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Intracerebroventricular beta-endorphin increases food intake of rats
Life Sciences, Vol. 29, pp. 1429~1434 Printed in the U.S.A.
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INTRACEREBROVENTRICULAR BETA-ENDORPHIN INCREASES FOOD INTAKE OF RATSI L. David McKay2, Nancy J. Kenney, Neile K. Edens3, Robert H. Williams 4 and Stephen C. Woods Departments of Psychology and of Medicine, U n i v e r s i t y of Washington, Seattle, Washington, USA, 98195 (Received in final form July 28, 1981) Sumar~ B-Endorphin (B-END), met-enkepalin (M-ENK), and D-Ala2-N-Me 5met-enkephalinamide were administered intracerebroventricularly to rats and e f f e c t s on the i n g e s t i o n of a l i q u i d d i e t were examined. B-END s i g n i f i c a n t l y increased food intake in a halfhour t e s t at a dose of 200 n g / r a t . Lower or higher doses did not affect food intake. Neither M-ENK or the synthetic enkephalin analog affected ingestion of the liquid diet. These findings demonstrate rapid action of an endorphin on food intake administered at a lower dose than has previously been reported and suggest a s p e c i f i c i t y f o r B-END in the e n d o r p h i n e r g i c a l l y mediated hyperphagic response.
Neuropeptides have become increasingly implicated in behavioral processes (1,2). Because many neuropeptides are found both w i t h i n the nervous and the gastrointestinal systems (3,4), research has focused upon behaviors controlled by both systems, food intake in this instance. A number of peptides reported to occur in both the brain and the gut have been suggested as possible satiety f a c t o r s in t h a t the 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 these peptides j u s t prior to a meal reduces the size of that meal (5-7). Cholecystokinin (CCK) has been studied the most extensively with regard to i t s a b i l i t y to reduce meal size (5-9), and there is also compelling evidence of a s a t i a t i n g e f f e c t f o r both somatostatin (i0) and bombesin (6,11). Several reviews of the influences of neuropeptides on food intake have recently been written (5-9). The discovery in 1975 of endogenous ]igands which i n t e r a c t with the morphine or o p i a t e - s e n s i t i v e receptors of the body (12,13) has opened new approaches f o r researching controls of food intake. Of particular importance to the study of food intake is the p o s s i b i l i t y of an endogenous opiate ligand which augments meal size. An early report suggested that the opiate receptor antagonist, naloxone, decreases food intake (14), and there have been a number of replications of that early finding (15-21). Naloxone also reduces operant responding f o r food (22-24), diazepam-induced eating (25), hyperphagia 1This research was p a r t i a l l y supported by NIH research grant AM 17844 to SCW and AM 22024 to NJK. 2Requests for reprints should be addressed to L.D. McKay, Dept. of Psychology, NI-25, University of Washington, Seattle, WA 98195 Presently at the Dept. of Psychology, University of Massachusetts. Deceased
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resulting from hypothalamic damage in rats (15) and body weight gain (20). The specificity of naloxone upon food intake controlling systems was brought to question by reports that naloxone also reduces water intake in several species (15,18,26-30), as well as affecting other types of motivated behavior (31,32). Finally, at some doses, naloxone reportedly elicited nausea and led to the formation of s p e c i f i c learned t a s t e aversions (17,21,33,34). Therefore, although considerable evidence suggests that an antagonist of the endogenous opiate system reduces food intake, implying in turn that the endogenous opiates might themselves increase food intake, whether this is due to a specific effect influencing only feeding or to a more general effect is not clear. In spite of the confounding factors which make the naloxone-related data d i f f i c u l t to interpret, the findings do raise the possibility that endogenous opiates themselves might increase food intake. Margules and his colleagues, in addition to showing that naloxone reduced food intake of genetically obese rats and mice, were able to correlate the reduction of food intake with an elevation of the levels of beta-endorphin (B-END) in the pituitary (16). They suggested that the elevated levels of B-END might play a role in the etiology of the obesity which is characteristic of these animals. There is one report that the direct administration of B-END into the ventromedial hypothalamus causes a small increment of food intake (35), but that experiment lacked controls for other peptides and for other areas of the brain. There is also a recent report that the administration of B-END into the paraventricular nucleus also increased food intake in rats (36). We now report that B-END administered in a narrow dose range into the cerebral ventricles of rats caused an increased intake of a liquid diet. Administration of the same amount of B-END peripherally had no effect on food intake and its central administration had no e f f e c t on water intake. Someof these experiments were reported in a preliminary abstract (37). Methods Subjects in all experiments were naive male Wistar-derived rats (280 to 380 g) obtained from the colony maintained by the Department of Psychology of the University of Washington. They were housed in standard individual hanging cages in a temperature-controlled room which was automatically illuminated each day from 0700 to 1900 hours. The rats had continuous access to water throughout the experiments. All rats were i n i t i a l l y adapted to a mildly restrictive feeding schedule on which food (Purina p e l l e t s ) was removed each day at 0900 hours. At 1500 hours (i.e, 6 hours l a t e r ) , a l i q u i d diet consisting of 50% tap water and 50% sweetened, condensed milk (Eagle brand) was given each rat in a calibrated drinking tube for 30 minutes. At the end of that i n t e r v a l , the tubes were removed, intake was recorded, and pellets were returned until the next morning. This schedule has proven successful for studying the effects of peptides on food intake in our lab (6,7,10,37). Rats were maintained on this schedule until a stable daily intake of the liquid diet occurred (approximately 10 to 15 days). Following this adaptation period, rats were f i t t e d wih unilateral 23gauge stainless steel cannulas s t e r e o t a x i c a l l y aimed at the l e f t l a t e r a l cerebral v e n t r i c l e ( from bregma: AP -1.2; ML +1.5; and -4.0 mm ventral to dura). Obturators (30-gauge) remained in place when the cannulas were not in use. Details of the cannulation method have been reported elsewhere (38). The animals were allowed 3 to 5 days for recovery from the surgery before testing began. They remained on the 6-hour daily deprivation schedule during this time.
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P r e l i m i n a r y v e r i f i c a t i o n of access to the v e n t r i c l e by each cannula was accomplished by a behavioral test. All t e s t i n g was done in 5-day blocks, and on the f i r s t and f i f t h days each rat was screened f o r drinking in response to an i n t r a v e n t r i c u l a r (IVT) i n j e c t i o n through the cannula of i00 ng of carbachol (carbamylcholine, Sigma) in I ul of physiological saline. This treatment is known to e l i c i t s i g n i f i c a n t water consumption. Rats that did not consume at l e a s t 3 ml of water over the 20 minutes subsequent to the i n j e c t i o n on both carbachol tests were not included in the analyses. At the end of the experiments, h i s t o l o g i c a l confirmation of access to the v e n t r i c l e s was made a f t e r the brains had been fixed in formalin in situ. For a l l IVT i n j e c t i o n s , the rats were hand-held and the actual i n j e c t i o n took I0 seconds. The r a t s were returned to t h e i r cages 30 seconds l a t e r . On Days 2, 3, and 4 the l i q u i d diet was placed on the cages during the 30-second i n t e r v a l . One ul of physiological saline was administered IVT just p r i o r to the presentation of l i q u i d diet on Days 2 and 4; on Day 3, the t e s t day, B-END or some other experimental compound was administered IVT in I ul of saline. Results B-END at dose of 200 n g / r a t caused a s i g n i f i c a n t increase of food i n t a k e . The average m i l k i n t a k e on the B-END t e s t day was 10.2 C 1.0 ml (Mean + SEM) as compared to 7.5 ~ 0.8 ml on the c o n t r o l days (P
P
Thus B-END, when administered into the l a t e r a l cerebroventricle at the s p e c i f i c dose of 200 n g / r a t , caused a mean increase of 83% in the s i z e of a 30-minute meal. A number of control conditions were also run. In one experiment, rats were adapted to a 23-hour water deprivation schedule. Water intake at the end of that i n t e r v a l was not r e l i a b l y influenced by 200 ng/rat of IVT B-END. In another control experiment, rats were given 200 ng of B-END i n t r a peritoneally. There was no s i g n i f i c a n t e f f e c t on food intake (Table I). The results of these experiments suggest that B-END is acting c e n t r a l l y to i n f l u ence meal size and that the e f f e c t is not one of generalized arousal. In o t h e r r a t s , p r e l i m i n a r y t e s t i n g of the s p e c i f i c i t y of the increased food intake among various endorphins was conducted. As seen in Table I , the IVT a d m i n i s t r a t i o n of m e t - e n k e p h a l i n at doses ranging from I to I00 n g / r a t (spanning the same molar range as the B-END t e s t s ) caused no s i g n i f i c a n t change of meal s i z e . Increases of 36% f o l l o w i n g I u g / r a t of M-ENK and 14% f o l l o w i n g the I0 n g / r a t dose were a t t a i n e d , but were not s i g n i f i c a n t l y d i f f e r e n t from control values. Likewise, the IVT administration of the longera c t i n g analog of m e t - e n k e p h a l i n , D-ala2-N-Me5-Met-Enkephalinamide, had no r e l i a b l e e f f e c t on food intake (Table I). Discussion These experiments show that B-END, when administered over a narrow dose range into the l a t e r a l cerebral v e n t r i c l e , increased intake of a l i q u i d d i e t by m i l d l y food-deprived rats. These results are consistent with the reports
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TABLE I
Peptide
Route
Dose
Average Milk Intake Iml)
Control B-END
Test
IVT
20 ng
5.2 ~ 1.7
5.2 ~ 1.8
IVT
100 ng
8.2t0.8
9.1t0.8
IVT
200 ng
7.5t0.8
I0.2~I.0
IVT
1
IVT a
200 ng
8.3 t 1.5
IP
200 ng
7.0~2.3
ug
ii.4
t I.I
iI.0
~ 1.0
15.2 t 2.1 8.0t2.0
IVT
I ng
8.5 _+ 1.7
11.6 + 1.4
IVT
10 ng
11.5 + 3.4
13.1 +_ 3.4
IVT
I00 ng
12.3 + 4.0
12.8 +_ 3.1
M-ENK ANALOG IVT
2 ng
11.7 + 3.4
9.4 _+ 0.6
IVT
1 ug
5.2 + 1.9
6.8 +_ 1.1
M-ENK
Table 1. E f f e c t s of I n t r a c e r b r o v e n t r i c u l a r l y I n j e c t e d B-Endorphin (B-END), Met-Enkephalin (M-ENK) and D-Ala2-N-Me5-Met-Enkephalinamide (M-ENK ANALOG) on M i l k I n t a k e . a i s the average of f o u r independent r e p l i c a t i o n s . that i n t r a h y p o t h a l a m i c B-END increases food intake, whether i n j e c t e d i n t o the p a r a v e n t r i c u l a r (36) or i n t o the ventromedial nucleus (35). In a d d i t i o n the present r e s u l t s are c o n s i s t e n t w i t h numerous r e p o r t s t h a t naloxone reduces food i n t a k e (14-25). The narrowness of the dose-effect curve is puzzling. I t may be t h a t the very low doses are simply i n s u f f i c i e n t to s t i m u l a t e the system w h i l e l a r g e r doses a c t i v a t e other, competing systems or perhaps even other receptors (e.g. 40). Some r e c e n t s u p p o r t f o r t h i s l a t t e r h y p o t h e s i s i s p r o v i d e d by the report that an increased latency to drink occurred in animals which received IVT B-END (39). This occurred even when the animals were water deprived and had been given strong dipsogenic s t i m u l i . The doses of c e n t r a l l y administered B-END s u c c e s s f u l l y increasing food i n t a k e in two p r e v i o u s e x p e r i m e n t s (35, 36) were l a r g e r than ours ( in general between 3 and 5 ug) and t h e y r e p o r t e d a l a t e n c y of at l e a s t 30 minutes before t h e i r animals would s t a r t to eat. The hyperphagia in our expe iments occurred during the f i r s t h a l f - h o u r f o l l o w i n g i n j e c t i o n . This d i f f e r ence may be a consequence of u s i n g a l i q u i d d i e t i n our e x p e r i m e n t , i n cont r a s t to the dry chow used in the other studies. Another possible explanation may be t h a t l a r g e doses of B-END d e l a y e a t i n g due to o t h e r , c o m p e t i t i v e e f f e c t s of t h e p e p t i d e . Evidence f o r t h i s comes from the o b s e r v a t i o n t h a t
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doses of B-END of 5 ug and h i g h e r , when given IVT to r a t s , can e l i c i t a c a t a t o n i c - l i k e s t a t e (40) which would c e r t a i n l y be a s s o c i a t e d w i t h reduced food intake. F i n a l l y , i t is possible that the latency observed in the other experiments was an a r t i f a c t of an i n a p p r o p r i a t e locus of i n j e c t i o n , thus requiring time f o r the peptide to d i f f u s e to a location were eating could be elicited. The f a i l u r e of met-enkephalin to increase food intake may imply that BEND actions occur at receptors other than those which are affected by M-ENK. The anatomical d i s t r i b u t i o n of these two peptides in the b r a i n are q u i t e d i f f e r e n t (41). I t may also be that giving the drug in l o g a r i t h m i c a l l y incremental doses may have resulted in our missing a c r i t i c a l , narrow e f f e c t i v e dose. F i n a l l y , i t may be t h a t the r a p i d degradation of m e t - e n k e p h a l i n , as compared to B-END, reduced i t s effectiveness as an agonist. This argument is, however, weakened by the ineffectiveness of the enkephalin analog to e l i c i t any changes of feeding behavior. In s t u d i e s which have s t r o n g l y suggested a s p e c i f i c role for the enkephalins, enkephalin a c t i v i t y can be discriminated from t h a t of B-END (42), and i t is p o s s i b l e t h a t the two peptide fragments may e f f e c t non-overlapping receptor populations. Margules et al. (16) suggested t h a t e l e v a t e d l e v e l s of B-END may be responsible f o r the overeating and obesity of g e n e t i c a l l y obese rodents since some doses of naloxone, in t h e i r hands, did not i n f l u e n c e food i n t a k e of normal-weight animals but did reduce food intake of the obese animals. I n t e r pretation of these data is problematical because, although the brain weights of the f a t t y and lean r a t s were s i m i l a r , the doses of naloxone given were calculated on the basis of t o t a l body weight. Our results are consistent with the h y p o t h e s i s t h a t endogenous B-END could play a r o l e in the e t i o l o g y of obesity, but i t must be mentioned that other i n v e s t i g a t o r s found suppressant effects on food intake of low doses of naloxone in normal-weight rats and mice (e.g. 17). Since naloxone seem to e f f e c t many other behaviors as well,not the least of which is water intake, evidence from these experiments remain equivocal. At present, the most parsimonious hypothesis of the involvement of B-END in ingestion may be to assume that i t increases food intake when released in the a p p r o p r i a t e q u a n t i t y and in the a p p r o p r i a t e b r a i n area. In t h i s l a t t e r regard, i t was r e c e n t l y reported t h a t the c o n c e n t r a t i o n of B-END in the hypothalamus,but not of t h a t in the p i t u i t a r y , was decreased in r a t s in response to starvation (43). In summary, we have found that the IVT administration of B-END increases i n t a k e of a l i q u i d d i e t by r a t s , and t h a t t h i s hyperphagia was more r a p i d l y induced than has previously been reported. These results provide a complement to numerous studies showing that other neuropeptides reduce meal size. References 1. 2. 3. 4. 5. 6. 7.
A.J. KASTIN, R.D. OLSON, A.V. SCHALLY, D. COY, L i f e Sciences 25 401-414 (1979). D.T. KRIEGER and J.C. HUGHES (Eds.), Neuroendocrinology , Sunderland: Sinauer Assoc., Inc., (1980). R. GUILLEMIN, Rec. Prog. Hor. Res. 33, 1-28 (1976). A.G.E. PEARSE, and T. TAKOR TAKOR, FTd. Proc. 38, 2288-2294 (1979). G.P. SMITH and J. GIBBS, Prog. Psychobiol. P h y s i o l . Psychol. _8, 179-242 (1979). S . C . WOODS, L.D. McKAY, L.J. STEIN, D.B. WEST, E.C. LOTTER and D.C. PORTE J r . , Brain Res. B u l l . 5 (Suppl. 4), 1-5 (1980). S.C. WOODS, D.B. WEST, L.J. ~ E I N , L.D. McKAY, E.C. LOTTER, S. G. PORTE,
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N. J. KENNEY and D.P. PORTE J r . , D i a b e t a l o g i a (In Press). K. MUELLER, and S. HSAIO, Neurosci. Behav. Rev._2, 79-87 (1978). J.E. MORLEY, L i f e Sci. 27, 355-368 (1980). E.C. LOTTER, R. KRINSKY, J.M. McKAY, C.M. TRENEER, D. PORTE Jr. and S.C. WOODS, J. Comp. P h y s i o l . Psychol. (In Press). J. GIBBS, D.J. FAUSER, E.A. ROWE, B.J. ROLLS, E.T. ROLLS, S.P. MADDISON, Nature 282, 208-210 (1979). J. HUGHES, Brain Res. 8__8_8,295-308 (1975). J. HUGHES, T.W. SMITH, H.W. KOSTERLITZ, L.A. FOTHERGILL, B.A. MORGENand H.R. MORRIS, Nature 258, (1975). S.G. HOLTZMAN, J. Pharmacol. Expr. Ther. 189, 51-60 (1974). G.H. RODGERS, H. FRENK, A.N. TAYLOR and J.C. LIEBESKIND, Proc. West. Pharm. Soc. 21, 457-460 (1978). D.L. MARGULES,B. MOISSET, M.J. LEWIS, H. SHIBUYA and C.B. PERT, Science 202, 988-991 (1978). H. FRENK, and G.H. ROGERS, Brain Behav. 26, 36-40 (1979). D.R. BROWN, and HOLTZMAN,, Pharmacol. Biochem. Behav. 11, 568-573 (1979). B.M. KING, F.X. CASTELLAVOS, A.J.KASTIN, M.C. BERZASC., M.D. MAUK, G.A. OLSON and R.D. OLSON, Pharmacol. Biochem. Behav. I_II, 729-732 (1979). B. BRANDS, J.A. THORNHILL, M. HIRST and C.W. GOWDEY, L i f e Sci. 24, 17731778 (1979). S.R. GOLDBERG, W.H. MORSE and D. M. GOLDBERG, J. Pharm. Exper. Ther. 196, 625-636 (1976). D.A. DOWNS and J.H. WOODS, J. Pharm. Exper. Ther. 196, 298-306 (1976). V.F. GELLERT and S.B. SPARBER, J. Pharm. Exper. Ther. 201, 44-54 (1977). R.T. KELLER and S.R. GOLDBERG, In: Opioid P e p t i d e ~ E . Usdin, Ed.), London: MacMillan & Co. (1798). J.M. STAPLETON, M.D. LIND, V.J. MERRIMAN and REID, L i f e Sci. 24, 24212426, (1979). J.M. STAPLETON, N.L. OSTRIWSKI, V.J. MERRIMAN, M.D. LIND and L.D. REID, Bull. Psychonomic. Soc. 13, 237-238 (1979). M. WU, S.E. CRUS-MORALES, J.R. QUINAN, J.M. STAPLETON and L.D. REID, Bull. Psychonom.Soc. 14, 323-325 (1979). S.G. HOLTZMAN, Life Sci. 26, 1465-1470 (1980) S.G. HOLTZMAN, L i f e Sci.___~, 219-226 (1979). R.P. MAICKEL, M.C. BRANDE and J.E. ZABIK, Neuropharm. 16, 863-866 (1977). J.D. BELLUZZI and L. STEIN, Nature 266, 556-558 (1977 .~J.D. BELLUZZI and L. STEIN, Soc. Neurosci. Abstr. 4, 405 (1978). I.P. STOLERMAN, C.W.T. PILCHER and G.D. D'MELLO, Li-fe Sci. 2__22,1755-1762 (1978). A.E. Le BLANC and H. CAPPELL, Pharmacol. Biochem. Behav. 3, 185-188 (1975). L. GRANDISON and A. GUIOOTTI, Neuropharm. 16, 533-536 (1977). S.F. LEIBOWITZ and G. HOR, Soc. Neurosci. A-b-str. 6, 318 (1980). N.J. KENNEY, L.D. McKAY, S.C. WOODS and R.H. WILLIAMS, Soc. Neurosci. Abstr. 4, 176 (1978). N.J. KEN-NEY and A.N. EPSTEIN, J. Comp. Physiol. Psychol. 9_22, 204-219 (1978). J.Y. SUMMY-LONG and L.M. ROSELLA, Soc. Neurosci. Abstr. 6, (1980). F. BLOOM, D. SEGAL, N. LING, and R. GUILLEMIN, Science 19-4, 630-632 (1976). E.J. SIMON and J.M. HILLER, Annul. Rev. Pharmacol. T o x i c o l . I_88, 371394 (1978). A. DUPONT, N. BARDEN, L. CUSAN, Y. MERAND, F. LABRIE and H. VAUDRY, Fed. Proc. 39, 2544-2550 (1980). S.R. GAMBERT, T.L. GARTHWAITE, C.H. PONTZER and T.C. HAGEN, Science 210, 1271-1272 (1980).
Pergamon Pres~
INTRACEREBROVENTRICULAR BETA-ENDORPHIN INCREASES FOOD INTAKE OF RATSI L. David McKay2, Nancy J. Kenney, Neile K. Edens3, Robert H. Williams 4 and Stephen C. Woods Departments of Psychology and of Medicine, U n i v e r s i t y of Washington, Seattle, Washington, USA, 98195 (Received in final form July 28, 1981) Sumar~ B-Endorphin (B-END), met-enkepalin (M-ENK), and D-Ala2-N-Me 5met-enkephalinamide were administered intracerebroventricularly to rats and e f f e c t s on the i n g e s t i o n of a l i q u i d d i e t were examined. B-END s i g n i f i c a n t l y increased food intake in a halfhour t e s t at a dose of 200 n g / r a t . Lower or higher doses did not affect food intake. Neither M-ENK or the synthetic enkephalin analog affected ingestion of the liquid diet. These findings demonstrate rapid action of an endorphin on food intake administered at a lower dose than has previously been reported and suggest a s p e c i f i c i t y f o r B-END in the e n d o r p h i n e r g i c a l l y mediated hyperphagic response.
Neuropeptides have become increasingly implicated in behavioral processes (1,2). Because many neuropeptides are found both w i t h i n the nervous and the gastrointestinal systems (3,4), research has focused upon behaviors controlled by both systems, food intake in this instance. A number of peptides reported to occur in both the brain and the gut have been suggested as possible satiety f a c t o r s in t h a t the 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 these peptides j u s t prior to a meal reduces the size of that meal (5-7). Cholecystokinin (CCK) has been studied the most extensively with regard to i t s a b i l i t y to reduce meal size (5-9), and there is also compelling evidence of a s a t i a t i n g e f f e c t f o r both somatostatin (i0) and bombesin (6,11). Several reviews of the influences of neuropeptides on food intake have recently been written (5-9). The discovery in 1975 of endogenous ]igands which i n t e r a c t with the morphine or o p i a t e - s e n s i t i v e receptors of the body (12,13) has opened new approaches f o r researching controls of food intake. Of particular importance to the study of food intake is the p o s s i b i l i t y of an endogenous opiate ligand which augments meal size. An early report suggested that the opiate receptor antagonist, naloxone, decreases food intake (14), and there have been a number of replications of that early finding (15-21). Naloxone also reduces operant responding f o r food (22-24), diazepam-induced eating (25), hyperphagia 1This research was p a r t i a l l y supported by NIH research grant AM 17844 to SCW and AM 22024 to NJK. 2Requests for reprints should be addressed to L.D. McKay, Dept. of Psychology, NI-25, University of Washington, Seattle, WA 98195 Presently at the Dept. of Psychology, University of Massachusetts. Deceased
~
0024-3205/81/141429-06502.00/0 Copyright (c) 1981 Pergamon Press Ltd.
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resulting from hypothalamic damage in rats (15) and body weight gain (20). The specificity of naloxone upon food intake controlling systems was brought to question by reports that naloxone also reduces water intake in several species (15,18,26-30), as well as affecting other types of motivated behavior (31,32). Finally, at some doses, naloxone reportedly elicited nausea and led to the formation of s p e c i f i c learned t a s t e aversions (17,21,33,34). Therefore, although considerable evidence suggests that an antagonist of the endogenous opiate system reduces food intake, implying in turn that the endogenous opiates might themselves increase food intake, whether this is due to a specific effect influencing only feeding or to a more general effect is not clear. In spite of the confounding factors which make the naloxone-related data d i f f i c u l t to interpret, the findings do raise the possibility that endogenous opiates themselves might increase food intake. Margules and his colleagues, in addition to showing that naloxone reduced food intake of genetically obese rats and mice, were able to correlate the reduction of food intake with an elevation of the levels of beta-endorphin (B-END) in the pituitary (16). They suggested that the elevated levels of B-END might play a role in the etiology of the obesity which is characteristic of these animals. There is one report that the direct administration of B-END into the ventromedial hypothalamus causes a small increment of food intake (35), but that experiment lacked controls for other peptides and for other areas of the brain. There is also a recent report that the administration of B-END into the paraventricular nucleus also increased food intake in rats (36). We now report that B-END administered in a narrow dose range into the cerebral ventricles of rats caused an increased intake of a liquid diet. Administration of the same amount of B-END peripherally had no effect on food intake and its central administration had no e f f e c t on water intake. Someof these experiments were reported in a preliminary abstract (37). Methods Subjects in all experiments were naive male Wistar-derived rats (280 to 380 g) obtained from the colony maintained by the Department of Psychology of the University of Washington. They were housed in standard individual hanging cages in a temperature-controlled room which was automatically illuminated each day from 0700 to 1900 hours. The rats had continuous access to water throughout the experiments. All rats were i n i t i a l l y adapted to a mildly restrictive feeding schedule on which food (Purina p e l l e t s ) was removed each day at 0900 hours. At 1500 hours (i.e, 6 hours l a t e r ) , a l i q u i d diet consisting of 50% tap water and 50% sweetened, condensed milk (Eagle brand) was given each rat in a calibrated drinking tube for 30 minutes. At the end of that i n t e r v a l , the tubes were removed, intake was recorded, and pellets were returned until the next morning. This schedule has proven successful for studying the effects of peptides on food intake in our lab (6,7,10,37). Rats were maintained on this schedule until a stable daily intake of the liquid diet occurred (approximately 10 to 15 days). Following this adaptation period, rats were f i t t e d wih unilateral 23gauge stainless steel cannulas s t e r e o t a x i c a l l y aimed at the l e f t l a t e r a l cerebral v e n t r i c l e ( from bregma: AP -1.2; ML +1.5; and -4.0 mm ventral to dura). Obturators (30-gauge) remained in place when the cannulas were not in use. Details of the cannulation method have been reported elsewhere (38). The animals were allowed 3 to 5 days for recovery from the surgery before testing began. They remained on the 6-hour daily deprivation schedule during this time.
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P r e l i m i n a r y v e r i f i c a t i o n of access to the v e n t r i c l e by each cannula was accomplished by a behavioral test. All t e s t i n g was done in 5-day blocks, and on the f i r s t and f i f t h days each rat was screened f o r drinking in response to an i n t r a v e n t r i c u l a r (IVT) i n j e c t i o n through the cannula of i00 ng of carbachol (carbamylcholine, Sigma) in I ul of physiological saline. This treatment is known to e l i c i t s i g n i f i c a n t water consumption. Rats that did not consume at l e a s t 3 ml of water over the 20 minutes subsequent to the i n j e c t i o n on both carbachol tests were not included in the analyses. At the end of the experiments, h i s t o l o g i c a l confirmation of access to the v e n t r i c l e s was made a f t e r the brains had been fixed in formalin in situ. For a l l IVT i n j e c t i o n s , the rats were hand-held and the actual i n j e c t i o n took I0 seconds. The r a t s were returned to t h e i r cages 30 seconds l a t e r . On Days 2, 3, and 4 the l i q u i d diet was placed on the cages during the 30-second i n t e r v a l . One ul of physiological saline was administered IVT just p r i o r to the presentation of l i q u i d diet on Days 2 and 4; on Day 3, the t e s t day, B-END or some other experimental compound was administered IVT in I ul of saline. Results B-END at dose of 200 n g / r a t caused a s i g n i f i c a n t increase of food i n t a k e . The average m i l k i n t a k e on the B-END t e s t day was 10.2 C 1.0 ml (Mean + SEM) as compared to 7.5 ~ 0.8 ml on the c o n t r o l days (P
P
Thus B-END, when administered into the l a t e r a l cerebroventricle at the s p e c i f i c dose of 200 n g / r a t , caused a mean increase of 83% in the s i z e of a 30-minute meal. A number of control conditions were also run. In one experiment, rats were adapted to a 23-hour water deprivation schedule. Water intake at the end of that i n t e r v a l was not r e l i a b l y influenced by 200 ng/rat of IVT B-END. In another control experiment, rats were given 200 ng of B-END i n t r a peritoneally. There was no s i g n i f i c a n t e f f e c t on food intake (Table I). The results of these experiments suggest that B-END is acting c e n t r a l l y to i n f l u ence meal size and that the e f f e c t is not one of generalized arousal. In o t h e r r a t s , p r e l i m i n a r y t e s t i n g of the s p e c i f i c i t y of the increased food intake among various endorphins was conducted. As seen in Table I , the IVT a d m i n i s t r a t i o n of m e t - e n k e p h a l i n at doses ranging from I to I00 n g / r a t (spanning the same molar range as the B-END t e s t s ) caused no s i g n i f i c a n t change of meal s i z e . Increases of 36% f o l l o w i n g I u g / r a t of M-ENK and 14% f o l l o w i n g the I0 n g / r a t dose were a t t a i n e d , but were not s i g n i f i c a n t l y d i f f e r e n t from control values. Likewise, the IVT administration of the longera c t i n g analog of m e t - e n k e p h a l i n , D-ala2-N-Me5-Met-Enkephalinamide, had no r e l i a b l e e f f e c t on food intake (Table I). Discussion These experiments show that B-END, when administered over a narrow dose range into the l a t e r a l cerebral v e n t r i c l e , increased intake of a l i q u i d d i e t by m i l d l y food-deprived rats. These results are consistent with the reports
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TABLE I
Peptide
Route
Dose
Average Milk Intake Iml)
Control B-END
Test
IVT
20 ng
5.2 ~ 1.7
5.2 ~ 1.8
IVT
100 ng
8.2t0.8
9.1t0.8
IVT
200 ng
7.5t0.8
I0.2~I.0
IVT
1
IVT a
200 ng
8.3 t 1.5
IP
200 ng
7.0~2.3
ug
ii.4
t I.I
iI.0
~ 1.0
15.2 t 2.1 8.0t2.0
IVT
I ng
8.5 _+ 1.7
11.6 + 1.4
IVT
10 ng
11.5 + 3.4
13.1 +_ 3.4
IVT
I00 ng
12.3 + 4.0
12.8 +_ 3.1
M-ENK ANALOG IVT
2 ng
11.7 + 3.4
9.4 _+ 0.6
IVT
1 ug
5.2 + 1.9
6.8 +_ 1.1
M-ENK
Table 1. E f f e c t s of I n t r a c e r b r o v e n t r i c u l a r l y I n j e c t e d B-Endorphin (B-END), Met-Enkephalin (M-ENK) and D-Ala2-N-Me5-Met-Enkephalinamide (M-ENK ANALOG) on M i l k I n t a k e . a i s the average of f o u r independent r e p l i c a t i o n s . that i n t r a h y p o t h a l a m i c B-END increases food intake, whether i n j e c t e d i n t o the p a r a v e n t r i c u l a r (36) or i n t o the ventromedial nucleus (35). In a d d i t i o n the present r e s u l t s are c o n s i s t e n t w i t h numerous r e p o r t s t h a t naloxone reduces food i n t a k e (14-25). The narrowness of the dose-effect curve is puzzling. I t may be t h a t the very low doses are simply i n s u f f i c i e n t to s t i m u l a t e the system w h i l e l a r g e r doses a c t i v a t e other, competing systems or perhaps even other receptors (e.g. 40). Some r e c e n t s u p p o r t f o r t h i s l a t t e r h y p o t h e s i s i s p r o v i d e d by the report that an increased latency to drink occurred in animals which received IVT B-END (39). This occurred even when the animals were water deprived and had been given strong dipsogenic s t i m u l i . The doses of c e n t r a l l y administered B-END s u c c e s s f u l l y increasing food i n t a k e in two p r e v i o u s e x p e r i m e n t s (35, 36) were l a r g e r than ours ( in general between 3 and 5 ug) and t h e y r e p o r t e d a l a t e n c y of at l e a s t 30 minutes before t h e i r animals would s t a r t to eat. The hyperphagia in our expe iments occurred during the f i r s t h a l f - h o u r f o l l o w i n g i n j e c t i o n . This d i f f e r ence may be a consequence of u s i n g a l i q u i d d i e t i n our e x p e r i m e n t , i n cont r a s t to the dry chow used in the other studies. Another possible explanation may be t h a t l a r g e doses of B-END d e l a y e a t i n g due to o t h e r , c o m p e t i t i v e e f f e c t s of t h e p e p t i d e . Evidence f o r t h i s comes from the o b s e r v a t i o n t h a t
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doses of B-END of 5 ug and h i g h e r , when given IVT to r a t s , can e l i c i t a c a t a t o n i c - l i k e s t a t e (40) which would c e r t a i n l y be a s s o c i a t e d w i t h reduced food intake. F i n a l l y , i t is possible that the latency observed in the other experiments was an a r t i f a c t of an i n a p p r o p r i a t e locus of i n j e c t i o n , thus requiring time f o r the peptide to d i f f u s e to a location were eating could be elicited. The f a i l u r e of met-enkephalin to increase food intake may imply that BEND actions occur at receptors other than those which are affected by M-ENK. The anatomical d i s t r i b u t i o n of these two peptides in the b r a i n are q u i t e d i f f e r e n t (41). I t may also be that giving the drug in l o g a r i t h m i c a l l y incremental doses may have resulted in our missing a c r i t i c a l , narrow e f f e c t i v e dose. F i n a l l y , i t may be t h a t the r a p i d degradation of m e t - e n k e p h a l i n , as compared to B-END, reduced i t s effectiveness as an agonist. This argument is, however, weakened by the ineffectiveness of the enkephalin analog to e l i c i t any changes of feeding behavior. In s t u d i e s which have s t r o n g l y suggested a s p e c i f i c role for the enkephalins, enkephalin a c t i v i t y can be discriminated from t h a t of B-END (42), and i t is p o s s i b l e t h a t the two peptide fragments may e f f e c t non-overlapping receptor populations. Margules et al. (16) suggested t h a t e l e v a t e d l e v e l s of B-END may be responsible f o r the overeating and obesity of g e n e t i c a l l y obese rodents since some doses of naloxone, in t h e i r hands, did not i n f l u e n c e food i n t a k e of normal-weight animals but did reduce food intake of the obese animals. I n t e r pretation of these data is problematical because, although the brain weights of the f a t t y and lean r a t s were s i m i l a r , the doses of naloxone given were calculated on the basis of t o t a l body weight. Our results are consistent with the h y p o t h e s i s t h a t endogenous B-END could play a r o l e in the e t i o l o g y of obesity, but i t must be mentioned that other i n v e s t i g a t o r s found suppressant effects on food intake of low doses of naloxone in normal-weight rats and mice (e.g. 17). Since naloxone seem to e f f e c t many other behaviors as well,not the least of which is water intake, evidence from these experiments remain equivocal. At present, the most parsimonious hypothesis of the involvement of B-END in ingestion may be to assume that i t increases food intake when released in the a p p r o p r i a t e q u a n t i t y and in the a p p r o p r i a t e b r a i n area. In t h i s l a t t e r regard, i t was r e c e n t l y reported t h a t the c o n c e n t r a t i o n of B-END in the hypothalamus,but not of t h a t in the p i t u i t a r y , was decreased in r a t s in response to starvation (43). In summary, we have found that the IVT administration of B-END increases i n t a k e of a l i q u i d d i e t by r a t s , and t h a t t h i s hyperphagia was more r a p i d l y induced than has previously been reported. These results provide a complement to numerous studies showing that other neuropeptides reduce meal size. References 1. 2. 3. 4. 5. 6. 7.
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