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Thermogenic responses to adrenoceptor agonists and brown fat adrenoceptors in overfed rats
European Journal of Pharmacology, 125 (1986) 313-323 Elsevier
313
T H E R M O G E N I C R E S P O N S E S TO A D R E N O C E P T O R A G O N I S T S A N D B R O W N FAT A D R E N O C E P T O R S IN OVERFED RATS NANCY J. ROTHWELL, MICHAEL J. STOCK * and DEEPAK K. SUDERA Department of Physiology, St. George's Hospital Medical School, Tooting, London SW17, U.K. Received 20 June, 1985, revised MS received 20 March 1986, accepted 1 April 1986
N.J. ROTHWELL, M.J. STOCK and D.K. SUDERA, Thermogenic responses to adrenoceptor agonists and brown fat adrenoceptors in overfed rats, European J. Pharmacol. 125 (1986) 313-323. Rats fed a cafeteria diet to produce hyperphagia showed increases in the maximal thermogenic responses (rise in oxygen consumption) to isoprenaline (mixed fl-agonist), prenalterol (ill-selective agonist) and clenbuterol (fl2-agonist), and left-shifts in the dose-response curves to the latter two. The maximal response to phenylephrine (a-agonist) was similar for control and cafeteria rats. Ligand binding studies revealed increases in fl-adrenoceptor density of 33-38% in brown fat cells and isolated membranes from cafeteria-fed rats, but a 30% reduction in fl-receptors in heart membranes. Cold-adaptation caused a 22% reduction in fl-receptor density in brown fat membranes, but no change in heart. The ratio of flx/fl2-receptors in brown fat was reduced from 59/45 in control to 47/54 in cafeteria-fed rats, but was not significantly altered in heart (58/44) or in brown fat from cold-adapted animals (64/30). a-Adrenoceptor density was increased above control values by 69 and 25% in brown adipose tissue from cafeteria and cold-adapted rats, respectively. Brown adipose tissue Hyperphagia
Cold-adaptation
a-Adrenoceptor
1. Introduction The primary function of brown adipose tissue (BAT) is heat production, and this tissue is largely responsible for the increases in metabolic rate associated with cold exposure (non-shivering thermogenesis, NST) or hyperphagia (diet-induced thermogenesis, DIT). Hyperphagic rats fed a palatable cafeteria diet, and cold-adapted animals show enhanced thermogenic responses to noradrenaline and increased sympathetic activity in brown fat (see Girardier, 1983; Nedergaard and Lindberg, 1982; Rothwell and Stock, 1983; 1984a for reviews). Activation of the tissue by diet or cold can be prevented by sympathetic denervation of the tissue (Girardier, 1983; Rothwell and Stock, 1984b). The thermogenic effects of the sympathetic * To whom all correspondence should be addressed. 0014-2999/86/$03.50 © 1986 Elsevier Science Publishers B.V.
fll-Adrenoceptor
fl2-Adrenoceptor
nervous system appear to be mediated largely by interaction of noradrenaline with fl-adrenoceptors (Bukowiecki et al., 1978; 1980; Feist, 1983; Mohell et al., 1983; Nedergaard and Lindberg, 1982; Svoboda et al., 1979; 1984) and a-receptors are thought to play only a minor role in the activation of brown fat (Minneman and Molinoff, 1980; Mohell et al., 1983; Raasmaja et al., 1984a,b). Ligand binding studies have identified a high density of fl-adrenoceptors on brown fat cell membranes and on isolated b r o w n adipocytes (Bukowiecki et al., 1978; 1980; Svoboda et al., 1979; 1984) and ligand displacement with adrenoceptor agonists has indicated that these are predominantly of the fit-subtype (Bukowiecki et al., 1978; 1980; Svoboda et al., 1979; 1984). However, both ill- and fl2-selective adrenoceptor agonists will stimulate thermogenesis and BAT activity (Rothwell et al., 1982) and recently we have reported the presence of both receptor subtypes
314 (60%/31, 40% f12) in isolated brown fat membranes (Rothwell et al., 1985). Chronic pharmacological or physiological sympathetic stimulation of most tissues usually causes desensitization to adrenoceptor agonists and 'down-regulation' of adrenoceptor density (see Minneman and Molinoff, 1980; Minneman et al., 1979; Nahorski, 1981 for reviews) and cold-adaptation, for example, causes a reduction in the density of /3-adrenoceptors on BAT. This is accompanied by a lower sensitivity to the thermogenic effects of adrenoceptor agonists, although the maximal capacity for catecholamine-induced thermogenesis is enhanced (Girardier, 1983; Nedergaard and Lindberg, 1982). Cafeteria-fed rats exhibiting diet-induced thermogenesis also show enhanced maximal thermogenic responses to noradrenaline, and DIT in these animals can be almost totally prevented by acute fl-adrenergic blockade with propranolol, whereas the aantagonist phentolamine has little effect (Rothwell and Stock, 1981; 1984b). Little is known about the changes in fl-receptor density a n d / o r receptor subtype during chronic hyperphagia, and similarly, variations in thermogenic sensitivity to receptor-selective adrenoceptor agonists have not yet been investigated in this situation. In the present study, we have therefore investigated the effects of cafeteria feeding on the thermogenic responses to selective ill-, /32- or a-adrenoceptors agonists, and on /3-receptor number and subtype and a-receptor density in BAT. Ligand binding studies were also performed on heart ventricle of cafeteria-fed rats, and on heart and BAT from cold-adapted animals for comparison.
foods (e.g. various biscuits, cakes, chocolates, meats) in addition to the stock diet. This treatment has previously been shown to stimulate energy intake and metabolic rate by 50-100% in young rats (Rothwell and Stock, 1983; 1984a). Cold-adapted rats were fed stock diet only and housed at 5 °C. All treatments were performed for at least 20 days, unless otherwise stated, and in many cases (particularly for controls) heart and BAT was pooled from 2-3 rats for each experiment.
2.2. Thermogenic responses to adrenoceptor agonists
2. Materials and methods
Resting oxygen consumption (~/Oz) was measured in control and cafeteria-fed rats at 29°C in closed-circuit respirometers (24) for 2 h before and 2-3 h after injections of either phenylephrine (a-agonist; 1, 2, 3.5, 5 mg/kg), isoprenaline (mixed /3-agonist; 0.01, 0.025, 0.05, 0.2 mg/kg), prenalterol (fll-agonist; 0.1, 0.25, 0.5, 1.0 mg/kg) or clenbuterol (fl2-agonist; 0.25, 0.5, 0.75, 1.0, 2.0 mg/kg). All drugs were injected subcutaneously (s.c.) in 0.2 ml 0.9% saline containing ascorbic acid (0.01%). Each animal was tested on up to four occasions with different drugs, but drugs were tested randomly, with at least 4 days between tests on any one animal. A total of 32 control and 36 cafeteria animals were used, yielding 4-6 values for each dose of each drug tested. Values associated with physical activity were discarded and results were expressed as ml O 2 consumed/rain per kg °7s, and presented graphically as the maximal increase in 902 as a percentage of the pre-injection value. Many of these rats were subsequently used for ligand binding studies (see below), but at least one week elapsed between measurements of 'JO 2 and receptor binding.
2.1. Animals
2.3. Preparation of membranes
Male Sprague-Dawley rats (Charles River, Kent, U.K.) were used at 40-60 days of age. Control animals were housed at 24°C (12 h light/dark cycle) and allowed free access to a pelleted stock diet (PRD, Christopher Hill Group Ltd., Dorset, U.K.). Cafeteria-fed rats were kept at 24°C and allowed a daily choice of four different human
All the dissectible brown adipose tissue (interscapular, cervical, subscapular, axillary, perirenal, periaortic) was taken from the animals immediately after decapitation. Heart ventricles were also removed, and tissues were finely minced in ice-cold 0.25 M sucrose buffer containing 1 mM EDTA and 10 mM Tris (pH 7.4), and then ho-
315 mogenized (Polytron, mark 6 for 7 s). The homogenate was centrifuged at 3500 x g for 5 min, and the supernatant filtered through surgical gauze to remove cell debris. Mitochondria were sedimented by centrifugation at 11000 x g for 15 rain and the supernatant was then recentrifuged at 100000 X g for 60 min to obtain a microsomal fraction, which was resuspended in buffer to achieve a protein concentration of approximately 150/~g/ml (equivalent to 120 ~g/ml in the final incubation assay). Protein was determined by a dye-reagent method (Bio-Rad) using bovine serum albumin (BSA) standards.
2.4. Preparation of isolated brown adipocytes Brown adipocytes were isolated by a method similar to that described by Svoboda et al. (1979; 1984). Tissue from all depots was minced in Krebs buffer (10 mM glucose, 10 mM fructose and 10 mM pyruvate) containing 4% BSA (fatty acid-free; Sigma, Poole, U.K.) and 3 mg/ml collagenase (Sigma Type II). The tissue was gassed with 95% 02-5% CO 2 and after 10 min preincubation (37 ° C), was filtered. The filtrate was discarded and the tissue re-incubated in fresh medium for 40 rain with shaking every 3-5 min. The isolated cells were filtered through nylon and allowed to stand for 40 min before removing the cell layer and resuspending in BSA-free Krebs buffer. The cells were washed once and resuspended to a final concentration of approximately 0.3 x 106 cells/ml. Cells were counted in a Neubauer chamber, but any cells which took up Trypan blue (usually less than 5% of total cells) were excluded from counting.
2.5 Radiofigand binding studies a- and fl-adrenoceptor number and affinity were assessed from the binding of [3H]dihydroergocryptine ([3H]DHE; 15.7 Ci/mmol, Amersham International, U.K., 0.2-10 nM/incubation) or [3H]dihydroalprenolol ([3H]-DHA; 83 Ci/mmol, Amersham International, U.K., 0.2-10 nM), respectively. Membranes or cells were incubated in 1 ml buffer (50 mM Tris, 10 mM MgC12, pH 7.4) containing the appropriate radio-
ligand for 15 min at 37°C. Incubations were terminated by adding 2 ml ice-cold incubation buffer followed by rapid vacuum filtration through Whatman G F / C filters followed by three 5 ml washes with buffer. Radioactivity retained on the filters was measured in a Beckman (LS-6800) liquid scintillation counter. Non-specific binding was assessed from parallel incubations containing either 10 ~M phentolamine (a-receptors) or 5/IM L-propanolol (B-receptors). Binding data were subjected to Scatchard analysis and the maximum number of binding sites (Bmax) and dissociation constant (Kd) calculated from regression analysis of linear Scatchard plots.
2.6. Assessment of/3-adrenoceptor subtype The relative proportions of/31- and /32-adrenoceptors was assessed from Hofstee plots of the displacement of [3H]DHA (2 nM) from heart and BAT membranes or brown adipocytes using selective/31 (atenolol)- or B2 (ICI 118551)antagonists as described previously (Rothwell et al., 1985). Curvilinear Hofstee plots were resolved into two lines by iterative computer analysis (Rothwell et al., 1985), and the dissociation constants for inhibition of binding (Kd) to each site was calculated from the slope of these lines and corrected using the equation of Cheng and Prusoff (1973). The percentage of each receptor subtype was determined from the ordinate intercept of each line. In addition, the effect of a non-selective antagonist (propranolol) was also tested. Mean values are presented+ S.E.M.-and the number of determinations refers to separate tissue preparations (1-4 animals per preparation). Since many of the experiments were performed over several weeks, with control and experimental animals being studied at the same time, data were usually compared by a Student's t-test for matched data, using two-tailed probabilities.
3. Results
Resting VO2 before injection of adrenoceptor agonists was significantly increased in cafeteria-fed rats compared to controls (table 1). The maximal incremental response to injection of phenylephrine
316 TABLE 1 Resting VO 2 and peak response ( m l / m i n per kg °75) to adrenoceptor agonists in control and cafeteria-fed rats. Average preinjection
Phenylephrine (5.0) * Isoprenaline (1.5) Prenalterol (1.0) Clenbuterol (2.0)
Peak increment
Control
Cafeteria
16.07 _+0.20 16.11 + 0.29 16.35 + 0.22 15.88 + 0.19
18.40 + 0.17 18.02 + 0.17 18.45 + 0.17 18.76 + 0.15
Control b b b b
4.56 + 13.37 + 8.53 + 9.52 +
Cafeteria 0.43 0.31 0.35 0.81
4.73 _+0.48 16.86 + 0.71 b 9.58 + 0.21 a 12.61 + 0.96 a
* Dose used for maximal response (mg/kg). Mean values+ S.E.M. (n = 4-6). a p < 0.05, b p < 0.001 vs. control.
did not differ between groups, but all three fladrenoceptor agonists provoked larger responses in cafeteria than in control rats (table 1). The maximal rise in "¢Oz, as a percentage of the pre-injection value, was greatest for isoprenaline ( > 80%, fig. 1), similar for prenalterol and clenbuterol (5065%) and smallest for phenylephrine (25-30%). Cafeteria-fed rats showed a smaller rise in "¢O2 following the lowest dose of phenylephrine than did controls, and the highest dose tested produced some behavioural changes and increased physical
activity in both groups. Isoprenaline provoked slightly greater increases in VO 2 in cafeteria-fed rats at all doses (fig. 1) but the difference between cafeteria and control rats was statistically significant only for the highest dose. Dose-response curves for clenbuterol and prenalterol were both shifted slightly to the left in cafeteria-fed rats, with these animals showing significantly greater responses than controls at the lower end of the dose range. The mass of total brown adipose tissue was
I$OPRENALINE
PI'IEN YLEPHRINEs
30 + 60
~
sJ
2040
10-
2
i"
g •> C ¢ :
V;( r 0.03
0.1
r 1.0
0-
I
4,0
~J I 5
60-
eO-
u c ~
I 0.5
PRENALTEROL 50-
30-
2"0-
CLENBUTEROL
1 30 ,/' ,~ '~
~ 50-
-<
40-
r 20
110
404 302010-
10-
O"
~(, 0
0.1
i+
v
r
i
011
1
2
4 nOlO
(i
O- l#,l 0
i
I
I
n
1
2
5
10
alilolo/lll]
Fig. 1. The effects of varying doses of adrenoceptor agonists on resting oxygen consumption (% increase above pre-injection value) in control (e) and cafeteria-fed ( I ) rats. Note the differing scales for each drug. Mean values+ S.E.M. (n = 4-6).
317
increased by approximately two-fold following 20 days of cafeteria feeding (1.42 + 0.11 g, n = 12) or cold-adaptation (1.58 + 0.14 g, n = 10) compared to control values (0.70 + 0.01 g, n = 15, P < 0.001). Approximately 350/~g membrane protein was obtained per 100 mg brown fat from control and cafeteria-fed rats, but up to 580 ~tg/100 g for cold-adapted rats. One gram of brown fat from control or cafeteria-fed rats yielded 4-5 × 10 6 BAT cells, and the total number of brown adipocytes was elevated by almost 100% in cafeteria-fed rats; cells were not prepared from cold-adapted animals. Approximately 200 /~g membranes were isolated per 100 mg heart ventricle in all groups.
The binding of [3H]DHE and [3H]DHA to isolated brown fat membranes and adipocytes showed saturation after approximately 6-10 min incubation and at a concentration of 5-6 nM radioligand (figs. 2-5). Non-specific binding varied from 25 to 70% of total binding at low to high concentrations of ligand, respectively, and binding to the G F / C filters represented less than 0.5% of total binding. Linear regressions were obtained for Scatchard analysis of binding data, and in all cases correlation coefficients were > 0.95 (e.g. figs. 2-5). For fl-adrenoceptors, dissociation constants (Kd) were approximately 2 nM for BAT cells or membranes and for heart membranes (figs. 2 and 4, table 2). The maximum number of binding sites for
160' CAFETERIA 12C
320
CONTROL
X "~
60
~ff'
240"
160"
4o,
i ~
80.
[ 3 ~ OHA conce41b~ation (nM) 0 ¸ 0
; o.o~CA 6-
[ q O . E . . . . e o t , . , . , o.) 0.4'
0.06--
0 .04 --
FETERIA 0.2"
CAFETERIA
0.02~ ~
0 0
50
100 ~H] D H A B o u n d
150 I f tool
200
mgP)
Fig. 2. Specific binding of [3H]dihydroalprenolol (DHA) to isolated brown adipose tissue (BAT) membranes from control and cafeteria-fed rats plotted as a function of ligand concentration (upper graph), and Scatchard analysis of [3H]DHA binding (lower graph) derived from the same data. Mean values+ S.E.M. (n = 10).
CONTROL
. 80
.
. 160
.
. 240
320
400
i 3 I~DHE bound (frnoles mg protein)
Fig. 3. Specific binding of [3H]dihydroergocryptine (DHE) to isolated brown adipose tissue (BAT) membranes from control and cafeteria-fed rats plotted as a function of bound concentration (upper graph), and Scatchard analysis of [3H]DHE binding (lower graph) derived from the same data. Mean values + S.E.M. (n ~ 10).
318 80 6o
~ 40
m
/~
~CONTROL
o
,-ff 2O
0 o
o
0
. 2
0 ~1~
.
. 4
'2 4 ~H] DHEconcentration (nM)
. 6
8
1'0 i
[3HI DHA concentration (nM)
0.02"
!
* 0.01
O
i
AFETERIA
0.02
m 0 0 o
0
, 20
, 40
60
80
20
Fig. 4. Specific binding of [3H]dihydroalprenolol (DHA) to isolated brown adipose tissue (BAT) cells from control and cafeteria-fed rats plotted as a function of ligand concentration (upper graph) and Scatchard analysis of [3H[DHA binding (lower graph) derived from the same data. Mean values + S.E.M. (n = 3).
[3H]DHA was increased by 38% in BAT membranes from cafeteria-fed rats and by 49% in BAT cells, when compared to control values (fig. 2, table 2). Hill plots of these data revealed linear regressions (r = 0.8-1.0) with slopes (Hill coefficients) very close to unity (e.g. BAT membranes: control 1.01, cafeteria 0.97; BAT cells: control 0.94, cafeteria 0.9). In a separate trial, the density of /3-adrenoceptors in BAT was not significantly different from controls after only seven days of cafeteria feeding. A higher density of /3-adrenoceptors was seen in heart ventricle membranes than in BAT membranes, but the maximum number of binding sites in heart was reduced by 30% after 20 days of cafeteria feeding (table 2). Hill
60
80
100
~3HIDHE bound (fmole$ 10 6 8ATcelle )
100
[3H~DHA bound (frnoles 106 BAT cells )
40
Fig. 5. Specific binding of [3H]dibydroergocryptine (DHE) to isolated brown adipose tissue (BAT) cells from control and cafeteria-fed rats plotted as a function of ligand concentration (upper graph) and Scatchard analysis of [3H]DHE binding (lower graph) derived from the same data. Mean values + S.E.M. (n = 3). TABLE 2 /3-Adrenoceptor number assessed from Scatchard analysis of [3H]DHA binding to BAT membranes, cells and heart membranes.
BAT Isolated membranes Bmax (fmol/mgP) K d (nM) Isolated cells Bmax (fmol/106 cells) K d (nM) Heart Isolated membranes Bmax ( f m o l / m g P ) K d (nM)
Control
Cafeteria
140 + 16 1.60+ 0.3
193 + 16 * 2.2 _+ 0.3
45
+ 6 2.5+ 0.2
323 + 36 1,83+ 0.09
67 + 6 * 3.3 + 0.4
226 + 38 * 1.74+ 0.08
Mean values:t:S.E.M., n = 7-10 membrane preparations, 4-5 cell preparations. * P < 0.05 paired t-test. Data shown in figs. 2 and 4.
319 coefficients for these d a t a were a g a i n close to u n i t y ( c o n t r o l 0.98, cafeteria 1.04). H o f s t e e plots ( o r d i n a t e : % i n h i b i t i o n of r a d i o l i g a n d b i n d i n g , abscissa: % i n h i b i t i o n / a n t a g o n i s t c o n c e n t r a t i o n ) of the d i s p l a c e m e n t b y p r o p r a n o l o l of [ 3 H ] D H A f r o m B A T m e m b r a n e s were l i n e a r for c o n t r o l (y i n t e r c e p t = 94% K h = 0.009 /~M, r = 0.99) a n d cafeteria-fed rats (y = 96%, K h = 0.002 # M , r = 0.96). H o w e v e r , d i s p l a c e m e n t b y the selective a n t a g o n i s t s a t e n o l o l (fig. 6) or I C I 118551 revealed c u r v i l i n e a r plots w h i c h were resolved i n t o two c o m p o n e n t lines. A t e n o l o l a n d I C I 118551 s h o w e d 50- to 100-fold selectivity for ill", or fl2-receptor s u b t y p e s respectively, a n d values o b t a i n e d for the relative p r o p o r t i o n s of the f l - r e c e p t o r s u b t y p e s w e r e s i m i l a r for b o t h
IOD'
•
80.
CONTROL
60' 40. 20.
oJ
t.° 10
IO0
. 20
30
40
•
50
6"0
•
•
80
CAFETERIA
40,
20' O' 100'
0 •
10
20
30
• 40
TABLE 3 Proportion of fl-adrenoceptor subtypes and Kh for atenolol inhibition of [3H]DHA binding to isolated BAT membranes derived from Hofstee analysis of displacement curves. Mean values-+S.E.M. Displacement curves were derived from 3-4 membrane preparations in each case; r = regression coefficients for resolution of curvilinear Hofstee plots into 2-site plots. Data shown in fig. 6. Control Proportion (%) fl] 59 _+1 f12 45 _+1 K~ (/~M) fll 0.56_+0.25 f12 60 _+8 r 0.95
Cafeteria
Cold-adapted
47 54
64 30
_+ 3 • _+ 3 *
0.56_+ 0 . 2 1 46 +__12 0.98
_+2 _+5
0.56_+0.15 49 -+7 0.96
* P < 0.05 compared to control.
a n t a g o n i s t s (tables 3 a n d 4). T h e K d value for the I C I 118551 d i s p l a c e m e n t f r o m the i l l - r e c e p t o r was slightly h i g h e r in the cafeteria g r o u p c o m p a r e d to the c o n t r o l a n d c o l d - a d a p t e d group. B A T m e m b r a n e receptors f r o m c o n t r o l rats were p r e d o m i n a n t l y of the f i t - s u b t y p e , b u t this was reversed b y cafeteria f e e d i n g (tables 3 a n d 4, fig 6). T h u s , cafeteria-fed rats h a d a s i g n i f i c a n t l y lower p r o p o r t i o n of /~t-receptors a n d a h i g h e r p r o p o r t i o n of fl2-receptors. T h e s e d a t a were o b t a i n e d o n separate m e m b r a n e p r e p a r a t i o n s to those p r e s e n t e d in table 2. H o w e v e r , w h e n S c a t c h a r d a n a l y s i s a n d d i s p l a c e m e n t curves for a t e n o l o l were s t u d i e d
I 50
60
TABLE 4 80.
COLD
Proportion of fl-adrenoceptor subtypes and K~ for ICI 118551 inhibition of [3H]DHA binding to isolated BAT membranes derived from Hofstee analysis of displacement curves. Mean values_+S.E.M. Displacement curves were derived from 2-4 membrane preparations; r = regression coefficients for resolution of curvilinear Hofstee plots into 2-site plots.
4O
2O O
• 0
10
20
30
40
50
60
Control
%I~ c
Fig. 6. Hofstee plots of [3H]DHA displacement data. Ordinate = % inhibition (I) of specific [3HIDttA binding by atenolok abscissa = I/concentration of antagonist (~M) for BAT membranes isolated from control, cafeteria or cold-adapted rats. Curves were resolved into two component lines by interactive computer analysis, and values obtained are shown in table 3 and in the text. Values represent means of 3-4 separate membrane preparations.
Proportion (%) /~] 57 + 4 f12 45 + 5 /3] /32 r
1.0 _+0.4 0.03 _+0.01 0.91
Cafeteria
Cold-adapted
44 53
66 30
+6 • +3•
3.1 _ + 1 . 8 0.03 _+0.02 0.88
* P < 0.05 compared to controls.
+2 _5
0.72_+0.16 0.02 _+0.01 0.89
320
using the same preparations, the results obtained for the maximum number of binding sites by Scatchard analysis were similar to those presented in table 2 (B. . . . f m o l / m g protein: control 160, cafeteria 200, n = 2). Displacement with ICI 118551 was not tested on these same preparations. Using the data from tables 2-4, it can be calculated that the total number of ill-receptors (fmol/mg protein) was approximately 83 + 9 for control and 91 + 8 for cafeteria (NS) and of flzreceptors was 60 + 6 for control and 106 + 9 for cafeteria (P < 0.01). Displacement curves for isolated BAT cells using atenolol (ICI 118551 was not tested) revealed values for the proportion of each receptor subtype which were similar to those obtained on membranes in control animals (fla = 58 + 4%, f12 = 4 2 + 5%), and experiments performed on two adipocyte preparations from cafeteria rats showed approximately equal proportions of ill- (5~%) and fl2-receptors (50%). fl-Adrenoceptor profile of heart was unaffected by cafeteria feeding (% fl~-receptors: control 58 + 2, cafeteria 59 + 4; % fl2-receptors: control 43 + 1, cafeteria 46 + 3, NS). Linear Scatchard plots were obtained for the binding of [3H]DHE to BAT and heart (figs. 3 and 5; r values > 0.95). Dissociation constants for binding were approximately 1.5 nM in both tissues, were unaffected by cafeteria feeding (table 4), and Hill plots revealed linear regressions (correlation coefficients of 0.86-1.0) with slopes close to unity (0.97-1.06) in all cases. The maximum number of binding sites for [3H]DHE was increased by 69% in BAT membranes (fig. 3) and by 72% in BAT cells (fig. 5) in cafeteria-fed rats compared to control values (table 5). A higher density of a-adrenoceptors was observed in heart membranes, but the density was unaffected by cafeteria feeding. Separate control animals were used for comparison with cold-adapted rats (table 6) and binding to isolated BAT cells was not assessed. ~xAdrenoceptor density in BAT membranes was slightly (25% increase), but not significantly affected by cold-adaptation (table 6), whereas fl-receptor number was significantly reduced by 22%. Binding affinity was unaffected by cold-adaptation, and neither a- nor fl-receptor density was
TABLE 5 a-Adrenoceptor number assessed from Scatchard analysis of [3H]DHE binding to BAT membranes and cells and heart membranes. Control
Cafeteria
208 +14 1.4 + 0.11
352 _+35 ** 1.3 ± 0.4
53 __+ 5 1.4 + 0.4
95 ± 13 * 2.0 ± 0.3
303 ± 72 1.5 + 0.5
355 ± 60 1.74± 0.08
BA T Isolated membranes BmaX (fmol/mgP) K d (nM) Isolated cells Bmax (fmol/mgP) K a (nM)
Heart Isolated membranes Bmax (fmol/mgP) K d (nM)
Mean values + S,E.M.; 5-7 membrane preparations; 4 cell preparations. * P < 0.05, ** P < 0.01 compared to control. Data shown in figs. 3 and 5.
significantly changed in heart membranes. The proportion of fl-receptor subtypes and K~ values for displacement (tables 3 and 4) were similar for controls and cold-adapted rats and these data indicate that the total number of ill-receptors in BAT membranes were unaffected by cold ex-
TABLE 6 c~- and /3-adrenoceptor number assessed from the binding of [3H]DHE and [3H]DHA to BAT and heart membranes from control and cold-adapted animals. Control
Cold-adapted
250 + 7 2.0 + 0.7
312 ±36 1.5+ 0.5
158 + +38 1.3 + 0.5
252 :t:76 1.4+ 0.8
158 + 12 1.97+ 0.18
124 + 14 * 2.6+ 0.25
155 1.0
135 0.9
ct-Receptors BAT Bmax (fmol/mgP) K~ (nm) Heart Bmax (fmol/mgP) K~ (nM)
fl -Receptors BAT B~a ~ (fmol/mgP) K~ (nM) Heart Bmax (fmol/mgP) K~ (nM)
Mean values+S.E.M.; n = 5-6 membrane preparations, apart from heart fl-receptors, n = 2. * P < 0.05 vs. control.
321 posure (control 92 _+ 8, cold 82 + 10 f m o l / m g protein, NS) but the density of fl2-receptor was reduced (control 82 + 10, cold 37 + 8 f m o l / m g protein, P < 0.01).
4. Discussion
We have previously reported enhanced thermogenic responses to noradrenaline in cafeteria-fed animals (Rothwell and Stock, 1981; 1983; 1984a), and in vivo measurements of blood flow and tissue oxygen extraction have indicated that this is largely due to increases in the oxygen consumption of BAT (Rothwell and Stock, 1981). In the present study, the resting ~rO2 at thermoneutrality was significantly increased in cafeteria-fed rats (table 1), and isoprenaline (fll/fl2-agonist), prenalterol (selective fll-agonist) and clenbuterol (f12) produced greater maximal responses (table 1) in cafeteria-fed rats, whereas the a-agonist, phenylephrine, elicited a similar response in both control and cafeteria groups. Other workers (e.g. Nedergaard and Lindberg, 1982) have claimed that approximately 20% of the sympathetic activation of heat production in BAT is mediated by the cq-receptor, but Foster (1984) reported that et-adrenoceptor agonists stimulated BAT thermogenesis only in the presence of a fl~-agonist. In the present study it was found that phenylephrine produced similar, or even slightly lower responses in cafeteria-fed rats compared to control animals, but it is possible that phenylephrine may have influenced metabolic rate in tissues other than BAT. The enhanced sensitivity to prenalterol and clenbuterol (indicated by the left shift in the dose-response curves) contrasts with the reduced effects of fl-agonists on in vitro respiration of brown adipocytes taken from coldadapted animals (Bukowiecki et al., 1980; Girardier, 1983). However, these fl-agonists influence many tissues in addition to brown fat, so direct comparisons of drug responses in the whole animal to those obtained in isolated cell preparations may be tenuous. Scatchard analysis of [3H]DHE or [3H]DHA binding to BAT membranes or cells has now been used by a number of groups to assess a- and
fl-adrenoceptor number, respectively (Bukowiecki et al., 1979; 1980; Mohell et al., 1979; Raasmaja et al., 1984a,b; Rothwell et al., 1985; Svoboda et al., 1979; 1984). In both cases, the binding is saturable, stereospecific, reversible, and apparently follows simple Michaelis-Menten kinetics (Hill coefficients close to unity). The similarity of dissociation constants obtained using BAT membranes (microsomal preparations) and isolated intact cells indicates that the use of either of these systems is valid. However, non-specific binding is usually higher in isolated cells, and fig. 4 indicates that in the Bmax of the [3H]DHA binding to cells from cafeteria-fed rats some non-specific binding is included. Heart ventricle was simply used as a reference tissue for binding studies, and agonist responses were not tested. Cold-adapted animals were also used for comparing receptor density, since these animals show changes in metabolic rate, noradrenaline turnover and BAT activity which are qualitatively similar, although greater in magnitude, to those seen in overfed rats. fl-Adrenoceptor density was increased by 3349% in BAT membranes and intact cells obtained from cafeteria-fed rats, but was significantly reduced in heart membranes from these animals and in the BAT membranes of cold-adapted rats. The reduction in fl-adrenoceptor density in brown fat and depressed sensitivity to fl-adrenoceptor agonists in cold-adapted animals has been ascribed to increased sympathetic activity causing 'down regulation' of receptor number (Girardier, 1983; Nedergaard and Lindberg, 1982). However, the increased noradrenaline turnover in BAT and heart from cafeteria-fed rats is as great as that seen in cold-adapted rats (Landsberg and Young, 1983), so a similar reduction in r-receptor density might be expected. It is now accepted that ill- and flz-adrenoceptors co-exist in many tissues (e.g. heart, lung), and that their relative proportions can be assessed from Eadie-Hofstee plots of the displacement of radioligand by selective antagonists, provided certain criteria are met (see Minneman and Molinoff, 1980; Minneman et al., 1979 for reviews). We have previously applied this method to BAT membranes and cells (Rothwell et al., 1985) and the results indicated that both contained ill- and f12"
322 receptors in the approximate ratio of 60/40. This ratio is similar to that found by us and other workers (see Minneman and Molinoff, 1980; Minneman et al., 1979; Nahorski, 1981) in heart, and was confirmed in control rats in the present study. This, and the fact that both atenolol and ICI 118551 gave similar results for receptor profile, would support the validity of these techniques in the present study. Cafeteria feeding caused a shift towards a greater proportion of fl2-receptors in BAT, whereas the proportions in heart were not significantly altered. Calculation of the total number of each receptor subtype indicated no change in the density of fll-adrenoceptors in BAT of cafeteria-fed or cold-adapted animals, but a 70% increase in fl2-receptors in the cafeteria group and a 65% reduction in cold-adapted rats. Cafeteria feeding also caused a significant increase in a-receptor density in BAT, but no change in heart. A small and non-significant rise in ~-receptor density was observed in BAT from coldadapted animals, but significant increases have previously been found in cold-adapted rats by other workers (Raasmaja et al., in press, a). The effects of cafeteria feeding are similar to those seen by Raasmaja et al. (in press, b) who found a two-fold increase in a-receptor number in BAT membranes from cafeteria-fed rats using the alselective ligand, prazosin. This observation, together with the consistent results we have obtained in brown fat cells and tissue membranes, suggests that cafeteria feeding influences only the post-synaptic al-receptors without affecting the pre-synaptic ~2-receptor number. The ratio of ~/fl-receptors was increased from about 1 to 1.5 in the brown fat cells and heart of cafeteria-fed animals, whereas in cold-adapted animals the ratio of a/fl-receptors was increased in BAT membranes, but unaffected in heart. The changes in thermogenic responses to fladrenoceptor agonists in vivo seen in cafeteria-fed rats could be partly explained by alterations in fl-receptor density and subtype, but there was no correlation between the effects of phenylephrine on 402 and or-receptor number in BAT. The marked increase in t~-adrenoceptor receptor density in brown fat from cafeteria-fed rats was not reflected by a greater thermogenic response to
phenylephrine, and in fact there was a slightly reduced sensitivity to the drug in these animals. However, Nedergaard and Lindberg (1982) have proposed that the al-adrenoceptors play only an indirect role in activating BAT and may not stimulate thermogenesis directly. The density of fl2-adrenoceptors in BAT and sensitivity to the thermogenic effects of a flzagonists in vivo were both enhanced by cafeteria feeding. However, sensitivity to the fll'agonist prenalterol was also slightly greater in cafeteria-fed animals in spite of the absence of any change in ill-receptor density. Nevertheless, it could be argued that total BAT ill-receptor number was increased by approximately two-fold after cafeteria feeding simply as a result of hypertrophy of the tissue and any correlations between wholebody responses to agonists and changes in adrenoceptor density in a single tissue must be treated with caution. However, it is obviously difficult to rationalise the increases in response to selective ill- and fl2-agonists with the fact that a mixed fl-agonist (isoprenaline) failed to induce larger thermogenic responses in cafeteria-fed rats, in spite of the changes in fl-receptor density and total BAT depot receptor number. There are numerous similarities between nonshivering and diet-induced thermogenesis (see Girardier, 1983; Landsberg and Young, 1983; Nedergaard and Lindberg, 1982; Rothwell and Stock, 1983; 1984a for reviews), but it now appears that cold-exposure causes a decrease in fl-receptor density and reduced sensitivity to fl-adrenoceptor agonist, whereas overfeeding apparently stimulates both of these parameters. Furthermore, in preliminary studies, we have found that the increase in fl-receptor number is still present in BAT from adrenalectomised cafeteria-fed animals, so it is unlikely that adrenal release of catecholamines or steroids is responsible for this difference (Rothwell, Stock and Sudera, unpublished data). Thus, these changes in fl-receptor number and profile appear to represent the first observation of a qualitative difference between the effects of cold and hyperphagia on BAT, although the thermogenic response of the tissue to adrenoceptor agonists still shows many similarities in the two situations.
323
Acknowledgements We are grateful to Mike Lacey and Ian Connoley for their excellent technical assistance. This work was supported by grants from ICI plc and the Royal Society.
References Bukowiecki, L., N. Follea, A. Paradis and A.J. Collet, 1980, Stereospecific stimulation of brown adipocyte respiration by catechalomines via /31-adrenoreceptors, Am. J. Physiol. 238, E552. Bukowiecki, L., N. Follea, J. Vallieres and J. Leblanc, 1978, fl-Adrenergic receptors in brown adipose tissue: characterization and alterations during acclimation of rats to cold, European J. Biochem. 92, 189. Cheng, Y.C. and W.H. Prusoff, 1973, Relationship between the inhibition constant (K i) and the concentration of inhibitor which causes 50 percent inhibition (Is0) of an enzymatic reaction, Biochem. Pharmacol. 22, 3099. Feist, D.D., 1983, Increased a-adrenergic receptors in brown fat of winter-acclimatized Alaskan voles, Am. J. Physiol. 245, R357. Foster, D.O., 1984, Evidence of a mediatory role of brown adipose tissue in the calorigenic response of cold-acclimated rats to noradrenaline, Can. J. Physiol. Pharmacol. 52, 1051. Girardier, L., 1983, Brown fat: An energy dissipating tissue, in: Mammalian Thermogenesis, eds. L. Girardier and M.J. Stock (Chapman & Hall, London) p. 50. Landsberg, L. and J.B. Young, 1983, Autonomic regulation of thermogenesis, in: Mammalian Thermogenesis, eds. L. Giradier and M.J. Stock (Chapman & Hall, London) p. 99. Minneman, K.P., L.R. Hegstrand and P.B. Molinoff, 1979, Simultaneous determination of beta-1 and beta-2 adrenergic receptors in tissues containing both receptor subtypes, Mol. Pharmacol. 16, 34. Minneman, K.P. and P.B. Molinoff, 1980, Classification and quantitation of B-adrenergic receptor subtypes, Biochem. Pharmacol. 29, 1317. Mohell, N., J. Nedergaard and B. Cannon, 1983, Quantitative
differentiation of a 1- and /~-adrenergic respiratory responses in isolated hamster brown fat cells; evidence for the presence of an al-adrenergic component, European J. Pharmacol. 93, 183. Nahorski, S.R., 1981, Identification and significance of betaadrenoreceptor subtypes, Trends Pharmacol. Sci. 4, 95. Nedergaard, J. and O. Lindberg, 1982, The brown fat cell, Int. Rev. Cytol. 74, 187. Raasmaja, A., N. Mitchell and J. Nedergaard, Increased a 1adrenergic receptors density in brown adipose tissue of cold-acclimated rats and hamsters, European J. Pharmacol. (in press, a). Raasmaja, A., N. Mitchell and J. Nedergaard, Increased a 1adrenergic receptor density in brown adipose tissue of cafeteria-fed rats, Biosci. Rep. (in press, b). Rothwell, N.J. and M.J. Stock, 1981, Influence of noradrenaline on blood flow to brown adipose tissue in rats exhibiting diet-induced thermogenesis, Pfli~gers Arch. 389, 237. Rothwell, N.J. and M.J. Stock, 1983, Diet-induced thermogenesis, in: Mammalian Thermogenesis, eds. L. Girardier and M.J. Stock (Chapman & Hall, London) p. 208. Rothwell, N.J. and M.J. Stock, 1984a, Brown adipose tissue, in: Recent Advances in Physiology - 10, ed. P.F. Baker (Churchill Livingstone, Edinburgh) p. 208. Rothwell, N.J. and M.J. Stock, 1984b, Effects of denervating brown adipose tissue on the response to cold, hyperphagia and noradrenaline treatment in the rat, J. Physiol. 355, 457. Rothwell, N.J., M.J. Stock and D. Stribling, 1982, Diet-induced thermogenesis, Pharmacol. Ther. 17, 251. Rothwell, N.J., M.J. Stock and D.K. Sudera, 1985, fl-Adrenoreceptors in rat brown adipose tissue: proportions of 13I and fl2-subtypes, Am. J. Physiol. 248, E397. Svoboda, P., J. Svartengren and Z. Drahota, 1984, The functional and structural reorganisation of the plasma membranes of brown adipose tissue induced by cold acclimation of the hamster, II, fl-Adrenergic receptor, Mol. Physiol. 5, 221. Svoboda, P., J. Svartengren, M. Snochowski, J. Houstek and B. Cannon, 1979, High number of high affinity sites for (-)3(H)-dihydroalprenolol in isolated hamster brown fat cells, European J. Biochem. 102, 203.
313
T H E R M O G E N I C R E S P O N S E S TO A D R E N O C E P T O R A G O N I S T S A N D B R O W N FAT A D R E N O C E P T O R S IN OVERFED RATS NANCY J. ROTHWELL, MICHAEL J. STOCK * and DEEPAK K. SUDERA Department of Physiology, St. George's Hospital Medical School, Tooting, London SW17, U.K. Received 20 June, 1985, revised MS received 20 March 1986, accepted 1 April 1986
N.J. ROTHWELL, M.J. STOCK and D.K. SUDERA, Thermogenic responses to adrenoceptor agonists and brown fat adrenoceptors in overfed rats, European J. Pharmacol. 125 (1986) 313-323. Rats fed a cafeteria diet to produce hyperphagia showed increases in the maximal thermogenic responses (rise in oxygen consumption) to isoprenaline (mixed fl-agonist), prenalterol (ill-selective agonist) and clenbuterol (fl2-agonist), and left-shifts in the dose-response curves to the latter two. The maximal response to phenylephrine (a-agonist) was similar for control and cafeteria rats. Ligand binding studies revealed increases in fl-adrenoceptor density of 33-38% in brown fat cells and isolated membranes from cafeteria-fed rats, but a 30% reduction in fl-receptors in heart membranes. Cold-adaptation caused a 22% reduction in fl-receptor density in brown fat membranes, but no change in heart. The ratio of flx/fl2-receptors in brown fat was reduced from 59/45 in control to 47/54 in cafeteria-fed rats, but was not significantly altered in heart (58/44) or in brown fat from cold-adapted animals (64/30). a-Adrenoceptor density was increased above control values by 69 and 25% in brown adipose tissue from cafeteria and cold-adapted rats, respectively. Brown adipose tissue Hyperphagia
Cold-adaptation
a-Adrenoceptor
1. Introduction The primary function of brown adipose tissue (BAT) is heat production, and this tissue is largely responsible for the increases in metabolic rate associated with cold exposure (non-shivering thermogenesis, NST) or hyperphagia (diet-induced thermogenesis, DIT). Hyperphagic rats fed a palatable cafeteria diet, and cold-adapted animals show enhanced thermogenic responses to noradrenaline and increased sympathetic activity in brown fat (see Girardier, 1983; Nedergaard and Lindberg, 1982; Rothwell and Stock, 1983; 1984a for reviews). Activation of the tissue by diet or cold can be prevented by sympathetic denervation of the tissue (Girardier, 1983; Rothwell and Stock, 1984b). The thermogenic effects of the sympathetic * To whom all correspondence should be addressed. 0014-2999/86/$03.50 © 1986 Elsevier Science Publishers B.V.
fll-Adrenoceptor
fl2-Adrenoceptor
nervous system appear to be mediated largely by interaction of noradrenaline with fl-adrenoceptors (Bukowiecki et al., 1978; 1980; Feist, 1983; Mohell et al., 1983; Nedergaard and Lindberg, 1982; Svoboda et al., 1979; 1984) and a-receptors are thought to play only a minor role in the activation of brown fat (Minneman and Molinoff, 1980; Mohell et al., 1983; Raasmaja et al., 1984a,b). Ligand binding studies have identified a high density of fl-adrenoceptors on brown fat cell membranes and on isolated b r o w n adipocytes (Bukowiecki et al., 1978; 1980; Svoboda et al., 1979; 1984) and ligand displacement with adrenoceptor agonists has indicated that these are predominantly of the fit-subtype (Bukowiecki et al., 1978; 1980; Svoboda et al., 1979; 1984). However, both ill- and fl2-selective adrenoceptor agonists will stimulate thermogenesis and BAT activity (Rothwell et al., 1982) and recently we have reported the presence of both receptor subtypes
314 (60%/31, 40% f12) in isolated brown fat membranes (Rothwell et al., 1985). Chronic pharmacological or physiological sympathetic stimulation of most tissues usually causes desensitization to adrenoceptor agonists and 'down-regulation' of adrenoceptor density (see Minneman and Molinoff, 1980; Minneman et al., 1979; Nahorski, 1981 for reviews) and cold-adaptation, for example, causes a reduction in the density of /3-adrenoceptors on BAT. This is accompanied by a lower sensitivity to the thermogenic effects of adrenoceptor agonists, although the maximal capacity for catecholamine-induced thermogenesis is enhanced (Girardier, 1983; Nedergaard and Lindberg, 1982). Cafeteria-fed rats exhibiting diet-induced thermogenesis also show enhanced maximal thermogenic responses to noradrenaline, and DIT in these animals can be almost totally prevented by acute fl-adrenergic blockade with propranolol, whereas the aantagonist phentolamine has little effect (Rothwell and Stock, 1981; 1984b). Little is known about the changes in fl-receptor density a n d / o r receptor subtype during chronic hyperphagia, and similarly, variations in thermogenic sensitivity to receptor-selective adrenoceptor agonists have not yet been investigated in this situation. In the present study, we have therefore investigated the effects of cafeteria feeding on the thermogenic responses to selective ill-, /32- or a-adrenoceptors agonists, and on /3-receptor number and subtype and a-receptor density in BAT. Ligand binding studies were also performed on heart ventricle of cafeteria-fed rats, and on heart and BAT from cold-adapted animals for comparison.
foods (e.g. various biscuits, cakes, chocolates, meats) in addition to the stock diet. This treatment has previously been shown to stimulate energy intake and metabolic rate by 50-100% in young rats (Rothwell and Stock, 1983; 1984a). Cold-adapted rats were fed stock diet only and housed at 5 °C. All treatments were performed for at least 20 days, unless otherwise stated, and in many cases (particularly for controls) heart and BAT was pooled from 2-3 rats for each experiment.
2.2. Thermogenic responses to adrenoceptor agonists
2. Materials and methods
Resting oxygen consumption (~/Oz) was measured in control and cafeteria-fed rats at 29°C in closed-circuit respirometers (24) for 2 h before and 2-3 h after injections of either phenylephrine (a-agonist; 1, 2, 3.5, 5 mg/kg), isoprenaline (mixed /3-agonist; 0.01, 0.025, 0.05, 0.2 mg/kg), prenalterol (fll-agonist; 0.1, 0.25, 0.5, 1.0 mg/kg) or clenbuterol (fl2-agonist; 0.25, 0.5, 0.75, 1.0, 2.0 mg/kg). All drugs were injected subcutaneously (s.c.) in 0.2 ml 0.9% saline containing ascorbic acid (0.01%). Each animal was tested on up to four occasions with different drugs, but drugs were tested randomly, with at least 4 days between tests on any one animal. A total of 32 control and 36 cafeteria animals were used, yielding 4-6 values for each dose of each drug tested. Values associated with physical activity were discarded and results were expressed as ml O 2 consumed/rain per kg °7s, and presented graphically as the maximal increase in 902 as a percentage of the pre-injection value. Many of these rats were subsequently used for ligand binding studies (see below), but at least one week elapsed between measurements of 'JO 2 and receptor binding.
2.1. Animals
2.3. Preparation of membranes
Male Sprague-Dawley rats (Charles River, Kent, U.K.) were used at 40-60 days of age. Control animals were housed at 24°C (12 h light/dark cycle) and allowed free access to a pelleted stock diet (PRD, Christopher Hill Group Ltd., Dorset, U.K.). Cafeteria-fed rats were kept at 24°C and allowed a daily choice of four different human
All the dissectible brown adipose tissue (interscapular, cervical, subscapular, axillary, perirenal, periaortic) was taken from the animals immediately after decapitation. Heart ventricles were also removed, and tissues were finely minced in ice-cold 0.25 M sucrose buffer containing 1 mM EDTA and 10 mM Tris (pH 7.4), and then ho-
315 mogenized (Polytron, mark 6 for 7 s). The homogenate was centrifuged at 3500 x g for 5 min, and the supernatant filtered through surgical gauze to remove cell debris. Mitochondria were sedimented by centrifugation at 11000 x g for 15 rain and the supernatant was then recentrifuged at 100000 X g for 60 min to obtain a microsomal fraction, which was resuspended in buffer to achieve a protein concentration of approximately 150/~g/ml (equivalent to 120 ~g/ml in the final incubation assay). Protein was determined by a dye-reagent method (Bio-Rad) using bovine serum albumin (BSA) standards.
2.4. Preparation of isolated brown adipocytes Brown adipocytes were isolated by a method similar to that described by Svoboda et al. (1979; 1984). Tissue from all depots was minced in Krebs buffer (10 mM glucose, 10 mM fructose and 10 mM pyruvate) containing 4% BSA (fatty acid-free; Sigma, Poole, U.K.) and 3 mg/ml collagenase (Sigma Type II). The tissue was gassed with 95% 02-5% CO 2 and after 10 min preincubation (37 ° C), was filtered. The filtrate was discarded and the tissue re-incubated in fresh medium for 40 rain with shaking every 3-5 min. The isolated cells were filtered through nylon and allowed to stand for 40 min before removing the cell layer and resuspending in BSA-free Krebs buffer. The cells were washed once and resuspended to a final concentration of approximately 0.3 x 106 cells/ml. Cells were counted in a Neubauer chamber, but any cells which took up Trypan blue (usually less than 5% of total cells) were excluded from counting.
2.5 Radiofigand binding studies a- and fl-adrenoceptor number and affinity were assessed from the binding of [3H]dihydroergocryptine ([3H]DHE; 15.7 Ci/mmol, Amersham International, U.K., 0.2-10 nM/incubation) or [3H]dihydroalprenolol ([3H]-DHA; 83 Ci/mmol, Amersham International, U.K., 0.2-10 nM), respectively. Membranes or cells were incubated in 1 ml buffer (50 mM Tris, 10 mM MgC12, pH 7.4) containing the appropriate radio-
ligand for 15 min at 37°C. Incubations were terminated by adding 2 ml ice-cold incubation buffer followed by rapid vacuum filtration through Whatman G F / C filters followed by three 5 ml washes with buffer. Radioactivity retained on the filters was measured in a Beckman (LS-6800) liquid scintillation counter. Non-specific binding was assessed from parallel incubations containing either 10 ~M phentolamine (a-receptors) or 5/IM L-propanolol (B-receptors). Binding data were subjected to Scatchard analysis and the maximum number of binding sites (Bmax) and dissociation constant (Kd) calculated from regression analysis of linear Scatchard plots.
2.6. Assessment of/3-adrenoceptor subtype The relative proportions of/31- and /32-adrenoceptors was assessed from Hofstee plots of the displacement of [3H]DHA (2 nM) from heart and BAT membranes or brown adipocytes using selective/31 (atenolol)- or B2 (ICI 118551)antagonists as described previously (Rothwell et al., 1985). Curvilinear Hofstee plots were resolved into two lines by iterative computer analysis (Rothwell et al., 1985), and the dissociation constants for inhibition of binding (Kd) to each site was calculated from the slope of these lines and corrected using the equation of Cheng and Prusoff (1973). The percentage of each receptor subtype was determined from the ordinate intercept of each line. In addition, the effect of a non-selective antagonist (propranolol) was also tested. Mean values are presented+ S.E.M.-and the number of determinations refers to separate tissue preparations (1-4 animals per preparation). Since many of the experiments were performed over several weeks, with control and experimental animals being studied at the same time, data were usually compared by a Student's t-test for matched data, using two-tailed probabilities.
3. Results
Resting VO2 before injection of adrenoceptor agonists was significantly increased in cafeteria-fed rats compared to controls (table 1). The maximal incremental response to injection of phenylephrine
316 TABLE 1 Resting VO 2 and peak response ( m l / m i n per kg °75) to adrenoceptor agonists in control and cafeteria-fed rats. Average preinjection
Phenylephrine (5.0) * Isoprenaline (1.5) Prenalterol (1.0) Clenbuterol (2.0)
Peak increment
Control
Cafeteria
16.07 _+0.20 16.11 + 0.29 16.35 + 0.22 15.88 + 0.19
18.40 + 0.17 18.02 + 0.17 18.45 + 0.17 18.76 + 0.15
Control b b b b
4.56 + 13.37 + 8.53 + 9.52 +
Cafeteria 0.43 0.31 0.35 0.81
4.73 _+0.48 16.86 + 0.71 b 9.58 + 0.21 a 12.61 + 0.96 a
* Dose used for maximal response (mg/kg). Mean values+ S.E.M. (n = 4-6). a p < 0.05, b p < 0.001 vs. control.
did not differ between groups, but all three fladrenoceptor agonists provoked larger responses in cafeteria than in control rats (table 1). The maximal rise in "¢Oz, as a percentage of the pre-injection value, was greatest for isoprenaline ( > 80%, fig. 1), similar for prenalterol and clenbuterol (5065%) and smallest for phenylephrine (25-30%). Cafeteria-fed rats showed a smaller rise in "¢O2 following the lowest dose of phenylephrine than did controls, and the highest dose tested produced some behavioural changes and increased physical
activity in both groups. Isoprenaline provoked slightly greater increases in VO 2 in cafeteria-fed rats at all doses (fig. 1) but the difference between cafeteria and control rats was statistically significant only for the highest dose. Dose-response curves for clenbuterol and prenalterol were both shifted slightly to the left in cafeteria-fed rats, with these animals showing significantly greater responses than controls at the lower end of the dose range. The mass of total brown adipose tissue was
I$OPRENALINE
PI'IEN YLEPHRINEs
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0-
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PRENALTEROL 50-
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CLENBUTEROL
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404 302010-
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011
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2
4 nOlO
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Fig. 1. The effects of varying doses of adrenoceptor agonists on resting oxygen consumption (% increase above pre-injection value) in control (e) and cafeteria-fed ( I ) rats. Note the differing scales for each drug. Mean values+ S.E.M. (n = 4-6).
317
increased by approximately two-fold following 20 days of cafeteria feeding (1.42 + 0.11 g, n = 12) or cold-adaptation (1.58 + 0.14 g, n = 10) compared to control values (0.70 + 0.01 g, n = 15, P < 0.001). Approximately 350/~g membrane protein was obtained per 100 mg brown fat from control and cafeteria-fed rats, but up to 580 ~tg/100 g for cold-adapted rats. One gram of brown fat from control or cafeteria-fed rats yielded 4-5 × 10 6 BAT cells, and the total number of brown adipocytes was elevated by almost 100% in cafeteria-fed rats; cells were not prepared from cold-adapted animals. Approximately 200 /~g membranes were isolated per 100 mg heart ventricle in all groups.
The binding of [3H]DHE and [3H]DHA to isolated brown fat membranes and adipocytes showed saturation after approximately 6-10 min incubation and at a concentration of 5-6 nM radioligand (figs. 2-5). Non-specific binding varied from 25 to 70% of total binding at low to high concentrations of ligand, respectively, and binding to the G F / C filters represented less than 0.5% of total binding. Linear regressions were obtained for Scatchard analysis of binding data, and in all cases correlation coefficients were > 0.95 (e.g. figs. 2-5). For fl-adrenoceptors, dissociation constants (Kd) were approximately 2 nM for BAT cells or membranes and for heart membranes (figs. 2 and 4, table 2). The maximum number of binding sites for
160' CAFETERIA 12C
320
CONTROL
X "~
60
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240"
160"
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i ~
80.
[ 3 ~ OHA conce41b~ation (nM) 0 ¸ 0
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0 .04 --
FETERIA 0.2"
CAFETERIA
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0 0
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100 ~H] D H A B o u n d
150 I f tool
200
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Fig. 2. Specific binding of [3H]dihydroalprenolol (DHA) to isolated brown adipose tissue (BAT) membranes from control and cafeteria-fed rats plotted as a function of ligand concentration (upper graph), and Scatchard analysis of [3H]DHA binding (lower graph) derived from the same data. Mean values+ S.E.M. (n = 10).
CONTROL
. 80
.
. 160
.
. 240
320
400
i 3 I~DHE bound (frnoles mg protein)
Fig. 3. Specific binding of [3H]dihydroergocryptine (DHE) to isolated brown adipose tissue (BAT) membranes from control and cafeteria-fed rats plotted as a function of bound concentration (upper graph), and Scatchard analysis of [3H]DHE binding (lower graph) derived from the same data. Mean values + S.E.M. (n ~ 10).
318 80 6o
~ 40
m
/~
~CONTROL
o
,-ff 2O
0 o
o
0
. 2
0 ~1~
.
. 4
'2 4 ~H] DHEconcentration (nM)
. 6
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[3HI DHA concentration (nM)
0.02"
!
* 0.01
O
i
AFETERIA
0.02
m 0 0 o
0
, 20
, 40
60
80
20
Fig. 4. Specific binding of [3H]dihydroalprenolol (DHA) to isolated brown adipose tissue (BAT) cells from control and cafeteria-fed rats plotted as a function of ligand concentration (upper graph) and Scatchard analysis of [3H[DHA binding (lower graph) derived from the same data. Mean values + S.E.M. (n = 3).
[3H]DHA was increased by 38% in BAT membranes from cafeteria-fed rats and by 49% in BAT cells, when compared to control values (fig. 2, table 2). Hill plots of these data revealed linear regressions (r = 0.8-1.0) with slopes (Hill coefficients) very close to unity (e.g. BAT membranes: control 1.01, cafeteria 0.97; BAT cells: control 0.94, cafeteria 0.9). In a separate trial, the density of /3-adrenoceptors in BAT was not significantly different from controls after only seven days of cafeteria feeding. A higher density of /3-adrenoceptors was seen in heart ventricle membranes than in BAT membranes, but the maximum number of binding sites in heart was reduced by 30% after 20 days of cafeteria feeding (table 2). Hill
60
80
100
~3HIDHE bound (fmole$ 10 6 8ATcelle )
100
[3H~DHA bound (frnoles 106 BAT cells )
40
Fig. 5. Specific binding of [3H]dibydroergocryptine (DHE) to isolated brown adipose tissue (BAT) cells from control and cafeteria-fed rats plotted as a function of ligand concentration (upper graph) and Scatchard analysis of [3H]DHE binding (lower graph) derived from the same data. Mean values + S.E.M. (n = 3). TABLE 2 /3-Adrenoceptor number assessed from Scatchard analysis of [3H]DHA binding to BAT membranes, cells and heart membranes.
BAT Isolated membranes Bmax (fmol/mgP) K d (nM) Isolated cells Bmax (fmol/106 cells) K d (nM) Heart Isolated membranes Bmax ( f m o l / m g P ) K d (nM)
Control
Cafeteria
140 + 16 1.60+ 0.3
193 + 16 * 2.2 _+ 0.3
45
+ 6 2.5+ 0.2
323 + 36 1,83+ 0.09
67 + 6 * 3.3 + 0.4
226 + 38 * 1.74+ 0.08
Mean values:t:S.E.M., n = 7-10 membrane preparations, 4-5 cell preparations. * P < 0.05 paired t-test. Data shown in figs. 2 and 4.
319 coefficients for these d a t a were a g a i n close to u n i t y ( c o n t r o l 0.98, cafeteria 1.04). H o f s t e e plots ( o r d i n a t e : % i n h i b i t i o n of r a d i o l i g a n d b i n d i n g , abscissa: % i n h i b i t i o n / a n t a g o n i s t c o n c e n t r a t i o n ) of the d i s p l a c e m e n t b y p r o p r a n o l o l of [ 3 H ] D H A f r o m B A T m e m b r a n e s were l i n e a r for c o n t r o l (y i n t e r c e p t = 94% K h = 0.009 /~M, r = 0.99) a n d cafeteria-fed rats (y = 96%, K h = 0.002 # M , r = 0.96). H o w e v e r , d i s p l a c e m e n t b y the selective a n t a g o n i s t s a t e n o l o l (fig. 6) or I C I 118551 revealed c u r v i l i n e a r plots w h i c h were resolved i n t o two c o m p o n e n t lines. A t e n o l o l a n d I C I 118551 s h o w e d 50- to 100-fold selectivity for ill", or fl2-receptor s u b t y p e s respectively, a n d values o b t a i n e d for the relative p r o p o r t i o n s of the f l - r e c e p t o r s u b t y p e s w e r e s i m i l a r for b o t h
IOD'
•
80.
CONTROL
60' 40. 20.
oJ
t.° 10
IO0
. 20
30
40
•
50
6"0
•
•
80
CAFETERIA
40,
20' O' 100'
0 •
10
20
30
• 40
TABLE 3 Proportion of fl-adrenoceptor subtypes and Kh for atenolol inhibition of [3H]DHA binding to isolated BAT membranes derived from Hofstee analysis of displacement curves. Mean values-+S.E.M. Displacement curves were derived from 3-4 membrane preparations in each case; r = regression coefficients for resolution of curvilinear Hofstee plots into 2-site plots. Data shown in fig. 6. Control Proportion (%) fl] 59 _+1 f12 45 _+1 K~ (/~M) fll 0.56_+0.25 f12 60 _+8 r 0.95
Cafeteria
Cold-adapted
47 54
64 30
_+ 3 • _+ 3 *
0.56_+ 0 . 2 1 46 +__12 0.98
_+2 _+5
0.56_+0.15 49 -+7 0.96
* P < 0.05 compared to control.
a n t a g o n i s t s (tables 3 a n d 4). T h e K d value for the I C I 118551 d i s p l a c e m e n t f r o m the i l l - r e c e p t o r was slightly h i g h e r in the cafeteria g r o u p c o m p a r e d to the c o n t r o l a n d c o l d - a d a p t e d group. B A T m e m b r a n e receptors f r o m c o n t r o l rats were p r e d o m i n a n t l y of the f i t - s u b t y p e , b u t this was reversed b y cafeteria f e e d i n g (tables 3 a n d 4, fig 6). T h u s , cafeteria-fed rats h a d a s i g n i f i c a n t l y lower p r o p o r t i o n of /~t-receptors a n d a h i g h e r p r o p o r t i o n of fl2-receptors. T h e s e d a t a were o b t a i n e d o n separate m e m b r a n e p r e p a r a t i o n s to those p r e s e n t e d in table 2. H o w e v e r , w h e n S c a t c h a r d a n a l y s i s a n d d i s p l a c e m e n t curves for a t e n o l o l were s t u d i e d
I 50
60
TABLE 4 80.
COLD
Proportion of fl-adrenoceptor subtypes and K~ for ICI 118551 inhibition of [3H]DHA binding to isolated BAT membranes derived from Hofstee analysis of displacement curves. Mean values_+S.E.M. Displacement curves were derived from 2-4 membrane preparations; r = regression coefficients for resolution of curvilinear Hofstee plots into 2-site plots.
4O
2O O
• 0
10
20
30
40
50
60
Control
%I~ c
Fig. 6. Hofstee plots of [3H]DHA displacement data. Ordinate = % inhibition (I) of specific [3HIDttA binding by atenolok abscissa = I/concentration of antagonist (~M) for BAT membranes isolated from control, cafeteria or cold-adapted rats. Curves were resolved into two component lines by interactive computer analysis, and values obtained are shown in table 3 and in the text. Values represent means of 3-4 separate membrane preparations.
Proportion (%) /~] 57 + 4 f12 45 + 5 /3] /32 r
1.0 _+0.4 0.03 _+0.01 0.91
Cafeteria
Cold-adapted
44 53
66 30
+6 • +3•
3.1 _ + 1 . 8 0.03 _+0.02 0.88
* P < 0.05 compared to controls.
+2 _5
0.72_+0.16 0.02 _+0.01 0.89
320
using the same preparations, the results obtained for the maximum number of binding sites by Scatchard analysis were similar to those presented in table 2 (B. . . . f m o l / m g protein: control 160, cafeteria 200, n = 2). Displacement with ICI 118551 was not tested on these same preparations. Using the data from tables 2-4, it can be calculated that the total number of ill-receptors (fmol/mg protein) was approximately 83 + 9 for control and 91 + 8 for cafeteria (NS) and of flzreceptors was 60 + 6 for control and 106 + 9 for cafeteria (P < 0.01). Displacement curves for isolated BAT cells using atenolol (ICI 118551 was not tested) revealed values for the proportion of each receptor subtype which were similar to those obtained on membranes in control animals (fla = 58 + 4%, f12 = 4 2 + 5%), and experiments performed on two adipocyte preparations from cafeteria rats showed approximately equal proportions of ill- (5~%) and fl2-receptors (50%). fl-Adrenoceptor profile of heart was unaffected by cafeteria feeding (% fl~-receptors: control 58 + 2, cafeteria 59 + 4; % fl2-receptors: control 43 + 1, cafeteria 46 + 3, NS). Linear Scatchard plots were obtained for the binding of [3H]DHE to BAT and heart (figs. 3 and 5; r values > 0.95). Dissociation constants for binding were approximately 1.5 nM in both tissues, were unaffected by cafeteria feeding (table 4), and Hill plots revealed linear regressions (correlation coefficients of 0.86-1.0) with slopes close to unity (0.97-1.06) in all cases. The maximum number of binding sites for [3H]DHE was increased by 69% in BAT membranes (fig. 3) and by 72% in BAT cells (fig. 5) in cafeteria-fed rats compared to control values (table 5). A higher density of a-adrenoceptors was observed in heart membranes, but the density was unaffected by cafeteria feeding. Separate control animals were used for comparison with cold-adapted rats (table 6) and binding to isolated BAT cells was not assessed. ~xAdrenoceptor density in BAT membranes was slightly (25% increase), but not significantly affected by cold-adaptation (table 6), whereas fl-receptor number was significantly reduced by 22%. Binding affinity was unaffected by cold-adaptation, and neither a- nor fl-receptor density was
TABLE 5 a-Adrenoceptor number assessed from Scatchard analysis of [3H]DHE binding to BAT membranes and cells and heart membranes. Control
Cafeteria
208 +14 1.4 + 0.11
352 _+35 ** 1.3 ± 0.4
53 __+ 5 1.4 + 0.4
95 ± 13 * 2.0 ± 0.3
303 ± 72 1.5 + 0.5
355 ± 60 1.74± 0.08
BA T Isolated membranes BmaX (fmol/mgP) K d (nM) Isolated cells Bmax (fmol/mgP) K a (nM)
Heart Isolated membranes Bmax (fmol/mgP) K d (nM)
Mean values + S,E.M.; 5-7 membrane preparations; 4 cell preparations. * P < 0.05, ** P < 0.01 compared to control. Data shown in figs. 3 and 5.
significantly changed in heart membranes. The proportion of fl-receptor subtypes and K~ values for displacement (tables 3 and 4) were similar for controls and cold-adapted rats and these data indicate that the total number of ill-receptors in BAT membranes were unaffected by cold ex-
TABLE 6 c~- and /3-adrenoceptor number assessed from the binding of [3H]DHE and [3H]DHA to BAT and heart membranes from control and cold-adapted animals. Control
Cold-adapted
250 + 7 2.0 + 0.7
312 ±36 1.5+ 0.5
158 + +38 1.3 + 0.5
252 :t:76 1.4+ 0.8
158 + 12 1.97+ 0.18
124 + 14 * 2.6+ 0.25
155 1.0
135 0.9
ct-Receptors BAT Bmax (fmol/mgP) K~ (nm) Heart Bmax (fmol/mgP) K~ (nM)
fl -Receptors BAT B~a ~ (fmol/mgP) K~ (nM) Heart Bmax (fmol/mgP) K~ (nM)
Mean values+S.E.M.; n = 5-6 membrane preparations, apart from heart fl-receptors, n = 2. * P < 0.05 vs. control.
321 posure (control 92 _+ 8, cold 82 + 10 f m o l / m g protein, NS) but the density of fl2-receptor was reduced (control 82 + 10, cold 37 + 8 f m o l / m g protein, P < 0.01).
4. Discussion
We have previously reported enhanced thermogenic responses to noradrenaline in cafeteria-fed animals (Rothwell and Stock, 1981; 1983; 1984a), and in vivo measurements of blood flow and tissue oxygen extraction have indicated that this is largely due to increases in the oxygen consumption of BAT (Rothwell and Stock, 1981). In the present study, the resting ~rO2 at thermoneutrality was significantly increased in cafeteria-fed rats (table 1), and isoprenaline (fll/fl2-agonist), prenalterol (selective fll-agonist) and clenbuterol (f12) produced greater maximal responses (table 1) in cafeteria-fed rats, whereas the a-agonist, phenylephrine, elicited a similar response in both control and cafeteria groups. Other workers (e.g. Nedergaard and Lindberg, 1982) have claimed that approximately 20% of the sympathetic activation of heat production in BAT is mediated by the cq-receptor, but Foster (1984) reported that et-adrenoceptor agonists stimulated BAT thermogenesis only in the presence of a fl~-agonist. In the present study it was found that phenylephrine produced similar, or even slightly lower responses in cafeteria-fed rats compared to control animals, but it is possible that phenylephrine may have influenced metabolic rate in tissues other than BAT. The enhanced sensitivity to prenalterol and clenbuterol (indicated by the left shift in the dose-response curves) contrasts with the reduced effects of fl-agonists on in vitro respiration of brown adipocytes taken from coldadapted animals (Bukowiecki et al., 1980; Girardier, 1983). However, these fl-agonists influence many tissues in addition to brown fat, so direct comparisons of drug responses in the whole animal to those obtained in isolated cell preparations may be tenuous. Scatchard analysis of [3H]DHE or [3H]DHA binding to BAT membranes or cells has now been used by a number of groups to assess a- and
fl-adrenoceptor number, respectively (Bukowiecki et al., 1979; 1980; Mohell et al., 1979; Raasmaja et al., 1984a,b; Rothwell et al., 1985; Svoboda et al., 1979; 1984). In both cases, the binding is saturable, stereospecific, reversible, and apparently follows simple Michaelis-Menten kinetics (Hill coefficients close to unity). The similarity of dissociation constants obtained using BAT membranes (microsomal preparations) and isolated intact cells indicates that the use of either of these systems is valid. However, non-specific binding is usually higher in isolated cells, and fig. 4 indicates that in the Bmax of the [3H]DHA binding to cells from cafeteria-fed rats some non-specific binding is included. Heart ventricle was simply used as a reference tissue for binding studies, and agonist responses were not tested. Cold-adapted animals were also used for comparing receptor density, since these animals show changes in metabolic rate, noradrenaline turnover and BAT activity which are qualitatively similar, although greater in magnitude, to those seen in overfed rats. fl-Adrenoceptor density was increased by 3349% in BAT membranes and intact cells obtained from cafeteria-fed rats, but was significantly reduced in heart membranes from these animals and in the BAT membranes of cold-adapted rats. The reduction in fl-adrenoceptor density in brown fat and depressed sensitivity to fl-adrenoceptor agonists in cold-adapted animals has been ascribed to increased sympathetic activity causing 'down regulation' of receptor number (Girardier, 1983; Nedergaard and Lindberg, 1982). However, the increased noradrenaline turnover in BAT and heart from cafeteria-fed rats is as great as that seen in cold-adapted rats (Landsberg and Young, 1983), so a similar reduction in r-receptor density might be expected. It is now accepted that ill- and flz-adrenoceptors co-exist in many tissues (e.g. heart, lung), and that their relative proportions can be assessed from Eadie-Hofstee plots of the displacement of radioligand by selective antagonists, provided certain criteria are met (see Minneman and Molinoff, 1980; Minneman et al., 1979 for reviews). We have previously applied this method to BAT membranes and cells (Rothwell et al., 1985) and the results indicated that both contained ill- and f12"
322 receptors in the approximate ratio of 60/40. This ratio is similar to that found by us and other workers (see Minneman and Molinoff, 1980; Minneman et al., 1979; Nahorski, 1981) in heart, and was confirmed in control rats in the present study. This, and the fact that both atenolol and ICI 118551 gave similar results for receptor profile, would support the validity of these techniques in the present study. Cafeteria feeding caused a shift towards a greater proportion of fl2-receptors in BAT, whereas the proportions in heart were not significantly altered. Calculation of the total number of each receptor subtype indicated no change in the density of fll-adrenoceptors in BAT of cafeteria-fed or cold-adapted animals, but a 70% increase in fl2-receptors in the cafeteria group and a 65% reduction in cold-adapted rats. Cafeteria feeding also caused a significant increase in a-receptor density in BAT, but no change in heart. A small and non-significant rise in ~-receptor density was observed in BAT from coldadapted animals, but significant increases have previously been found in cold-adapted rats by other workers (Raasmaja et al., in press, a). The effects of cafeteria feeding are similar to those seen by Raasmaja et al. (in press, b) who found a two-fold increase in a-receptor number in BAT membranes from cafeteria-fed rats using the alselective ligand, prazosin. This observation, together with the consistent results we have obtained in brown fat cells and tissue membranes, suggests that cafeteria feeding influences only the post-synaptic al-receptors without affecting the pre-synaptic ~2-receptor number. The ratio of ~/fl-receptors was increased from about 1 to 1.5 in the brown fat cells and heart of cafeteria-fed animals, whereas in cold-adapted animals the ratio of a/fl-receptors was increased in BAT membranes, but unaffected in heart. The changes in thermogenic responses to fladrenoceptor agonists in vivo seen in cafeteria-fed rats could be partly explained by alterations in fl-receptor density and subtype, but there was no correlation between the effects of phenylephrine on 402 and or-receptor number in BAT. The marked increase in t~-adrenoceptor receptor density in brown fat from cafeteria-fed rats was not reflected by a greater thermogenic response to
phenylephrine, and in fact there was a slightly reduced sensitivity to the drug in these animals. However, Nedergaard and Lindberg (1982) have proposed that the al-adrenoceptors play only an indirect role in activating BAT and may not stimulate thermogenesis directly. The density of fl2-adrenoceptors in BAT and sensitivity to the thermogenic effects of a flzagonists in vivo were both enhanced by cafeteria feeding. However, sensitivity to the fll'agonist prenalterol was also slightly greater in cafeteria-fed animals in spite of the absence of any change in ill-receptor density. Nevertheless, it could be argued that total BAT ill-receptor number was increased by approximately two-fold after cafeteria feeding simply as a result of hypertrophy of the tissue and any correlations between wholebody responses to agonists and changes in adrenoceptor density in a single tissue must be treated with caution. However, it is obviously difficult to rationalise the increases in response to selective ill- and fl2-agonists with the fact that a mixed fl-agonist (isoprenaline) failed to induce larger thermogenic responses in cafeteria-fed rats, in spite of the changes in fl-receptor density and total BAT depot receptor number. There are numerous similarities between nonshivering and diet-induced thermogenesis (see Girardier, 1983; Landsberg and Young, 1983; Nedergaard and Lindberg, 1982; Rothwell and Stock, 1983; 1984a for reviews), but it now appears that cold-exposure causes a decrease in fl-receptor density and reduced sensitivity to fl-adrenoceptor agonist, whereas overfeeding apparently stimulates both of these parameters. Furthermore, in preliminary studies, we have found that the increase in fl-receptor number is still present in BAT from adrenalectomised cafeteria-fed animals, so it is unlikely that adrenal release of catecholamines or steroids is responsible for this difference (Rothwell, Stock and Sudera, unpublished data). Thus, these changes in fl-receptor number and profile appear to represent the first observation of a qualitative difference between the effects of cold and hyperphagia on BAT, although the thermogenic response of the tissue to adrenoceptor agonists still shows many similarities in the two situations.
323
Acknowledgements We are grateful to Mike Lacey and Ian Connoley for their excellent technical assistance. This work was supported by grants from ICI plc and the Royal Society.
References Bukowiecki, L., N. Follea, A. Paradis and A.J. Collet, 1980, Stereospecific stimulation of brown adipocyte respiration by catechalomines via /31-adrenoreceptors, Am. J. Physiol. 238, E552. Bukowiecki, L., N. Follea, J. Vallieres and J. Leblanc, 1978, fl-Adrenergic receptors in brown adipose tissue: characterization and alterations during acclimation of rats to cold, European J. Biochem. 92, 189. Cheng, Y.C. and W.H. Prusoff, 1973, Relationship between the inhibition constant (K i) and the concentration of inhibitor which causes 50 percent inhibition (Is0) of an enzymatic reaction, Biochem. Pharmacol. 22, 3099. Feist, D.D., 1983, Increased a-adrenergic receptors in brown fat of winter-acclimatized Alaskan voles, Am. J. Physiol. 245, R357. Foster, D.O., 1984, Evidence of a mediatory role of brown adipose tissue in the calorigenic response of cold-acclimated rats to noradrenaline, Can. J. Physiol. Pharmacol. 52, 1051. Girardier, L., 1983, Brown fat: An energy dissipating tissue, in: Mammalian Thermogenesis, eds. L. Girardier and M.J. Stock (Chapman & Hall, London) p. 50. Landsberg, L. and J.B. Young, 1983, Autonomic regulation of thermogenesis, in: Mammalian Thermogenesis, eds. L. Giradier and M.J. Stock (Chapman & Hall, London) p. 99. Minneman, K.P., L.R. Hegstrand and P.B. Molinoff, 1979, Simultaneous determination of beta-1 and beta-2 adrenergic receptors in tissues containing both receptor subtypes, Mol. Pharmacol. 16, 34. Minneman, K.P. and P.B. Molinoff, 1980, Classification and quantitation of B-adrenergic receptor subtypes, Biochem. Pharmacol. 29, 1317. Mohell, N., J. Nedergaard and B. Cannon, 1983, Quantitative
differentiation of a 1- and /~-adrenergic respiratory responses in isolated hamster brown fat cells; evidence for the presence of an al-adrenergic component, European J. Pharmacol. 93, 183. Nahorski, S.R., 1981, Identification and significance of betaadrenoreceptor subtypes, Trends Pharmacol. Sci. 4, 95. Nedergaard, J. and O. Lindberg, 1982, The brown fat cell, Int. Rev. Cytol. 74, 187. Raasmaja, A., N. Mitchell and J. Nedergaard, Increased a 1adrenergic receptors density in brown adipose tissue of cold-acclimated rats and hamsters, European J. Pharmacol. (in press, a). Raasmaja, A., N. Mitchell and J. Nedergaard, Increased a 1adrenergic receptor density in brown adipose tissue of cafeteria-fed rats, Biosci. Rep. (in press, b). Rothwell, N.J. and M.J. Stock, 1981, Influence of noradrenaline on blood flow to brown adipose tissue in rats exhibiting diet-induced thermogenesis, Pfli~gers Arch. 389, 237. Rothwell, N.J. and M.J. Stock, 1983, Diet-induced thermogenesis, in: Mammalian Thermogenesis, eds. L. Girardier and M.J. Stock (Chapman & Hall, London) p. 208. Rothwell, N.J. and M.J. Stock, 1984a, Brown adipose tissue, in: Recent Advances in Physiology - 10, ed. P.F. Baker (Churchill Livingstone, Edinburgh) p. 208. Rothwell, N.J. and M.J. Stock, 1984b, Effects of denervating brown adipose tissue on the response to cold, hyperphagia and noradrenaline treatment in the rat, J. Physiol. 355, 457. Rothwell, N.J., M.J. Stock and D. Stribling, 1982, Diet-induced thermogenesis, Pharmacol. Ther. 17, 251. Rothwell, N.J., M.J. Stock and D.K. Sudera, 1985, fl-Adrenoreceptors in rat brown adipose tissue: proportions of 13I and fl2-subtypes, Am. J. Physiol. 248, E397. Svoboda, P., J. Svartengren and Z. Drahota, 1984, The functional and structural reorganisation of the plasma membranes of brown adipose tissue induced by cold acclimation of the hamster, II, fl-Adrenergic receptor, Mol. Physiol. 5, 221. Svoboda, P., J. Svartengren, M. Snochowski, J. Houstek and B. Cannon, 1979, High number of high affinity sites for (-)3(H)-dihydroalprenolol in isolated hamster brown fat cells, European J. Biochem. 102, 203.