Fatty acid mobilization to 2-deoxyglucose is blocked by globus pallidus lesions

Journal of the Autonomic Nervous System, 11 (1974) 161-171

161

Elsevier JAN 00373

Fatty acid mobilization to 2-deoxyglucose is blocked by globus pallidus lesions * M a r k W. G u n i o n 1, Carlos V. Grijalva 1, D o n a l d N o v i n 1 and F. Xavier Pi-Sunyer 2 I Department of Psychology and Brain Research Institute, University of California, Los Angeles, Los Angeles, CA 90024 and 2 Division of Endocrinology, St. Luke's- Roosevelt Hospital Center, Columbia University, New York, N Y 10025 (U.S.A.)

(Received May 17th, 1983) (Revised version received March 23rd, 1984) (Accepted April 15th, 1984)

K e y words." glucose - free fatty acids - 2-deoxyglucose - globus pallidus - b r a i n -

i n s u l i n - glycerol - rat

Abstract

Lesions of the lateral h y p o t h a l a m u s or discrete bilateral transections on the lateral border of the lateral h y p o t h a l a m u s disrupt the m o b i l i z a t i o n of metabolic fuels to i n t r a p e r i t o n e a l a d m i n i s t r a t i o n of the glucose analog 2-deoxyglucose. T o better define the pathways involved in these responses, the effects of globus pallidus lesions o n b o d y fuel m o b i l i z a t i o n were investigated. G l o b u s "pallidus lesions blocked the increase in p l a s m a free fatty acids n o r m a l l y caused b y 2-deoxyglucose, b u t did n o t d i m i n i s h the c o n c o m i t a n t hyperglycemia. The data indicate that a p a t h w a y r u n n i n g t h r o u g h the globus pallidus, crossing the dorsoanterolateral h y p o t h a l a m i c border, a n d t u r n i n g caudally in the dorsolateral h y p o t h a l a m u s is i m p o r t a n t i n the i m m e d i a t e release of free fatty acids following 2-deoxyglucose a d m i n i s t r a t i o n .

* A preliminary report of this work was presented at the symposium 'The Neural Basis of Feeding and Reward', Los Angeles, 1981 Correspondence: M.W. Gunion, C.U.R.E., Bldg. 115, Rm. 203, Wadsworth V.A., Los Angeles, CA 90073, U.S.A. 0165-1838/84/$03.00 © 1984 Elsevier Science Publishers B.V.

162 Introduction Under conditions of acute stress, body fuel availability is assured by substantial increases in circulating glucose and free fatty acid (FFA) concentrations. While it has recently become clear that the brain exerts control over these metabolic substrates, relatively little is known about the brain regions and systems involved in this control. Much of the work to date has centered on the hypothalamic level. Initial studies at this level showed that cutting along the rostral hypothalamic border suppressed the mobilization of FFA both to 2-deoxyglucose (2DG), and to more physiological stressors, without altering glucose release [9,25]. This suggested that rostro-caudal fibers of passage were important in the release of FFA, but not glucose, to these stimuli. Later studies specifically implicated the ventromedial hypothalamic area in the mobilization of fats from storage. Lesions centered in this area suppressed F F A increases to several different types of stress [20] and blocked the normal decrement in fat pad weight during fasting [2]. In neither study was the level of blood glucose affected. The lateral hypothalamus (LaH) has also been examined. Results of preliminary work showed that LaH lesions blocked FFA but not glucose increases due to 2DG [25]. This effect was confirmed in a full-scale study in another laboratory [8]. In the latter report it was also shown that parasagittal knife cuts along the entire lateral border of the hypothalamus blocked both the FFA and glucose increases to 2DG [8]. This result suggested that fibers crossing the lateral border of the hypothalamus at some rostro-caudal level are important in regulating the glucose and FFA responses to 2DG. This appeared to be the first central manipulation found to block the glucose response to 2DG. Lesions of the globus pallidus (GP) on the anterolateral hypothalamic border have produced effects on food intake and body weight quite similar to those caused by LaH lesions or knife cuts along the lateral border of the LaH (i.e. transient aphagia/hypophagia, maintenance of a lowered body weight, and loss of the feeding response to acute glucoprivation [12,16,18,19]). Presumably this is because GP lesions disrupted fibers leaving a n d / o r entering the LaH, fibers which were also disrupted by LaH lesions or knife cuts. The experiment reported here was conducted to test the possibility that destruction of the GP can produce metabolic deficits similar to those that were seen in LaH lesioned rats tested with 2DG.

Methods

Subjects and housing Male albino rats (Simonsen, Gilroy, CA), weighing 333-404 g at surgery, were housed singly in stainless steel cages under a 12/12 h light-dark cycle. Food pellets (Lab Blox, Wayne, Chicago, IL) and tap water were available ad libitum except as noted below.

Surgery Animals were anesthetized with sodium pentobarbital (50 m g / k g i.p., Nembutal;

163 Abbott Labs, North Chicago, IL). Bilateral G P lesions were made by passing a 2.0 mA direct anodal current for 20 s through a stainless steel electrode (size 00 insect pin insulated with Epoxylite no. 6001-M except for the cross section of the cut tip; Epoxylite, South E1 Monte, CA). The circuit was completed by clamping the cathode to a saline-soaked gauze pad wrapped around the tail. The electrode tip was placed 0.9 m m posterior to bregma, 2.6 m m to each side of the midline, and 5.7 m m below the dura, with the skull flat between bregma and lambda. Control groups received sham operations consisting of incision and closure of the scalp. Animals were deprived of food and water for 24 h before and after surgery. Procedure and groups

Baseline body weights were obtained during the 5 days prior to surgery. Three groups were then formed, which were matched for baseline body weights. Rats in one of the groups were subjected to G P lesions, while rats in the remaining two groups received sham surgery. One of the groups receiving sham surgery was designated the normal weight control group, and the other was designated the restricted weight control group. Following surgery, both the normal weight control group and the GP-lesioned group received food and water ad libitum until sacrifice. Rats in the restricted weight control group also received water ad libitum until sacrifice, but were given limited amounts of food once daily in the middle of the light period. This group was used as a control for any effects of the hypophagia, hypodipsia, and body weight loss which often follow G P lesions. Enough food was delivered so that the mean body weight of the restricted weight control group would be the same as the mean body weight of the GP-lesioned group. Hypophagic lesioned rats typically began eating pellets from the cage floor 2 - 3 days after surgery. Animals taking more than 3 days to recover food intake were offered wet pellet mash or wet chocolate chip cookie mash, dry pellets, and water in Petri dishes until pellet intake and normal drinking returned. By the 39th day after surgery all lesioned rats had maintained stable food intakes for over 30 days. At this point, each of the 3 groups was divided into halves, matched for body weight; one half of each group would be given 2 D G at sacrifice, and the other half would receive saline. The rats were killed by decapitation on the 40th or 41st day after surgery during the 4th-6th h of the light period. They had food ad libitum for the first 5 h of the preceding dark period; food was withheld during the remaining 11-13 h before sacrifice. Exactly 30 min before death, they were given intraperitoneal injections of 2 D G (500 m g / 2 m l / k g ; Sigma, St. Louis, MO) or saline (0.9%; 2 ml/kg), as appropriate. Cervical blood was collected on ice, allowed to clot, and centrifuged at 4 ° C ; the serum was saved and stored at - 1 5 °C. Brains from lesioned rats were removed and placed in 10% formaldehyde. A ssays

Plasma glucose was assayed by a hexokinase method [22] and F F A were measured by a copper complex method [6]. Glycerol levels were assayed by a modified enzymatic technique [4], and insulin was measured by radioimmunoassay using rat insulin standards [10].

164

165

Statistical analysis One-tail t-tests were planned a priori to test the effect of 2 D G on glucose and F F A in each surgical group, and to examine the effect of GP lesions on body weight. A two-tail t-test was planned a priori to compare body weights of the restricted weight control and G P groups. All other differences were examined by Tukey's H S D test [14] following weighted means least squares regression.

Results

Histology The G P lesions resembled those described elsewhere (Fig. 1) [16]. Referring to the atlas of Krnig and Klippel [15], the average lesion extended from plates 17 to 29 (range, plates 16 to 30) with the center of the lesions located between plates 20 and 25. Rostrally, the lesions damaged the ventromedial portion of the caudate-putamen. At the rostro-caudal midpoint, the lesions included a majority of the GP, a large portion of the internal capsule, and occasionally the stria terminalis medially or the caudate-putamen laterally. Posteriorly, the lesions typically encroached upon the reticular thalamic nucleus, the superior thalamic radiation, and the internal capsule. There were no detectable differences in the center or extent of the lesions among lesioned rats given saline or 2DG. After discarding the data from rats having lesions not falling within the above description, the cell nos. were: GP-saline, 9; GP-2DG, 11; normal weight controlsaline, 8; normal weight control-2DG, 8; restricted weight control-saline, 7; and restricted weight control-2DG, 8.

Body weight Fig. 2 shows the changes in body weight which occurred postoperatively. As expected, rats with GP lesions lost weight for several days after surgery, then began a slow recovery. By day 15, GP-lesioned rats showed a normal rate of weight gain, although their body weights remained consistently below those of the normal weight controls. On day 39, the GP-lesioned rats weighed 92% of the normal weight controls (t 30 = 3.619, P < 0.01). The mean body weight of the restricted weight control group was not significantly different from that of GP lesioned group on that day (t 33 = 0.955, P > 0.3).

Metabolic measures G P lesions abolished the rise in serum F F A caused by 2 D G administration (Fig. 3; 6% mean increase, t 18 = 0.464, P > 0.2). Both the normal weight and restricted

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Fig. 1. Reconstructionsof the globus pallidus lesions at the rostrocaudal midpoint of each lesion. Sections in the left column axe from saline-treated rats, while those in the right column are from 2DG-treated rats. The number to the right of each section corresponds to the K~Snigand Klippel [15] atlas plate on which the reconstruction was drawn.

166

weight control groups showed substantial increases in F F A following 2 D G (normal weight control: 26%, t 14 = 2.091, P < 0.05; and restricted weight controls: 66%, t 13 = 2.724, P < 0.01). Regression analysis showed a significant effect of drug ( F 1,45 = 14.4; P < 0.0004) and a drug × surgery interaction ( F 2,45 = 3.41; P < 0.05), although no reliable effect of surgery itself ( F 2,45 = 2.75, P < 0.08). Tukey's test showed no differences a m o n g the saline (basal) values (all P > 0.05). 460

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167 G P lesions d i d n o t affect the b l o o d glucose response to 2 D G a d m i n i s t r a t i o n (Fig. 4). 2 - D e o x y g l u c o s e significantly elevated b l o o d glucose in all 3 surgical t r e a t m e n t s ( n o r m a l weight control: t 14 = 4.20, P < 0.0005; globus pallidus: t 18 = 4.38; P < 0.0005; restricted weight controls: t 18 = 4.039, P < 0.0005). Regression analysis s h o w e d no effect of surgery ( F 5,45 = 0.39, P > 0.6) on glucose levels n o r a n y i n t e r a c t i o n o f surgery a n d d r u g ( F 2,45 = 0.22, P > 0.8). A s expected, T u k e y ' s test showed no differences a m o n g the saline-treated g r o u p s (all P > 0.05). T h e insulin a n d glycerol d a t a are c h a r a c t e r i z e d m o r e b y the a p p e a r a n c e o f t r e n d s t h a n b y statistical reliability. Both c o n t r o l groups showed a small m e a n increase i n insulin after 2 D G , while the G P lesioned g r o u p showed a small m e a n decrease ( T a b l e I). Regression analysis showed a statistically reliable effect of surgery ( F 2,45 = 5.88; P < 0.006), b u t no reliable effect of d r u g t r e a t m e n t ( F 1,45 = 1.60; P > 0.2), a n d no reliable surgery x d r u g i n t e r a c t i o n ( F 2,45 = 2.63; P > 0.09). T h e d a t a were further e x a m i n e d by c o n v e r t i n g the insulin values from rats given 2 D G to a p e r c e n t a g e of the m e a n of s a m e - s u r g e r y saline treated rats. A statistically reliable effect of surgical t r e a t m e n t was o b t a i n e d after o n e - w a y regression of these percentages ( F 2,24 = 4.20; P < 0.03). This suggests the presence of an i n t e r a c t i o n n o t clearly seen in the raw data; that is, that G P lesions b l o c k e d a n y increase in insulin which m a y have o c c u r r e d after 2 - D G . T h e r e was no c h a n g e in serum glycerol levels after 2 D G ( T a b l e I). R e g r e s s i o n analysis of the raw glycerol values showed no effect of surgical t r e a t m e n t ( F 2,45 = 1.00; P > 0.3) or d r u g a d m i n i s t r a t i o n ( F 1,45 = 0.15; P > 0.7), a n d no surgery x d r u g i n t e r a c t i o n ( F 2,45 = 0.18; P > 0.8). Even when the d a t a from 2 D G - t r e a t e d a n i m a l s were a n a l y z e d as a p e r c e n t a g e of the m e a n of s a m e - s u r g e r y saline rats, n o significant effect o f surgical t r e a t m e n t a p p e a r e d ( F 2,24 = 0.43, P > 0.6).

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168 TABLE I G R O U P INSULIN A N D GLYCEROL VALUES. DATA ARE PRESENTED AS THE MEAN + S T A N D A R D ERROR. Sal, saline; 2DG, 2-deoxyglucose. Surgery Normal weight control Globus pallidus Restricted weight control

Drug

Insulin ( n g / m l )

Glycerol (mg/dl)

Sal 2DG Sal 2DG Sal 2DG

Mean + SEM 0.65:0.1 0.85:0.1 0.8 5:0.1 0.5 + 0.1 1.0 + 0.2 1.65:0.4

Mean + SEM 1.1 + 0.1 1.0 5:0.1 0.9 5:0.1 0.9 5:0.1 1.2 + 0.2 1.1 +0.2

Discussion Lesioning the G P abolished F F A mobilization following intraperitoneal administration of 2DG. In combination with previously published reports, this finding allows some preliminary conclusions about the pathways of fibers concerned with F F A mobilization as they transit the hypothalamus. Prior to this study, it was known that these fibers must transit the LaH, since lesions of the L a H blocked F F A release to 2 D G [8,25]. Further, it seemed that these fibers must cross the lateral border of the hypothalamus, as knife cuts along the lateral borders of the L a H also blocked F F A release to 2 D G [8]. On the basis of the data presented here, it now appears that these fibers enter the G P after crossing the lateral border of the LaH. Whether these fibers terminate in the G P or merely traverse this area (or both) remains to be determined. This suggested route for fibers involved in FFA release must be considered in light of two other findings. First, previous work has shown that damage along the anterior border of the hypothalamus blocks FFA mobilization to 2 - D G [9,25]. In these studies, a cut was made along the anterior border of the hypothalamus just caudal to the optic chiasm. It is possible that these cuts transected fibers crossing the hypothalamo-pallidal border. Unfortunately, review of the histological descriptions and figures contained in those reports does not clearly indicate whether or not this was the case. Thus, this point remains unresolved. Second, it has been suggested that the ventromedial hypothalamic area is involved in F F A release. This suggestion is based on a partial suppression of F F A mobilization to 2 D G following electrolytic destruction in this region [20]. It is difficult to judge the extent of neural destruction in that study, however, because no histological assessment was provided in the report. Experience in this laboratory with the same current parameters and type of electrode suggests that the effective lesions in that study probably penetrated far beyond the ventromedial hypothalamus and the fornix, and well into the LaH. In a more recent study, the effect of lesions in the ventromedial hypothalamus on F F A release has been reexamined [21]. Histological examination showed that the lesions in this study destroyed the entire ventromedial hypothalamus but did not extend

169 beyond that region. F F A mobilization following intraperitoneal administration of 2 D G was not impaired by these lesions. This clearly suggests that ventromedial hypothalamic lesions in the first study [20] suppressed F F A release to 2 D G only because they encroached upon the LaH. The fact that L a H lesions are completely effective in blocking F F A release [8] while the (presumably) very large ventromedial hypothalamic lesions gave only a partial effect [20], is consonant with this hypothesis. Thus, there is good reason to regard ventromedial hypothalamic involvement in the F F A release to 2 D G with some scepticism. The brief food deprivation before sacrifice caused higher control levels of serum F F A than expected (normal rats fed ad lib would have plasma levels of about 10-25 ttmol/dl, compared to about 5 2 / ~ m o l / d l for saline-treated normal controls in this experiment). This proved to be fortuitous, however, because it showed another aspect of F F A mobilization in G P lesioned rats. That is, that GP rats apparently can mobilize F F A normally to the prolonged, gradual demand of food deprivation. This contrasts with their inability to mobilize F F A (present study) or increase their food intake [19] to acute demands created by 2 D G administration. This is an interesting parallel to what is already known about the feeding response of rats with L a H lesions in similar situations. LaH-lesioned rats do not increase their food intake to the acute challenges provided by regular insulin or 2 D G administration [5,17]. However, they do increase their food intakes normally to challenges with more gradual onsets: low temperatures, protamine zinc insulin administration, or caloric dilution of the diet [13,17,24]. It is possible then, that disruption of a common system underlies the deficits in both the behavioral and metabolic responses to acute metabolic stress in these two preparations. G P lesions left intact the normal glucose mobilization to 2 D G despite the marked blockade of F F A release. This selective disruption of metabolic fuel responses is consistent with other findings. It was previously shown that L a H lesions did not alter the glucose response to 2DG, although this response was greatly impaired by knife cuts on the lateral border of the L a H [8]. This combination of findings indicates that fibers crossing the ventrolateral hypothalamic border are required for the fast release of glucose following 2DG. The knife cuts would have severed such fibers; both L a H and G P lesions would have left these fibers intact. These cuts seem to be the only forebrain manipulation which disrupts the glucose response to 2DG. The knife cuts along the rostral hypothalamic borders completely blocked F F A release while leaving the glucose rise to 2 D G intact [9]. The same effect followed the very large ventromedial hypothalamic lesions which diminished F F A release (Ref. 20; the study using lesions restricted to the ventromedial hypothalamus did not report the glucose response to 2 D G in these animals). Neither insulin nor glycerol rose appreciably in rats treated with 2-DG. A decrease in circulating insulin, or a lack of a rise in insulin levels, in the face of hyperglycemia is the norm when the hyperglycemia is induced by 2 - D G (e.g. refs. 3, 7, 20, 23). This is due to a release of adrenal catecholamines caused by 2 D G which inhibits insulin release [3,7,11,23]. The absence of a rise in glycerol levels is mildly surprising, given that most manipulations which increase F F A levels also increase glycerol. It is not known what significance this finding might have.

170 A c a u t i o n a r y n o t e m u s t b e r a i s e d at t h i s j u n c t u r e . B e c a u s e s a m p l e s w e r e t a k e n o n l y a t o n e t i m e p o i n t i n t h i s e x p e r i m e n t , we d o n o t k n o w w h e t h e r G P l e s i o n s p e r m a n e n t l y b l o c k e d t h e F F A r e s p o n s e t o 2 D G , o r w h e t h e r t h e y m e r e l y d e l a y e d it beyond the time of sampling. The same can be said of LaH lesions and knife cut e f f e c t s o n t h e h y p e r g l y c e m i c r e s p o n s e to 2 - D G [8]. F u r t h e r w o r k t o d e t e r m i n e t h e t i m e c o u r s e o f t h e s e e f f e c t s w o u l d b e h e l p f u l , s i n c e it w o u l d d e l i n e a t e t h e t y p e o f effect (delay or permanent blockade) these manipulations produce. In summary, lesions of the GP disrupted the release of FFA caused by injection of 2DG. The same lesions did not affect the hyperglycemia caused by the same injections. In combination with previous data, these results suggest that a fiber s y s t e m t r a v e l i n g t h r o u g h t h e G P a n d e n t e r i n g t h e a n t e r o d o r s a l L a H is c r u c i a l in t h e r a p i d r e l e a s e o f F F A to 2 D G .

Acknowledgements W e w o u l d like t o t h a n k J. T e a g u e for d e m o n s t r a t i n g t h e free f a t t y a c i d a s s a y , a n d C. N o v i n f o r h e r h e l p w i t h t h e h i s t o l o g y . S u p p o r t e d b y N I H G r a n t N S 7 6 8 7 to D . N . a n d U C L A U n i v e r s i t y R e s e a r c h G r a n t 3820 t o C . V . G .

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