54

Brain Research. 512 119911) 54 5~J Elsevie~

BRES 15288

Obesity-prone and -resistant rats differ in their brain [3H]paraminoclonidine binding Barry E. Levin Neurology Service 1127), Veterans Administration Medical Center E. Orange, NJ 07019 (U.S.A.), Department of Neurosciences, New Jersey Medical School, Newark, NJ 07103 (U.S.A.)

(Accepted 8 August 1989) Key words: Adrenoceptor; a-Receptor; Diet-induced obesity; Brain catecholamine; Sympathetic nervous system; Glucose; Insulin; Norepinephrine; Prazosin

Half the rats fed a high-energy diet develop diet-induced obesity (DIO); the remainder are diet-resistant (DR). Since a-adrenoceptors modulate both food intake and body weight, this study was conducted to identify potential differences in brain a-receptor binding which might predispose some animals to become DIO (DIO-prone) and others DR (DR-prone) when fed a high energy diet. DIO-prone rats can be prospectively identified by high and DR-prone rats by a low plasma norepinephrine (NE) response to i.v. glucose. Here 28 chow-fed rats were tested for glucose-induced NE release and the 6 highest and 6 lowest plasma NE responders were identified as being most likely to be DIOand DR-prone, respectively. Binding to brain a-adrenoceptors was studied in these 12 rats by receptor autoradiography using 1 nM [3H]prazosin (PRZ; ax-) and 1 nM [3H]paraminoclonidine (PAC; a2-). There were no differences in [3H]PRZ binding in any of 18 brain areas examined. However, DIO-prone [3H]PAC binding was only 14-39% of DR-prone levels in 9 areas including 4 amygdalar nuclei, the lateral area, dorsoand ventromedial nuclei of the hypothalamus, median eminence and medial dorsal thalamic n. Although it is unclear whether this widespread decrease in [3H]PAC binding implicates brain a2-adrenoceptors in the pathophysiology of DIO, it does correlate with a phenotypic marker (increase glucose-induced NE release) which predicts the subsequent develoment of DIO on a high-energy diet.

INTRODUCTION Obesity has been viewed largely as a p r o b l e m of p e r i p h e r a l metabolic dysfunction 36. Increasingly, however, n u m e r o u s abnormalities of central and peripheral nervous system function have been identified in obese individuals and animals. Some obese humans have decreased sympathetic nervous system function 32 while rats with diet-induced obesity ( D I O ) show selective decreases in pancreatic sympathetic tone associated with hyperinsulinemia and insulin resistance 2°'25. In addition to decreased pancreatic sympathetic tone and hyperinsulinemia, genetically obese Z u c k e r rats also have more widespread abnormalities of peripheral sympathetic function 24. Clearly, the central nervous system plays some role in mediating these sympathetic defects. The association of obesity, abnormalities of sympathetic function and glucose metabolism led to studies which examined this interrelationship. Only about half of the adults male S p r a g u e - D a w l e y rats chronically fed a high energy diet develop D I O 19'2°'25. The r e m a i n d e r become diet resistant ( D R ) , i.e., they gain the same amount of carcass lipid and body weight on such diets as chow-fed controls. This b i m o d a l weight gain pattern is not seen if rats are kept on

chow for up to 9 months of age 2° and thus represents a diet-induced induction of a specific pattern of weight gain and carcass compositional changes. H o w e v e r , in o r d e r to identify underlying abnormalities which might play a causative role in the d e v e l o p m e n t of D I O , one must first prospectively be able to separate animals likely to develop D I O ( D I O - p r o n e ) from those likely to develop D R ( D R - p r o n e ) before they are actually exposed to a dietary challenge. We have found that these genotypic differences can be identified prospectively using the fact that rats increase their plasma norepinephrine (NE) levels in response to i.v. glucose 21. D I O - p r o n e rats show high levels of plasma norepinephrine (NE) to i.v. glucose, while D R - p r o n e rats have little NE response 19. Thus, using the interrelationship of glucose metabolism and sympathetic function as a phenotypic m a r k e r for the D I O - and D R - p r o n e states, rats can be investigated before dietary exposure for factors which might predispose them to these two patterns of weight gain which are only seen if they are e x p o s e d to a high-energy diet 2°. Brain catecholamines play an i m p o r t a n t role in the regulation of food intake, glucose m e t a b o l i s m and body weight gain 7"14"1s'26"2~'35"38. In particular, a - a d r e n o c e p t o r s

Correspondence: B.E. Levin, Neurology Service (127), VA Medical Center, E. ()range, NJ I)7019, U.S.A.

0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

55 play an i m p o r t a n t role in t h e s e processes 3'7-1°'15'27'38'42. T h e p r e s e n t studies w e r e u n d e r t a k e n to test the h y p o t h esis that the t e n d e n c y to b e c o m e D I O exposed

to

high-energy

diets

of D R

when

is a m a n i f e s t a t i o n

of

u n d e r l y i n g g e n e t i c d i f f e r e n c e s in a - a d r e n o c e p t o r f u n c t i o n in b r a i n areas critical to the r e g u l a t i o n of b o d y wight. T h u s , c h o w - f e d rats w e r e i d e n t i f i e d as D I O - or D R p r o n e a c c o r d i n g to t h e i r high o r low p l a s m a N E r e s p o n s e to i.v. g l u c o s e , r e s p e c t i v e l y , and w e r e t h e n e x a m i n e d for in v i t r o a - a d r e n o c e p t o r binding by r e c e p t o r a u t o r a d i o graphic methods. MATERIALS AND METHODS

Animals and experimental manipulation Twenty-eight male Sprague-Dawley rats (Charles River Labs) at 376--484 g were tested initially. They were housed on a 12:12 h light-dark schedule at 21-23 °C with Purina rat chow and water provided ad libitum for at least 2 weeks prior to testing. Animals were anesthetized with Chloropent (3 ml/kg). PE 50 catheters were inserted into the right atrium via the right jugular vein23. Distal ends of the catheters were tunneled to the back of the neck, filled with heparinized saline, plugged and flushed twice a day with 500U heparinized saline. When body weight and food intake had regained pre-operative levels, rats were tested for their response to intravenous glucose. Food was removed at 08.00 h and the catheter attached to an additional length of tubing which ran outside the home cage for remote sampling from the freely moving animal. After 4 h, a baseline venous sample (0.5 ml) was taken from the atrial catheter and then 0.5-1.0 ml of 50% glucose (1 g/kg) was given as an i.v. bolus. The tubing was flushed and further 0.5 ml samples were taken at 2, 5, 10, 30 and 60 min. Plasma volume was maintained by reinfusion of an equal amount of 0.9% saline and the washed red cells were returned to the animals after the 30 and 60 min samples. Following these injections, the catheters were tied off and the animals allowed 1 week to recover.

MgCI2, 0.01% ascorbic acid, 10/~M pargyline, pH 7.6. Sections were then incubated with 1 nM [3H]PAC (45.6 Ci/mmol; New England Nuclear) for 60 min at 23-25 °C. This was followed by two 10 min ice-cold buffer washes, a dip in ice-cold distilled water and drying. Non-specific binding was defined as binding of 1 nM [3H]PAC seen in the presence of 100/~M NE 4~. Labeled sections were apposed to Ultrofilm for 10-12 weeks.

Autoradiographic image analysis The resulting autoradiograms were analyzed with the DUMAS computer-assisted image analysis system (Drexal University). Cresyl violet-stained sections were superimposed upon the corresponding autoradiogram for anatomical definition and outlining of the boarders of the individual areas according to the atlas of Paxinos and Watson 3l. Brain areas were chosen for reading based on their known involvement in autonomic nervous system activity, food intake and body weight regulation or because there were especially high levels of binding present in that area for a given ligand. Density readings were taken within these areas and converted to binding activity using 3H-embedded polymer standards (American Radiochemicals). For each brain region or nucleus examined in each rat, 4-8 readings were averaged.

Statistics Area under the curve for plasma NE, glucose and insulin were calculated using the trapazoidal rule 12 comparing change postinjection to baseline levels. Out of the initial 28 rats tested, the 6 with the highest areas under the NE curve were assigned as DIO-prone and the 6 lowest as DR-prone 19. Specific binding for [3H]PRZ and [3H]PAC was determined for multiple brain areas by subtracting activity present in the presence of excess unlabeled competing ligand from total binding in its absence. Comparisons between DR- and DIO-prone rats were made for all parameters by one-way analysis of variance with further analysis by NewmanKeuls test for post-hoc comparisons at the P < 0.05 level. Correlations were done using Pearson's correlation coefficient. RESULTS R a t s w e r e c h o s e n p r o s p e c t i v e l y by t h e i r p l a s m a N E r e s p o n s e to an i n t r a v e o u s glucose load. F o l l o w i n g 1 g/kg

Assay of plasma constituents Blood was placed into ice-cold, heparinized tubes and the plasma removed. Samples for plasma catecholamine determinations were brought to 0.2 N perchloric acid and frozen for up to 4 weeks until assayed by radioenzymatic assay 23. This assay has an interassay variability of 5-7% and intra-assay variability of 7-8% for NE. Plasma insulin levels were assayed by double antibody radioimmunoassay using authentic rat insulin (Novo) as the standard 16 and glucose by automated glucose oxidase method (Beckman).

glucose, i.v., areas u n d e r the p l a s m a g l u c o s e c u r v e for the entire g r o u p of 28 rats r a n g e d f r o m 5912 to 7178 mg/dl/60 min and for insulin f o r m 500 to 2962 ~tU/ml/60 min. N E areas in r e s p o n s e to i.v. glucose r a n g e d f r o m - 9 7 5 0 to 72366 pg/ml/60 min. T h e r e w e r e , h o w e v e r , no c o r r e l a t i o n s a m o n g b o d y w e i g h t , insulin, glucose o r N E areas for the overall g r o u p of 28 rats. F o r c t - a d r e n o c e p t o r

Receptor autoradiography

studies, the 6 highest N E r e s p o n d e r s w e r e d e s i g n a t e d as

The brains from the 6 highest and 6 lowest NE responders (see Statistics) were removed following decapitation and quick frozen on dry ice. They were stored for no more than 2 weeks prior to sectioning. Brain sections (20 /~m) were cut alternately for [all] prazosin (PRZ) 33 and paraminoclonidine (PAC) 41 binding, placed on gel-coated slides and processed by standard procedures 33m'4a. a~- and a2-adrenoceptor binding was determined as previously described 3am'42. Briefly, [3H]PRZ binding to al-receptors was run with a 5 min preincubation in 50 mM Tris, 10 nM Na2EDTA, pH 7.4 buffer and then incubated with 1 nM [3H]PRZ (82.0 Ci/mmol; New England Nuclear), for 45 min at 23-25 °C. This was followed by two 5 min washes in ice-cold buffer, a dip in ice-cold distilled water and drying with cool air. Non-specific binding was defined as binding of 1 nM [3H]PRZ seen in the presence of 100 /~M phentolamine 33. Sections were apposed to LKB Ultrofilm for 6-8 weeks [3H]PAC binding to high-affinity a2-receptors was assessed in brain sections with a 30 min preincubation in 170 mM Tris, 10 mM

D I O - p r o n e and the 6 l o w e s t N E r e s p o n d e r s as D R - p r o n e (Fig. 1) in k e e p i n g with p r e v i o u s studies 19 s h o w i n g that such r e s p o n s e s (but n o t insulin o r glucose r e s p o n s e s ) predict the s u b s e q u e n t d e v e l o p m e n t of t h e D I O of D R states after 3 m o n t h s on a h i g h - e n e r g y diet. A r e a s u n d e r the N E c u r v e for the 6 high r e s p o n d e r s

(DIO-prone;

27,803 + 4095 pg/ml/60 m i n ) w e r e significantly g r e a t e r than t h o s e for the 6 low r e s p o n d e r s ( D R - p r o n e ; - 2 3 3 0 + 1127 pg/ml/60 m i n ; P = 0.001). W h e n c h o s e n in this way, t h e r e w e r e no o t h e r significant d i f f e r e n c e s b e t w e e n t h e s e g r o u p s with r e s p e c t to b o d y w e i g h t ( D I O = 431 _+ 11 g vs D R = 444 + 13 g) or the areas u n d e r t h e glucose ( D I O = 5953 + 706 vs D R = 5307 + 843 mg/dl/60 rain) o r

56 insulin ( D 1 0 = 1335 + 354 vs D R = 1077 + 163 pU/ml/60 min) curves for these 12 rats following 1 g/kg of i.v. glucose (Fig. 1). Neither was there a significant correlation between NE areas and either insulin, glucose or body weight for the 12 final rats used in this study. There were no differences in specific binding of 1 nM [3H]PRZ between D I O - and D R - p r o n e rats in any of the brain regions examined (Fig. 2). There was a tendency

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towards lower [3H]PRZ binding in the median eminence (ME) of D I O - p r o n e rats but this did not reach statistical significance (P = 0.064) because of the highly variable levels of binding in this area. Neither were there significant correlations between [3H]PRZ binding and body weight, NE, glucose or insulin areas. In contrast to the lack of difference in [3H]PRZ binding, 1 nM [3H]PAC binding was reduced in the brains of D I O - p r o n e as compared to DR-prone rats in 9 of the 16 areas assessed for specific binding of this ligand (Fig. 3). Lower levels of [3H]PAC binding in D I O - p r o n e , as a percent of D R - p r o n e rats, occurred in the dorsomedial n. (DMN; 26%), lateral area (LH; 35%), ME (19%) and ventromedial n. (VMN; 26%) of the hypothalamus, medial ( M A N ; 23%), basolateral (ABL; 32%), basomedial ( A B M ; 39%) and central (ACN; 14%) amygdalar nuclei and the medial dorsal thalamic n. (MDT; 32%). There were no differences in [3H]PAC binding found in either the paraventricular hypothalamic n. (PVN) or n. tractus solitarius (NTS) between D I O and D R - p r o n e rats. When data from both D I O - and D R - p r o n e rats were analyzed together, there were

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Fig. 1. Plasma glucose, insulin and norepinephrine (NE) responses to an intravenous glucose load (1 g/kg). Of 28 rats initially tested, the 6 highest (DIO-prone) and 6 lowest (DR-prone) NE responders were selected for brain a-adrenoceptor binding. Data from their plasma responses are presented here. Data are mean + S.E.M. (vertical bars).

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Fig. 2. Rats represented in Fig. 1 were assayed for specific brain [3H]prazosin binding (1 nM) in groups of 6 DR- and 6 DIO-prone animals. Data are mean + S.E.M. specific binding (fmol/mg protein) in: hypothalamic areas- All, anterior hypothalamus; DMN, dorsomedial n.; ME, median eminence; LH, lateral hypothalamic area; PVN, paraventricular n.; VMN, ventromedial n.; ARC, arcuate n.; amygdalar areas- ACN, central n.; ABL. basolateral n.; ABM, basomedial n.; neocortical area- SmI, layer IV of first somatosensory cortex; thalamic areas- VPM, ventroposteromedial n.; MDT, medial dorsal n.; VPL, ventroposterolaterm n., REN, n. reuniens; and medullary area- NTS, n. tractus solitarius.

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Fig. 3. The same rats as shown in Figs. I and 2 were assayed for brain [3H]paraminoclonidine binding (1 nM) in groups of 6 DR- and DIO-prone animals. Data are mean + S.E.M. specific binding (fmoi/mg protein). Abbreviations are the same as Fig. 2 plus: MAN, medial amygdalar n.; MVN, medial vestibular n. *P ~

Obesity-prone and -resistant rats differ in their brain [3H]paraminoclonidine binding.

Half the rats fed a high-energy diet develop diet-induced obesity (DIO); the remainder are diet-resistant (DR). Since alpha-adrenoceptors modulate bot...
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