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Decreased beta-adrenergic receptor binding in obese female zucker rats
Journal of the Autonomic Nervous System, 14 (1985) 81-87 Elsevier
81
JAN00464
Decreased beta-adrenergic receptor binding in obese female Zucker rats S h a r o n Bass and Sue R i t t e r Department of Veterinary and ComparativeAnatomy, Pharmacologyand Physiology, WashingtonState University, Pullman, WA 99164-6520 (U.S.A.) (Received January 21st, 1985) (Revised version received May 21st, 1985) (Accepted May 28th, 1985)
Key words: cardiac beta-adrenergic receptors - Zucker rats - genetic obesity sympathetic nervous system -- cardiac hypertrophy
Among the many endocrine and physiological disorders described for genetically obese [28] Zucker rats is a generalized disturbance of autonomic function. Studies of the peripheral sympathoadrenal system in Zucker rats have identified abnormalities in several parameters of catecholamine function which indicate that portions of this system are chronically underactive in animals expressing the obese trait [13,14,16]. The extensiveness of the defects which have been observed in catecholamine neurons of obese Zucker rats suggests that catecholamine receptors might also be abnormal in these animals. Although a reduction in number of beta receptors in brown adipocytes has, in fact, been noted in obese Zucker rats [11], beta receptor binding has not been extensively investigated in these animals. Therefore, we examined the beta-adrenergic receptors in cardiac ventricles of adult obese and lean female Zucker rats of two different ages. Since preliminary investigation of the hearts of female Zucker rats showed that obese rats displayed significant increase in heart weight as compared to lean controls, we also included in our study hearts of obese rats in which rate of weight gain and cardiac hypertrophy were reduced by food restriction. Beta receptor binding was measured in cardiac membranes of 4- and 9-month-old lean and obese female Zucker rats. The weight gains of five 9-month-old obese rats was reduced by caloric restriction (50% of ad libitum intake) imposed for 6 months prior to sacrifice. Rats were housed individually in wire mesh cages in a temperature controlled room on a 12 : 12 light-dark cycle. Membranes were prepared for assay by Correspondence: S. Ritter, Department of VCAPP, College of Veterinary Medicine, Washington State University, Pullman, WA 99164-6520, U.S.A. 0165-1838/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
82 a modification of the methods described by Baker and Potter [4]. Briefly, ventricles were weighed, minced and homogenized (Brinkman polytron) in 30 vols of ice-cold Tris buffer (50 mM, pH 7.2). Cardiac membranes were then filtered through organdy and centrifuged at 120 g to remove cellular debris and connective tissue. The supernatant was centrifuged at 30,000 g; the resulting pellet was washed and re-centrifuged two more times. The final pellet was resuspended in a volume of 18 ml Tris buffer per gram original tissue. Beta-adrenergic receptor binding was conducted with 6 different concentrations of [3H]dihydroalprenolol ([3H]DHA) from 0.5-10 nM, and 0.200-0.350 mg membrane protein in the presence or absence of 10/~M _+propranolol. Total assay volume was 0.3 ml. Tissues were incubated at 37 ° for 20 min, rapidly filtered over a millipore filter apparatus and washed with 10 ml Tris buffer (4°C). Filters were dried, placed in scintillation vials' with 8 ml toluene-based cocktail and counted for the presence of [3H]DHA binding. Specific binding at each concentration of [3H]DHA was determined from triplicate samples incubated with and without propranolol. Dissociation constants of [3H]DHA and receptor numbers were determined by least squares linear regression of Scatchard plots of each heart. Receptor concentrations were estimated by assaying membrane protein [19]. Data were analyzed by a general linear model using a two-by-two factorial for a completely randomized design, with type III sum of squares which adjusts for unequal numbers. Significant differences were isolated by post hoc application of Student's t-test. The receptor binding data are summarized in Table I, where t-test analysis was applied to compare means within 4- and 9-month-old groups. Analysis of variance of 4- and 9-month-old animals showed no significant effect of age on any of the parameters analyzed. Therefore, data from the two age groups were pooled. Lean and obese animals differed Significantly (P < 0.05) with respect to cardiac beta receptor concentration, body weight and cardiac ventricular weight. However, no differences were found in binding affinity (KD)- The F value, degrees of freedom and probability for these variables are as follows: beta receptor concentration, F(1,24) ~--" 4.80, P < 0.050; b. wt., F(1,34) = 934.5, P < 0.001; ventricle weight, F(1,24) = 65.0, P<0.001; K D, F~1.24)=0.58, P<0.45. Table I indicates that total beta receptor number per ventricle and membrane protein did not differ between lean and obese animals at either age. Food restriction successfully reduced rate of weight-gain in obese rats (430.0 g and 686.0 g, restricted vs obese) although they still weighed significantly more than lean controls (264.0 g). Ventricle weights were also reduced in food-restricted obese rats to levels that did not differ significantly from those of the lean rats (0.701 g vs 0.658 g, respectively). Beta receptor concentrations, however, were significantly-lower in food-restricted rats than in lean rats (38.64 vs 55.84 fmol/mg prot., respectively) despite similar heart sizes. Beta receptor binding was even lower in food-restricted rats than in obese ad libitum fed rats (46.65 fmol/mg prot.). Scatchard plots shown in Fig. 1 depict the binding data from the lean and obese 4-month-old animals and the lean, obese and obese food-restricted 9-month-old animals with the median beta receptor concentration for each group. Correlation coefficients were 0.95 or greater for all hearts used in the study. Blood pressure was measured in separate groups of obese and lean female Zucker
51.58_+ 6.39* 2.75_+ 0.27 350 _+ 19.1 * 0.69_+ 0.05 * 867.3 _+82.5 24.95-+ 1.37
63.22+ 2.29 3.16_+ 0.19 205 _+ 2.8 0.53_+ 0.014 761.1 +42.6 23.02_+ 1.23
*, Obese vs lean; P < 0.05, Student's t-test. **, Obese food restricted vs lean, P < 0.05, Student's t-test. *** Obese vs obese food restricted; P < 0.05, Student's t-test
Beta receptor ( f m o l / m g protein) K a (nM) Body weight(g) Ventricle weight (g) Beta receptors (fmol/ventricle) Membrane protein (rag) 22.69_+ 1.75
2.80_+ 0.36 264 _+ 3.8 0.66_+ 0.02 836.4 -+96.3
55.84_+ 3.25
Lean (n = 4)
Lean (n = 15)
Obese (n = 5)
9-Month
4-Month
Cardiac beta receptor binding in female Zucker rats (means +_ S.E.M.)
TABLE I
2.81 *
20.58_+
0.84
2.75_+ 0.21 686 _+ 13.3 * 0.97_+ 0.02 * 939.7 -+105.1
46.65_+
Obese (n = 4)
3.43 **
23.91+
0.77"**
2.27_+ 0.32 430 _+ 6.0 **'*** 0.70_+ 0.02 *** 636.1 _+113.3 **'***
38.44_+
Obese (food restricted) (n = 5)
~4 4 Month Old Zucker Rats (9)
26
26
"f 22
2O
22 •
20
18
m° X
14
if_
12
9 Month Old Zucker Rats (9)
24
×
16
~s 14
~Lean ~
8
6
6
4
4
2
2
o.\\\
(Iood t esltieted)
10
20
30
3H-DHA
Bound
40
50
(fmol/mg protein)
60
7O
TO
2O
30
•
40
50
60
3 H - D H A Bound (fmol/rng protein)
Fig. 1. Representative Scatchard plots of (-)[3H]dihydroaiprenolol binding to cardiac ventricle membranes. Each plot represents the lean and obese 4-month-old animal and lean, obese and obese food-restricted 9-months-old animal displaying the median beta receptor concentration for its group. Each point represents the mean of triplicate determinations.
rats between 7 and 9 months of age and a group of adult female Sprague-Dawley rats. Blood pressure was measured remotely in freely moving animals from the animals' home cage by connecting a femoral artery catheter to a Statham pressure transducer and associated recording equipment. Pressures were read from a Foregger monitor. After basal pressures were established, pressor responses to infused norepinephrine were determined. Norepinephrine was delivered over a 40-s period into a jugular catheter from a Sage variable speed syringe pump. Doses of 0.5, 1.0, 2.0, 5.0 and 10.0 # g / k g / m i n were infused sequentially by adjusting the infusion rate. N o more than 20 s were allowed to elapse between doses. Obese Zuckers rats did not differ from lean Zucker rats or Sprague-Dawley rats with respect to either basal blood pressures or pressor responses to infused norepinephrine. Plasma volume was determined in separate groups of obese and lean animals 6 months of age by the T-1824 dye dilution method [25], from jugular blood collected at 0, 10, 30 and 60 rain after dye injection. Dye concentration was corrected for blood loss due to sampling using hematocrit values and volumes per unit body weight were computed. Blood volume was significantly expanded in obese animals compared to lean (values represent mean _+ S.E.M. obese: 31.5 + 2.6 ml, n = 3 vs lean: 18.5 _+ 1.8 ml, n = 2" P < 0.05). Our results reveal that cardiac beta receptor concentrations are reduced in obese female Zucker rats of two age groups compared to their lean litter mates. Receptor binding affinity did not differ between obese and lean rats at either age, indicating that the decreased binding in obese animals was brought about by a quantitative, not a qualitative change in the cardiac beta receptor population. Food restriction in obese rats attenuated weight gain and prevented cardiac hypertrophy, but resulted in a further deficit in cardiac beta receptor concentration and a significantly lower total
85 number of beta receptors per ventricle than observed in obese and lean ad libitum fed controls. The plasma volume of the obese animals was significantly expanded, as expected from their greater body weights, but they were normotensive with respect to both lean littermates and Sprague-Dawley rats as reported previously for male Zucker rats [12]. Thus, the cardiac hypertrophy observed in the obese animals appears to be attributable to volume, rather than pressure overload. The factors responsible for the decreased cardiac beta receptor concentrations in obese Zucker rats have not been determined. Genetic factors are certainly involved, but available evidence does not indicate whether reduced beta receptor concentration in the obese animals represents a primary genetic influence or if such changes result secondarily from their altered metabolism, endocrine function or the obesity itself. Alterations of sympathetic function can be observed in other rat strains, when obesity is induced dietarily [17]. In addition, the numerous endocrine abnormalities described in obese Zucker rats include several, such as hypothyroidism [5,15], insulin resistance [7] and elevated plasma catecholamine levels [13] with potential for altering beta receptor concentrations [6,22, 23]. One factor which might contribute significantly to the reduction of cardiac beta receptor concentrations in obese Zucker rats is dilution of the receptor population by cardiac hypertrophy. The increase in ventricular weights in the obese rats ranged from 29% in the 4-month-old rats to 47% in the 9-month-old animals. The fact that the total number of receptors per ventricle was similar in obese and lean littermates strongly indicates that dilution by hypertrophy plays an important role in reduction of the receptor concentrations in our study. In other hypertrophic heart models, including spontaneously hypertensive rats [18], renovascular hypertensive rats [2,3] and exercised rats [24], cardiac hypertrophy is also accompanied by decreased beta receptor concentrations and in renovascular hypertensive rats this decrease has been shown to be reversible with regression of the hypertrophy [2]. A role for dilution in accounting for decreased beta receptor binding is supported by the fact that Levin et al. [12] found no differences in cardiac beta receptor concentrations in obese and lean male Zucker rats of approximately 5-6 months of age, in which the ventricular hypertrophy was much less than in our animals. In contrast, when body weight gain was restricted in obese animals, preventing cardiac hypertrophy, beta receptor concentrations were further reduced. These results suggest that other mechanisms besides hypertrophy may contribute to the alteration of beta receptor concentration. Food restriction itself alters many physiological systems which may influence beta receptor concentrations. For example, both plasma T3 and insulin levels have been shown to be reduced in food-restricted Zucker rats [21]. Our results indicate, however, that loss of membrane protein does not contribute to decreased beta receptor concentration in food-deprived Zucker rats. Down-regulation of receptors is also unlikely, since plasma catecholamines and neuronal sympathetic activity are either unchanged [26] or decreased in the fasted or food-restricted state [1,10,20,27]. Our results reveal that cardiac ventricular weight is increased and beta receptor concentration is reduced in obese adult female Zucker rats and that this concentration is further reduced when heart weight is diminished by food restriction in obese
86
rats. Studies are now underway in our laboratory to determine the extent to which the beta receptor changes and cardiac hypertrophy alter pumping properties of the heart and its responsiveness to catecholamines. Human studies indicate clearly that obesity is associated with greatly increased risk of cardiovascular morbidity and mortality, even in the absence of hypertension [8,9]. Since they are normotensive, obese Zucker rats might provide a useful model for studying the hemodynamic consequences of cardiac hypertrophy and beta receptor reduction in the absence of hypertension-induced cardiovascular pathology. We gratefully acknowledge Drs. Diane Figlewicz and Ruth Young for assistance in obtaining Zucker rats.
1 Avakian, E.V. and Horvath, S.M., Starvation suppresses sympathoadrenal medullary response to cold exposure in rats, Am. J. Physiol., 241 (1981) E316-E320. 2 Ayobe, M.H. and Tarazi, R.C., Reversal of changes in myocardial ]]-receptors and inotropic responsiveness with regression of cardiac hypertrophy in renal hypertensive rats (RHR), Circ. Res., 54 (1984) 125-134. 3 Ayobe, M.H. and Tarazi, R.C., Beta-receptors and contractile reserve in left ventricular hypertrophy, Hypertension, 5, Suppl. I (1983) 192-197. 4 Baker, S.P. and Potter, L.T., Purification and partial characterization of cardiac plasma membranes rich in beta-adrenoreceptors, Membr~ Biochem., 3 (1980) 185-205. 5 Bray, G.A. and York, D.A., Thyroid function of genetically obese rats, Endocrinology, 88 (1971), 1095-1099. 6 Ciaraldi, T. and Marinetti, G.V., Thyroxine and propylthiouracil effects in vivo on alpha and beta adrenergic receptors in rat heart, Biochem. Biophys, Res. Commun, 74 (1977) 984-991. 7 Crettaz, M., Zaninetti, D. and Jeanrenand, B, Insulin-resistance in heart and skeletal muscles of genetically obese Zucker rats, Biochzm. So<:. Trans., 9 (1981) 524-525. 8 Dnstan, H.P., Tarazi, R.C. and Mujais, S., A comparison of hemodynamic and volume characteristics of obese and non- obese hyp~ensive patients, Int. J. Obes., 5, Suppl i (t981) 19-25. 9 Frohlich, E.D., Messerli, F.H., Reisin, E. and Dunn, F.G., The problem of obesity and hypertension, Hypertension, 5, Suppl. III (1983) 71-78. 10 Landsberg, L. and Young, J.B., Fasting, feeding and regulation of the sympathetic nervous system, N. Engl. J. Med,, 298 (1978) 1295-1301. 11 Levin, B.E., Comai, K., O'Brien, R.A, and Sullivan, A.C., Abnormal brown adipose composition and ~8-adrenoreceptor binding in obese Zucker rats, Am. J. Physiol., 243 (1982) E217-224. 12 Levin, B.E., Stoddard-Apter, S. and Sullivan, A.C., Central activation and peripheral function of sympatho-adrenal and cardiovascular systems in the Zucker rat, Physiol. Behav., 32 (1984) 295-299. 13 Lcvin, B.E., Triscari, J. and Sullivan, A.C., Abnormal sympatho-adrenal function and plasma catecholamines in obese Zucker rats, Pharmacol. Biochem. Behav., 13 (1980) 107-113. 14 Levin, B.E., Triscari, J. and Sullivan, A.C., Defective catecholamin¢ metabolism in peripheral organs of genetically obese Zucker rats, Brain Res., 244 (1981) 353-366. 15 Levin, B.E.; Triscari, J. and Sullivan, A.C., Sympathetic activity in thyroid-treated Zucker rats, Am. J. Physiol., 243 (1982) R170-178. 16 Levin, B.E., Triscari, J. and Sullivan, A.C., Studies of origins of abnormal sympathetic function in obese Zucker rats, Am. J. Physiol., 245 (1983) E87-93. 17 Levin, B.E., Triscari, J. and Sullivan, A.C., Altered sympathetic activity during development of diet-induced obesity in rat, Am. J. Physiol:, 244 (1983) R347-355. 18 Limas, C. and Limas, C.J., Reduced number of ~8-adrenergic receptors in the myocardium of spontaneously hypertensive rats, Biochem. Biophys. Res. Commun., 83 (1978) 710-714.
87 19 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 20 Reisin, E., Frohlich, E.D., Messerli, F.H., Preslinski, G.R., Dunn, F.G., Jones, M.M. and Batson, H.M., Cardiovascular changes after weight reduction in obesity hypertension, Ann. Int. Med., 98 (1983) 315-319. 21 Rothwell, N.J., Saville, M.E. and Stock, M.J., Metabolic responses to fasting and refeeding in lean and genetically obese rats, Am. J. Physiol., 244 (1983) R615-620. 22 Scarpace, P.J. and Abrass, I.B., Thyroid hormone regulation of rat heart, lymphocyte and lung /]-adrenergic receptors, Endocrinology, 108 (1981) 1007-1011. 23 Stiles, G.L. and Lefkowitz, R.J., Thyroid hormone modulation of agonist-beta-adrenergic receptor interactions in the rat heart, Life Sci., 28 (1981) 2529-2536. 24 Sylvestre-Gervais, L., Nadeau, A., Nguyen, M.H., Tancrede, G. and Rousseau-Migneron, S., Effects of physical training on p-adrenergic receptors in rat myocardial tissue, Cardiovasc. Res., 16 (1982) 530-534. 25 Wang, L., Plasma volume, cell volume, total blood volume and F cells factor in the normal and splenectomized Sherman rat, Am. J. Physiol., 196 (1959) 188-192. 26 Weick, B.G., Ritter, S. and McCarty, R., Plasma catecholamines in fasted and sucrose supplemented rats, Physiol. Behav., 30 (1983) 247-252. 27 Young, J.B. and Landsberg, L., Suppresion of sympathetic nervous system during fasting, Science, 196 (1977) 1473-1475. 28 Zucker, L.M. and Zucker, T.F., Fatty, a new mutation in the rat, J. Hered., 52 (1961) 275-278.
81
JAN00464
Decreased beta-adrenergic receptor binding in obese female Zucker rats S h a r o n Bass and Sue R i t t e r Department of Veterinary and ComparativeAnatomy, Pharmacologyand Physiology, WashingtonState University, Pullman, WA 99164-6520 (U.S.A.) (Received January 21st, 1985) (Revised version received May 21st, 1985) (Accepted May 28th, 1985)
Key words: cardiac beta-adrenergic receptors - Zucker rats - genetic obesity sympathetic nervous system -- cardiac hypertrophy
Among the many endocrine and physiological disorders described for genetically obese [28] Zucker rats is a generalized disturbance of autonomic function. Studies of the peripheral sympathoadrenal system in Zucker rats have identified abnormalities in several parameters of catecholamine function which indicate that portions of this system are chronically underactive in animals expressing the obese trait [13,14,16]. The extensiveness of the defects which have been observed in catecholamine neurons of obese Zucker rats suggests that catecholamine receptors might also be abnormal in these animals. Although a reduction in number of beta receptors in brown adipocytes has, in fact, been noted in obese Zucker rats [11], beta receptor binding has not been extensively investigated in these animals. Therefore, we examined the beta-adrenergic receptors in cardiac ventricles of adult obese and lean female Zucker rats of two different ages. Since preliminary investigation of the hearts of female Zucker rats showed that obese rats displayed significant increase in heart weight as compared to lean controls, we also included in our study hearts of obese rats in which rate of weight gain and cardiac hypertrophy were reduced by food restriction. Beta receptor binding was measured in cardiac membranes of 4- and 9-month-old lean and obese female Zucker rats. The weight gains of five 9-month-old obese rats was reduced by caloric restriction (50% of ad libitum intake) imposed for 6 months prior to sacrifice. Rats were housed individually in wire mesh cages in a temperature controlled room on a 12 : 12 light-dark cycle. Membranes were prepared for assay by Correspondence: S. Ritter, Department of VCAPP, College of Veterinary Medicine, Washington State University, Pullman, WA 99164-6520, U.S.A. 0165-1838/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
82 a modification of the methods described by Baker and Potter [4]. Briefly, ventricles were weighed, minced and homogenized (Brinkman polytron) in 30 vols of ice-cold Tris buffer (50 mM, pH 7.2). Cardiac membranes were then filtered through organdy and centrifuged at 120 g to remove cellular debris and connective tissue. The supernatant was centrifuged at 30,000 g; the resulting pellet was washed and re-centrifuged two more times. The final pellet was resuspended in a volume of 18 ml Tris buffer per gram original tissue. Beta-adrenergic receptor binding was conducted with 6 different concentrations of [3H]dihydroalprenolol ([3H]DHA) from 0.5-10 nM, and 0.200-0.350 mg membrane protein in the presence or absence of 10/~M _+propranolol. Total assay volume was 0.3 ml. Tissues were incubated at 37 ° for 20 min, rapidly filtered over a millipore filter apparatus and washed with 10 ml Tris buffer (4°C). Filters were dried, placed in scintillation vials' with 8 ml toluene-based cocktail and counted for the presence of [3H]DHA binding. Specific binding at each concentration of [3H]DHA was determined from triplicate samples incubated with and without propranolol. Dissociation constants of [3H]DHA and receptor numbers were determined by least squares linear regression of Scatchard plots of each heart. Receptor concentrations were estimated by assaying membrane protein [19]. Data were analyzed by a general linear model using a two-by-two factorial for a completely randomized design, with type III sum of squares which adjusts for unequal numbers. Significant differences were isolated by post hoc application of Student's t-test. The receptor binding data are summarized in Table I, where t-test analysis was applied to compare means within 4- and 9-month-old groups. Analysis of variance of 4- and 9-month-old animals showed no significant effect of age on any of the parameters analyzed. Therefore, data from the two age groups were pooled. Lean and obese animals differed Significantly (P < 0.05) with respect to cardiac beta receptor concentration, body weight and cardiac ventricular weight. However, no differences were found in binding affinity (KD)- The F value, degrees of freedom and probability for these variables are as follows: beta receptor concentration, F(1,24) ~--" 4.80, P < 0.050; b. wt., F(1,34) = 934.5, P < 0.001; ventricle weight, F(1,24) = 65.0, P<0.001; K D, F~1.24)=0.58, P<0.45. Table I indicates that total beta receptor number per ventricle and membrane protein did not differ between lean and obese animals at either age. Food restriction successfully reduced rate of weight-gain in obese rats (430.0 g and 686.0 g, restricted vs obese) although they still weighed significantly more than lean controls (264.0 g). Ventricle weights were also reduced in food-restricted obese rats to levels that did not differ significantly from those of the lean rats (0.701 g vs 0.658 g, respectively). Beta receptor concentrations, however, were significantly-lower in food-restricted rats than in lean rats (38.64 vs 55.84 fmol/mg prot., respectively) despite similar heart sizes. Beta receptor binding was even lower in food-restricted rats than in obese ad libitum fed rats (46.65 fmol/mg prot.). Scatchard plots shown in Fig. 1 depict the binding data from the lean and obese 4-month-old animals and the lean, obese and obese food-restricted 9-month-old animals with the median beta receptor concentration for each group. Correlation coefficients were 0.95 or greater for all hearts used in the study. Blood pressure was measured in separate groups of obese and lean female Zucker
51.58_+ 6.39* 2.75_+ 0.27 350 _+ 19.1 * 0.69_+ 0.05 * 867.3 _+82.5 24.95-+ 1.37
63.22+ 2.29 3.16_+ 0.19 205 _+ 2.8 0.53_+ 0.014 761.1 +42.6 23.02_+ 1.23
*, Obese vs lean; P < 0.05, Student's t-test. **, Obese food restricted vs lean, P < 0.05, Student's t-test. *** Obese vs obese food restricted; P < 0.05, Student's t-test
Beta receptor ( f m o l / m g protein) K a (nM) Body weight(g) Ventricle weight (g) Beta receptors (fmol/ventricle) Membrane protein (rag) 22.69_+ 1.75
2.80_+ 0.36 264 _+ 3.8 0.66_+ 0.02 836.4 -+96.3
55.84_+ 3.25
Lean (n = 4)
Lean (n = 15)
Obese (n = 5)
9-Month
4-Month
Cardiac beta receptor binding in female Zucker rats (means +_ S.E.M.)
TABLE I
2.81 *
20.58_+
0.84
2.75_+ 0.21 686 _+ 13.3 * 0.97_+ 0.02 * 939.7 -+105.1
46.65_+
Obese (n = 4)
3.43 **
23.91+
0.77"**
2.27_+ 0.32 430 _+ 6.0 **'*** 0.70_+ 0.02 *** 636.1 _+113.3 **'***
38.44_+
Obese (food restricted) (n = 5)
~4 4 Month Old Zucker Rats (9)
26
26
"f 22
2O
22 •
20
18
m° X
14
if_
12
9 Month Old Zucker Rats (9)
24
×
16
~s 14
~Lean ~
8
6
6
4
4
2
2
o.\\\
(Iood t esltieted)
10
20
30
3H-DHA
Bound
40
50
(fmol/mg protein)
60
7O
TO
2O
30
•
40
50
60
3 H - D H A Bound (fmol/rng protein)
Fig. 1. Representative Scatchard plots of (-)[3H]dihydroaiprenolol binding to cardiac ventricle membranes. Each plot represents the lean and obese 4-month-old animal and lean, obese and obese food-restricted 9-months-old animal displaying the median beta receptor concentration for its group. Each point represents the mean of triplicate determinations.
rats between 7 and 9 months of age and a group of adult female Sprague-Dawley rats. Blood pressure was measured remotely in freely moving animals from the animals' home cage by connecting a femoral artery catheter to a Statham pressure transducer and associated recording equipment. Pressures were read from a Foregger monitor. After basal pressures were established, pressor responses to infused norepinephrine were determined. Norepinephrine was delivered over a 40-s period into a jugular catheter from a Sage variable speed syringe pump. Doses of 0.5, 1.0, 2.0, 5.0 and 10.0 # g / k g / m i n were infused sequentially by adjusting the infusion rate. N o more than 20 s were allowed to elapse between doses. Obese Zuckers rats did not differ from lean Zucker rats or Sprague-Dawley rats with respect to either basal blood pressures or pressor responses to infused norepinephrine. Plasma volume was determined in separate groups of obese and lean animals 6 months of age by the T-1824 dye dilution method [25], from jugular blood collected at 0, 10, 30 and 60 rain after dye injection. Dye concentration was corrected for blood loss due to sampling using hematocrit values and volumes per unit body weight were computed. Blood volume was significantly expanded in obese animals compared to lean (values represent mean _+ S.E.M. obese: 31.5 + 2.6 ml, n = 3 vs lean: 18.5 _+ 1.8 ml, n = 2" P < 0.05). Our results reveal that cardiac beta receptor concentrations are reduced in obese female Zucker rats of two age groups compared to their lean litter mates. Receptor binding affinity did not differ between obese and lean rats at either age, indicating that the decreased binding in obese animals was brought about by a quantitative, not a qualitative change in the cardiac beta receptor population. Food restriction in obese rats attenuated weight gain and prevented cardiac hypertrophy, but resulted in a further deficit in cardiac beta receptor concentration and a significantly lower total
85 number of beta receptors per ventricle than observed in obese and lean ad libitum fed controls. The plasma volume of the obese animals was significantly expanded, as expected from their greater body weights, but they were normotensive with respect to both lean littermates and Sprague-Dawley rats as reported previously for male Zucker rats [12]. Thus, the cardiac hypertrophy observed in the obese animals appears to be attributable to volume, rather than pressure overload. The factors responsible for the decreased cardiac beta receptor concentrations in obese Zucker rats have not been determined. Genetic factors are certainly involved, but available evidence does not indicate whether reduced beta receptor concentration in the obese animals represents a primary genetic influence or if such changes result secondarily from their altered metabolism, endocrine function or the obesity itself. Alterations of sympathetic function can be observed in other rat strains, when obesity is induced dietarily [17]. In addition, the numerous endocrine abnormalities described in obese Zucker rats include several, such as hypothyroidism [5,15], insulin resistance [7] and elevated plasma catecholamine levels [13] with potential for altering beta receptor concentrations [6,22, 23]. One factor which might contribute significantly to the reduction of cardiac beta receptor concentrations in obese Zucker rats is dilution of the receptor population by cardiac hypertrophy. The increase in ventricular weights in the obese rats ranged from 29% in the 4-month-old rats to 47% in the 9-month-old animals. The fact that the total number of receptors per ventricle was similar in obese and lean littermates strongly indicates that dilution by hypertrophy plays an important role in reduction of the receptor concentrations in our study. In other hypertrophic heart models, including spontaneously hypertensive rats [18], renovascular hypertensive rats [2,3] and exercised rats [24], cardiac hypertrophy is also accompanied by decreased beta receptor concentrations and in renovascular hypertensive rats this decrease has been shown to be reversible with regression of the hypertrophy [2]. A role for dilution in accounting for decreased beta receptor binding is supported by the fact that Levin et al. [12] found no differences in cardiac beta receptor concentrations in obese and lean male Zucker rats of approximately 5-6 months of age, in which the ventricular hypertrophy was much less than in our animals. In contrast, when body weight gain was restricted in obese animals, preventing cardiac hypertrophy, beta receptor concentrations were further reduced. These results suggest that other mechanisms besides hypertrophy may contribute to the alteration of beta receptor concentration. Food restriction itself alters many physiological systems which may influence beta receptor concentrations. For example, both plasma T3 and insulin levels have been shown to be reduced in food-restricted Zucker rats [21]. Our results indicate, however, that loss of membrane protein does not contribute to decreased beta receptor concentration in food-deprived Zucker rats. Down-regulation of receptors is also unlikely, since plasma catecholamines and neuronal sympathetic activity are either unchanged [26] or decreased in the fasted or food-restricted state [1,10,20,27]. Our results reveal that cardiac ventricular weight is increased and beta receptor concentration is reduced in obese adult female Zucker rats and that this concentration is further reduced when heart weight is diminished by food restriction in obese
86
rats. Studies are now underway in our laboratory to determine the extent to which the beta receptor changes and cardiac hypertrophy alter pumping properties of the heart and its responsiveness to catecholamines. Human studies indicate clearly that obesity is associated with greatly increased risk of cardiovascular morbidity and mortality, even in the absence of hypertension [8,9]. Since they are normotensive, obese Zucker rats might provide a useful model for studying the hemodynamic consequences of cardiac hypertrophy and beta receptor reduction in the absence of hypertension-induced cardiovascular pathology. We gratefully acknowledge Drs. Diane Figlewicz and Ruth Young for assistance in obtaining Zucker rats.
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