig. I. Brown adipocyte diameter and locule distribution in WKY rats. Locularity frequency distribution P = ~0.01 by x2 analysis; ** < 0.01 for cell diameters or individual cell types (control vs treated) by unpaired comparison with Student’s t-test.

The results of the drug treatment on pulse, blood pressure and body wt are outlined in Table I. The pulse rate of SHR rats was significantly greater than that of WKY and was unchanged with the drug treatment in either strain. Mean arterial blood pressure, however, decreased by over 25% in WKY and 40”/0 or more in the SHR rats. Initial body wt was comparable within both groups of either strain. The

I. Biometry,

Diameter and Lode d WKY Rats

l

RESULTS

Table

Adipcyte

NS NS 313k24 < 0.05

315* x.7 * 3.0 + 0.83 + 4

NS NS

NS NS

450 ? 22

NS

< 0.01

< 0.05

NS NS < 0.05 < 0.05

14 0.2 0.2 0.05

WKY-C = Wlstar-Kyoto control; WKY-T = Wistar-Kyoto treated; SHR-C = spontaneously T = spontaneously hypertensive treated. *Between strain effects determined prior to treatment. tBW = body wt. fPulse and SAP (systolic arterial pressure) measurements taken post-treatment. SIBAT = interscapular brown adipose txsuc.


hypertensive

control:

SHR-

Antihypertensives and brown fat morphology Broom Adipccyte Diameter and Locule Distributiar ot SHR Rats

.dl .* **

PXXP

control

PIPIPP

Tmsted

Fig. 2. Brown adipocyte diameter and locule distribution in rats. Locularity frequency distribution P = ~0.01 by x2 analysis; *P = < 0.05 and **P = CO.01for comparisons of cell diameter or locularity type (control vs treated) by Student’s f-test (unpaired). SHR

less prominent in SHR than in WKY animals. BAT depots were visibly similar in gross appearance in both strains. The morphologic characteristics of the interscapular BAT depots of the WKY and SHR animals are depicted in Figs 1 and 2, respectively. In both strains, adipocyte diameter increased an average of 3pm or approx. 1574 with drug treatment. These increases in brown adipocyte diameter were associated with a redistribution of the size and frequency of intracellular fat locules as depicted in the right panel of each figure. In WKY rats the frequency of adipocyte locularity in types II and III cells in untreated rats was nearly 60:? of the total but in treated animals the combined frequency of these cell types was less than 10%. Types IV and V represented more than 90% of the total after treatment, which was consistent with the larger adipocyte diameters and with the greater accumulation of locule-bound lipid in those cells. Brown adipocytes from treated SHR rats showed similar increases in the frequency of cell types IV and V accompanied with corresponding decreases in the frequency of types II and III when compared to IBAT cells from the untreated animals. These drug-associated increases in intracellular lipid content were also consistent with the greater cell diameters observed in those animals. The redistribution of adipocyte locule frequency in both strains following treatment was highly significant by x” analysis. The differences in frequency of individual cell types II-V in WKY and III-V in SHR were also highly significant when subjected to the unpaired t-test in separate comparisons.

DISCUSSION

The results of this study represent clear observations of pharmacologically-induced changes in brown adipose tissue mo~hology. These morphologic changes were consistent with transition from a thermogenically more active to a less active tissue, characterized by intracellular accumulation of lipid and enlarging locule and cell diameters. These observations were confirmed in a second strain of rat with different characteristics of body fat accumulation, but subjected to the same treatment regimen. In both

319

strains of rat, body wt was generally maintained during the period of drug treatment. The function of brown adipose tissue is thermogenesis, and several investigators have suggested that this tissue is largely if not entirely responsible for the thermogenic changes associated with diet-induced thermogenesis in man and animals (Rothwell and Stock, 1979; Perkins et al., 1981). In addition, a thermogenic defect in brown adipose tissue which results in decreased energy expenditure has been implicated in the development of obesity in animals (Himms-Hagen, 1980). The thermogenic activity of BAT is mediated by the sympathetic component of the antonomic nervous sytem, and pharmacologic inactivation of brown adipose tissue via sympathoplegic mechanisms, therefore, would be expected to result in morphologic evidence of thermogenic inactivity. In normally active BAT, resting cells accumulate lipid within their intracellular locules, and when activated by noradrenergic mechanisms, the stored triglycerides become metabolized during the thermogenic response (Rothwell and Stock, 1979; Mrosovsky and Rowlatt, 1968; Tanuma et al., 1975; Smith and Horwitz, 1969). During periods of cold acclimatization, brown adipose tissue mass and brown adipocyte lipid locularity have been shown to become increased, while in states of experimental protein malnutrition, undernutrition and acute starvation the IBAT mass and cellular locularity are maintained in an apparent the~ogenically active state (~rosovsky, 1962; Perkins ei al., 1981; Tulp et al., 1982). Tanuma et al. (1975), Mrosovsky (1964) and Mrosovsky and Rowlatt (1968) have noted an inverse association between brown adipocyte locule diameter and frequency and recent thermogenic activity, with the lesser rates of thermogenic activity being most associated with a greater frequency of unilo~ular type V adipocytes. An additional observation of this study suggests that neither IBAT weight, or its ratio to body wt are accurate predictors of thermogenic activity of an animal’s propensity to gain weight as fat. In the two strains of rat examined, body fat content as observed visually during dissection as well as with measurements of the epididymal depot differed significantly in the absence of drug treatment. IBAT weight, however, was similar in both strains, whether expressed as an absolute weight or as a proportion to body wt. IBAT is commonly expressed as a proportion to body wt in order to illustrate changes due to dietary or environmental factors, and the inference has been made that the IBAT:BW ratio may be correlated to thermogenic activity. Although post-treatment IBAT weights were not obtained in the present study, we have recently observed that the increased IBAT weight following dietary and surgical treatments was accounted for by changes in the number and volume of adipocytes (Tulp et al., 1981). Thus, based on the cell diameter data, IBAT weight following the present treatments would be predicted to increase by 25-357; without a significant change in body wt, but accompanied by morphologic evidence of decreased thermogenic activity. Antihypertensive regimens pharmacologically

320

ORIEN L. TULP et al.

comparable to the regimen of the present study are often administered during the treatment of hypertension. It would seem appropriate, therefore, to accompany hypertensive therapy with occasional anthropometric measurements so as to monitor any changes in body fat accumulation which may occur during long-term therapy, and which may aggravate the clinical effectiveness of the treatments. Brown adipose tissue is presumed to be active in adult man and to contribute to the economy of energy expenditure, particularly during periods of dietary excess (Rothwell and Stock, 1979). Chronic sympathoplegic hypertension regimens may result in concurrent decreases in the thermogenic activity of brown adipose tissue, blood pressure and peripheral blood flow, and may result in altered states of energy balance and greater gains of body fat than may be desirable. Surgical removal of IBAT and P-adrenergic blockade both result in a decreased capacity for energy expenditure and greater fat accumulation in the rat (Tulp, 1981; Tulp and Blaisdell, 1982). Although measurements of energy balance were not included in the present study, the frequency redistribution of brown adipocytes from a more active to a less active thermogenic state would be consistent with decreased thermogenic activity, and with gradual increases in body fat content. Garrow (1974) has suggested that a quantitatively small impairment in thermogenic capacity of only 5% over a prolonged period could often account for the gradual development of obesity in man. The net contribution of brown adipose tissue to energy expenditure and obesity has not been clearly established, but in concert with other thermogenie mechanisms is likely to represent a substantial proportion of the total energy expenditure over months or years. Regardless of the mechanisms involved, the observations of the present study indicate that sympathoplegic pharmacologic intervention during treatment of hypertension in the rat are associated with morphologic changes in brown adipose tissue that are consistent with decreased thermogenic activity, and if maintained over a long period could result in significant increases in the accumulation of body fat. Careful periodic anthropometric observations may be in order, therefore, when similar regimens are employed for the treatment of hypertension in man. Acknowledgements-Supported by USDA 0410-9-0289 and PHS HL-17335. The authors wish to thank Dr. William Halpern, of the Department of Physiology and Biophysics

of the University of Vermont for his generous contribution of SHR and WKY rats and for his assistance in the measurements of pulse and blood pressure and Penny Spear and Karen Bourassa of Colby College, for their exce!lent technical assistance in the preparation of this manuscript. REFERENCES Brooks S. L., Rothwell N. J., Stock M. J., Goodbody A. E. and Travhurn P. (1980) Nature. Lond. 286. 214276. Cameron i. and Smith R: (1964) Cytological’responses of brown adipose tissue in cold-exposed rats. J. Cell Biol. 23, 89-100. Garrow J. S. (1974) Enerw Balance and Obesity in Man. North Holland, Amsterdam. Hahn P. and Novak M. f 1975) Develoument of brown and white adipose tissue. i. Lipid Res. i6, 79-91.

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