Brain Research, 558 (1991) 273-279 © 1991 Elsevier Science Publishers B.V. All fights reserved. 0006-8993/91/$03.50 ADONIS 0006899391169495
273
BRES 16949
Effect of hypothalamic administration of growth hormone-releasing factor (GRF) on feeding behavior in rats Yasuo Tanaka, Masato Egawa, Shuji Inoue and Yutaro Takamura Third Department of Internal Medicine, Yokohama City University, Yokohama (Japan) (Accepted 23 April 1991)
Key words: Ventromedial hypothalamic nucleus; Lateral hypothalamic area; Paraventricular nucleus; Medial preoptic area; Microinjection; Insulin; Food intake; Glucose; Fatty acid
To examine the role and working site of growth hormone-releasing factor (GRF) in feeding behavior, we first tested the effect of the intracerebroventficular (i.c.v.) injection of GRF on food intake after 24 h of food deprivation. Cumulative food intake was measured 1, 3 and 6 h after injection. A lower dose of GRF stimulated food intake in a dose dependent manner (3 h; GRF 100 pmol 8.64 ± 1.06 g vs saline 5.50 ± 0.60 g, P < 0.05), while a higher dose (1 nmol, 500 pmol) suppressed food intake (3 h; GRF 1 nmol 2.65 ± 0.70 g vs saline 5.50 ± 0.60 g, P < 0.01). Second, the effect of i.c.v, injection of 100 pmol of GRF on peripheral metabolites was examined. The subsequent levels of plasma insulin, glucagon, glucose and non-esterified fatty acid showed no significant difference from those of saline administration. Third, the effect of microinjection of GRF (5 pmol) into several hypothalamic areas on food intake was examined. Injection into the ventromedial hypothalamic nucleus (VMN) stimulated food intake (3 h; GRF 5 pmol 10.32 ± 1.04 g vs saline 6.92 ± 0.32 g, P < 0.05), but no significant effect was observed following injection either into the lateral hypothalamie area (LHA), paraventricular nucleus (PVN) or medial preoptic area (MPOA). Finally, we tested the stimulatory effect of GRF on food intake in bilateral VMN lesioned rats. I.c.v. injection in these animals had no more significant effect on food intake than did saline injection in VMN lesioned rats (3 h; GRF 100 pmol 6.27 ± 0.87 g vs saline 5.34 ± 0.44 g). These results suggest that administration of a low dose of GRF centrally enhances feeding behavior and that the VMN is one of the working sites of this effect. INTRODUCTION Growth hormone-releasing factor ( G R F ) is k n o w n to be a physiological stimulant of growth h o r m o n e s'12. Recently, it has also b e e n reported to be a stimulator 23'28 or suppressor 1° of food intake when administered centrally. Vaccarino et al. 28 reported that intracerebroventricular (i.c.v.) injection of a low dose of G R F stimulated food intake in fasted rats, whereas Imaki et al. 10 reported that i.c.v, injection of a high dose of G R F suppressed food intake u n d e r the same conditions. The cause of this difference in results is u n k n o w n , however, there is a possibility that the effect of G R F is different according to the dose administered. Many brain-gut peptides are also k n o w n as food intake regulators. Some are stimulators 4"5, others are suppressors 14, and most of them have b e e n well discussed with regard ulates food lar nucleus (VMN), or
to their working sites. N e u r o p e p t i d e Y stimintake when injected into the paraventricu(PVN), ventromedial hypothalamic nucleus the lateral hypothalamic area ( L H A ) 16'25
Corticotropin-releasing h o r m o n e suppresses food intake
when injected into the PVN 14. Little is known, however, regarding the working site of GRF. Vaccarino's group reported that this site was within the medial preoptic area ( M P O A ) and that there was no effect within the PVN 7"29, but we found no reports of microinjection studies of feeding behavior into other hypothalamic areas. In the present study, we examined the effect of G R F on food intake with regard to dose d e p e n d e n c y and time course after its injection into the 3rd ventricle or hypothalamic nuclei, which are k n o w n to be feeding regulat o r y areas, for the purpose of investigating its working m a n n e r and sites. We also examined peripheral metabolites which may be related to feeding behavior to learn whether or not changes in their levels mediate the effect of GRF. MATERIALS AND METHODS
Animals Female Sprague-Dawley rats weighing 200-250 g were used in this study. Rats were housed in inidividual stainless steel cages under a controlled temperature (24 - 2 °C) and a 12-h diurnal light cycle (lights on at 08.00 h/lights off at 20.00 h) with free access to powdered chow which was placed in a stainless steel food box with
Correspondence: S. Inoue, Third Department of Internal Medicine, Yokohama City University, 3-9 Fukuura, Kanazawa-ku, Yokohama, Japan.
274 an acrylic holder to prevent spillage. Each rat was subjected to only one administration test at 10.00 h.
Experimental Procedures Experiment 1: effect of i.c.v, administration of GRF on food intake. Rats were anesthetized with 100 mg/kg i.p. hexobarbital, then stereotaxically implanted with a 23-gauge 15 mm stainless steel guide cannula aimed at the 3rd cerebroventriele. Each rat was handled for a minimum of 5 mirdday. After 7 days of recovery, rats were deprived of food for 24 h, and then each animal was injected over a 5-min period with 5/~1 saline or GRF dissolved in 5/A saline through a cannula using a 28-gauge stainless steel injector connected to a silastic tube and microinjeetor. To avoid backflow through the tract, the injector was left in place for 10 min following this procedure. It was then pulled out and rats were returned to their cages with pre-weighed powdered chow. At 1, 3, 6 h after injection, the cumulative amount of food intake was calculated.
Experiment 2: effect of i. c.v. administration of GRF on peripheral metabolites. Rats were implanted with a guide cannula aimed at the 3rd cerebroventricle as described in Exp. 1 under hexobarbital anesthesia. At the same time, a blood sampling catheter (silastic) was installed at the depth of the right atrium through the jugular vein and filled with heparinized saline (50 U/ml) to avoid occlusion. After 7 days of recovery, saline or GRF was administered i.c.v, under a freely moving condition. A sample of 1 ml of blood was taken through the catheter 0, 15, 30, 60 and 120 min after saline or GRF (100 pmol) injection. Immediately after each blood sampling, the same dose of heparinized blood was supplemented to avoid blood depletion 2. Determination of concentrations of plasma glucose, insulin, glucagon and non-esterified fatty acid (NEFA) was made from the samples.
Experiment 3: effect of hypothalamic nuclei administration of GRF on food intake. Under hexobarbital anesthesia (100 mg/kg i.p.), rats were implanted with a guide cannula aimed into the unilateral hypothalamic nucleus, VMN, LHA, PVN or MPOA using stereotaxic coordinates according to the brain map 22. An incisor bar was set 3.3 mm below the interauricular line (VMN: 2.8 mm AP (posterior to the bregma), 0.7 mm L (lateral to sagittal sinus) and 9.0 mm H (below the surface of the skull). LHA: 2.8 mm AP, 1.9 mm L and 9.0 mm H. PVN: 1.8 mm AP, 0.3 mm L and 8.0 mm H. MPOA: 0.5 mm AP, 0.5 mm L and 9.0 mm H). After 7 days of recovery, rats were deprived of food for 24 h, and 0.5 gl of saline for control or 5 pmol of GRF dissolved in 0.5/~1 saline was administered at each site. This dose was about 10% that of the i.c.v. administration of GRF, thus we considered it appropriate for microinjection into hypothalamic nuclei. Cumulative food intake was measured 1, 3 and 6 h after the injection.
Experiment 4: effect of i. c.v. administration of GRF on food in-
**
take in VMN lesioned rats. Under hexobarbital anesthesia, the rat's head was fixed using a stereotaxic instrument. An electrode with just the tip exposed was inserted into the VMN, and the bilateral VMN was destroyed by passing a direct cathodal current through it (2 mA, 20 s) as previously described 11. In sham-operated rats, the electrode was inserted into the VMN but no current was passed through. After 7 days, VMN-lesioned rats remarkably increased in body weight, and those with an increase of more than 40 g a week were used in this study. A guide cannula was implanted into the 3rd ventricle by the method described in Exp. 1. After 7 more days of recovery, saline or GRF was injected into the 3rd ventricle and cumulative food intake was measured 1, 3 and 6 h thereafter.
Histology After the experiment, all rats were injected with dye (5 Hi Methylene blue for i.c.v, and 0.2 Hl for intra hypothalamic nucleus) through the same injectors. One hundred ml of a 1(~ formalin solution was then perfused through the animals' hearts under an overdose of anesthesia, and the brains were removed and placed in 10% formalin solution. They were sectioned to confirm that the VMN had been lesioned and to determine the location of the injection (Fig. 3). Rats which had not been injected in the target nucleus (VMN, n = 11; LHA, n = 10; PVN, n = 8; MPOA, n = 5) were excluded from the statistical analysis.
Statistical Analysis All results were expressed as mean -+ S.E.M. Data were analyzed by one-way analysis of variance (ANOVA). Comparisons between the two groups were made by post-hoe test (P < 0.05 was taken as the criterion for significance).
RESULTS
E x p e r i m e n t 1: effect o f i. c.v. administration o f G R F on
food intake. Fig. la shows the dose dependent increase of cumulative food intake after i.c.v, injection of GRF at a lower dose (50 pmol, 100 pmol). The group injected with 100 pmol of GRF (n = 4) increased its food intake significantly compared to the saline-injected group (n = 5) 1, 3 and 6 h after GRF injection (P < 0.05, P < 0.05 and P < 0.01). Although the group injected with 50 pmol of GRF (n = 5) revealed an increasing tendency compared to the saline-injected group at each of the 3
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Fig. 1. Cumulative food intake 1, 3 and 6 h after 50, 100, 500 pmol and 1 nmol of GRF i.c.v, administration. **P < 0,01, *P < 0,05 compared to the saline-injected rats. a: 0, 50, 100 pmol. b: 0, 500 pmol, 1 nmol.
275 time points, there was no significant difference. Fig. lb shows the dose dependent decrease of cumulative food intake with administration of higher doses of G R F (500 pmol, 1 nmol). The group injected with 500 pmol (n = 5) significantly decreased food intake at 1 and 3 h compared to the saline-injected group (P < 0.01, P < 0.01), but no significant difference was observed at 6 h. Groups injected with 1 nmol of G R F (n = 5) significantly decreased food intake at 1, 3 h (P < 0.01, P < 0.01) and showed a lower tendency at 6 h.
Experiment 2: effect of i.c.v, administration of GRF on peripheral metabolites. To examine the effect of i.c.v. G R F injection on peripheral metabolites which may affect feeding behavior, several metabolites in plasma were measured after G R F administration. Fig. 2 shows plasma glucose, insulin, glucagon and N E F A levels after i.c.v. injection of G R F (100 pmol, n = 5) compared to saline injection (n = 5). There was a slight decreasing tendency of G R F on plasma glucose and NEFA, although no significant difference was observed between the GRF-injected groups and the saline-injected group. There was also no significant difference in plasma insulin or glucagon at any time point between the two groups following G R F injection.
Experiment 3: effect of the hypothalamic nuclei administration of GRF on food intake. To examine the stimulatory effect of G R F on food intake in the hypothalamus, 5 pmol of G R F was injected into several hypothalamic areas. Fig. 3 illustrates microinjection sites in each nucleus for rats which were successfully injected in the target nuclei. Fig. 4a shows the effect of administration into the VMN. Compared to the saline-injected group (n = 5), food intake was increased at 1, 3 and 6 h in the group injected with 5 pmol of G R F (n = 5). The difference in values between the two groups was statistically significant at 1 and 3 h (P < 0.01 and P < 0.05). Fig. 4b shows the effect of 5 pmol of G R F administered to the L H A . The GRF-injected group (n = 5) revealed a tendency to decrease food intake compared to the saline-injected group (n = 5) at the 3 time points, but the differences were not significant. Fig. 4c, d shows the effect of 5 pmol of G R F injected into the PVN and the MPOA. The GRF-injected group (PVN, n = 5; MPOA, n = 5) revealed a tendency to increase food intake compared to the saline-injected group (PVN, n = 5; MPOA, n = 5) 3, 6 h after the injection in the PVN, and 1, 3, 6 h after injection in the MPOA, but no significant difference was observed between the GRF-injected groups
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Fig. 2. Changes in plasma concentration of glucose, insulin, glucagon, and NEFA after 100 pmol of GRF i.c.v, administration. No significant differences were observed from those of saline-injected, a: glucose, b: insulin, c: glucagon, d: non-esterified fatty acid.
276
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and saline-injected groups in either hypothalamic area. Experiment 4: effect of i. c.v. administration of GRF on food intake in V M N lesioned rats. To confirm whether or not the VMN is a working site of GRF, VMN-lesioned rats were used for this experiment. The increase in their body weight one week after the VMN lesions was significantly greater than sham-operated rats (sham 3.5 --- 2.6 g vs VMN-lesioned 49.4 - 4.3 g, P < 0.01). Fig. 5 illustrates the destroyed areas of VMN lesions which included the entire VMN. Fig. 6 indicates that the food intake of VMN-lesioned rats injected with 100 pmol of GRF (n = 5) was not significantly different from that of VMN-lesioned rats injected with saline (n = 5), although there was an increasing tendency at the 3 time points.
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Fig. 6. Cumulative food intake in VMN-lesioned and sham-operated rats 1, 3 and 6 h after 1013pmol of GRF i.c.v, administration.
GRF was reported to be a feeding stimulator when administered through the lateral cerebroventricle in sheep (100 ng/kg) z3 and in male rats in a low dose (0.2-20 pmol) 28, while i.c.v, administration of GRF in a high dose (1 nmol) reportedly decreased food intake 1°. The contrasting results of these previous studies may have been due to the difference in dosages. The same result was reported regarding fl-endorphin 27. Therefore, we examined the effect of GRF using low and high doses in this study, and confirmed the factor's stimulatory effect on feeding behavior at low dose and suppressive effect at high dose in female rats. We also found that GRF has a feeding stimulating and suppressing effect when administered through the 3rd ventricle as well as through the lateral ventricle. We speculate, however, that the dose eliciting the suppressive effect is high considering the physiological state. Therefore, there is a possibility of there being a non-specific toxic effect with high dosage, The VMN, PVN and L H A are reported to be the working sites involved in the feeding behavior of other peptides. Vaccarino and Hayward 29 reported that the working site of GRF was within the MPOA and that GRF had no effect on food intake when injected into the PVN 7. We examined the GRF working site by injecting it into the VMN, PVN, LHA, and MPOA, and found that it stimulated feeding behavior only when injected into the VMN. The injections into the L H A had a tendency to decrease food intake and injections into the PVN had a tendency to increase it, although there was no statistical difference. We also found that the stimulatory effect of GRF into the MPOA was not significant, although injections showed a slight tendency to increase food intake. The reason for the difference between our results and those of Vaccarino and Hayward is not known at present; however, our reference search revealed that the MPOA has not previously been known to be an important area of feeding behavior 16'21. On the other hand, the VMN has been established as a feeding regulatory area, and has responded to microinjection with norepinephrine, neuropeptide Y, fl-endorphin and GABA, resuiting in increased food intake in rats 9'13A5'25. Furthermore, immunohistochemical studies of GRF showed that neurons containing this factor exist around the VMN 3'6' 19,20,24. However, we used a single dose of 5 pmol of GRF, and thus the possibility remains that another dose, 0.1 or 1 pmol is effective when administered into the MPOA 7. To further confirm that the VMN is a working site of the stimulatory effect of GRF, we injected GRF i.c.v, in VMN-lesioned rats and found that these animals showed
278 no significant response in feeding behavior. This result supports that the V M N is a major working site of the stimulating action of G R F . The possibility remains, however, that h y p e r p h a g i a already caused by the V M N lesions m a y affect this result. H o w e v e r , we are inclined to deny this possibility, since the effect of G R F was greater at 1 h in s h a m - o p e r a t e d rats, and the food intake at 1 h surpassed the levels of V M N - l e s i o n e d rats. The mechanism of stimulating action within the V M N by the administration of G R F is not well u n d e r s t o o d , but it is reasonable to consider that G R F may inhibit the satiety role o f the V M N . This speculation is compatible with the stimulatory effect of microinjections of p e n t o b a r b i t a l into the V M N on feeding b e h a v i o r TM. Cholecystokinin ( C C K ) is known to be a feeding suppressor, and its action is said to be due to the hyperglycemia it produces 17. Therefore, a n o t h e r possibility is that the effect of i.c.v, a d m i n i s t e r e d G R F is m e d i a t e d by changes of p e r i p h e r a l metabolites such as plasma glucose, insulin or glucagon. T h e r e was, however, no significant effect on these 3 metabolites or on N E F A level by i.c.v, administration of G R F at an equivalent dose to that used in the feeding experiment. Thus, the stimulatory effect of G R F on feeding b e h a v i o r is not due to the changes of these metabolites or h o r m o n e levels that
REFERENCES 1 Aponte, G., Leung, P., Gross, D. and Yamada, T., Effects of somatostatin on food intake in rats, Life Sci., 35 (1984) 741746. 2 Berthoud, H.R., Laughton, W.B. and Powley, T.L., A method for large volume blood sampling and transfusion in rats, Am. J. Physiol., 250 (1986) 331-337. 3 Bloch, B., Brazeau, E, Ling, N., Bohlen, P., Esch, E, Wehrenberg, W.B., Benoit, R., Bloom, F. and Guillemin, R., Immunohistochemical detection of growth hormone-releasing factor in brain, Nature, 301 (1983) 607-608. 4 Clark, J.T., Kalra, ES., Crowley, W.R. and Kalra, S.E, Neuropeptide Y and human pancreatic polypeptide stimulate feeding behavior in rats, Endocrinology, 115 (1984) 427-429. 5 Cooper, S.J., GABA and endorphin mechanisms in relation to the effects of benzodiazepins on feeding and drinking, Prog. Neuro-Psychopharmacol. Biol. Psychiatry, 7 (1983) 495-503. 6 Ciofi, E, Croix, D. and Tramu, G., Coexistence of hGHRF and NPY immunoreactivities in neurons of the arcuate nucleus of the rat, Neuroendocrinology, 45 (1987) 425-428. 7 Dickson, P.R. and Vaccarino, EJ., Characterization of feeding behavior induced by central injection of GRF, Am. J. Physiol., 259 (1990) 651-657. 8 Frohman, L.A. and Jansson, J.O., Growth hormone-releasing hormone, Endocr. Rev., 7 (1986) 223-253. 9 Grandison, L. and Guidotti, A., Stimulation of food intake by muscimol and fl endorphin, Neuropharmacology, 16 (1977) 533536. 10 Imald, T., Shibasaki, T., Hotta, M., Masuda, A., Demura, H., Shizume, K. and Ling, N., The satiety effect of growth hormone-releasing factor in rats, Brain Research, 340 (1985) 186188. 11 Inoue, S., Campfield, L.A. and Bray, G.A., Comparisons of metabolic alterations in hypothalamic and high fat diet-induced
might affect this behavior. We did not measure plasma growth h o r m o n e , but it is r e p o r t e d that the peripheral injection of growth h o r m o n e which p r o d u c e d an equivalent level of plasma growth h o r m o n e to that released by e n d o g e n o u s G R F secretion showed no significant effect on feeding behavior 2~. W h a t role does this stimulatory effect of G R F have on feeding behavior under physiological conditions? G R F is well known to be a physiological stimulator of growth h o r m o n e secretion. O n the other hand, somatostatin is a suppressor of the growth h o r m o n e in the hypothalamus 26. I.c.v. injection of somatostatin also decreases feeding behavior 1, although there is no r e p o r t of the effect of hypothalamic injection of somatostatin on food intake. Therefore, it is possible that G R F and somatostatin act on feeding behavior integratively in the same way that they m o d u l a t e growth h o r m o n e secretion. Many o t h e r p e p t i d e s also play a role in appetite regulation. A s M o r l e y suggested, the final integration of food intake m a y be brought about by a delicate balance m a i n t a i n e d in the concentrations of a n u m b e r of interacting peptides and m o n o a m i n e s 21. G R F may be one of these peptides. In summary, the results suggested that G R F has a stimulatory effect on feeding behavior and that one of its working sites in the h y p o t h a l a m u s is the VMN.
obesity, Am. J. Physiol., 233 (1977) 162-168. 12 Karashima, T., Olsen, D. and Schally, A.V., Effect of long-term administration of an analog of growth hormone-releasing factor on the GH response in rats, Life Sci., 40 (1987) 2437-2444, 13 Kelly, J., Alheid, G.F., Newberg, A. and Grossman, S.P., GABA stimulation and blockade in the hypothalamus and midbrain: effects on feeding and locomotor activity, Pharmacol. Biochem. Behav., 7 (1977) 537-541. 14 Krahn, D.D., GosneU, B.A., Levine, A.S. and Morley, J.E., Behavioral effects of corticotropin-releasing factor: localization and characterization of central effects, Brain Research, 443 (1988) 63-69. 15 Leibowitz, S.F., Pattern of drinking and feeding produced by hypothalamic norepinephrine injection in the satiated rat, Physiol. Behav., 14 (1975) 731-742. 16 Leibowitz, S.F., Brain monoamines and peptides: role in the control of eating behavior, Fed. Proc., 45 (1986) 1396-1403. 17 Levine, A.S. and Morley, J.E., Cholecystokinin-octapeptide suppresses stress-induced eating by inducing hyperglycemia, Reg. Peptides, 2 (1981) 353-357. 18 Maes, H., Time course of feeding induced by pentobarbital injections into the rat's VMN, Physiol. Behav., 24 (1980) 11071114. 19 Merchenthaler, I., Vigh, S., Schally, A.V. and Petrusz, P., lm munocytochemical localization of growth hormone-releasing factor in the rat hypothalamus, Endocrinology, 114 (1984) 10821085. 20 Merchenthaler, I., Thomas, C.R. and Arimura, A., Immunocytochemical localization of growth hormone releasing factor (GHRF)-containing structures in the rat brain using anti-rat GHRF serum, Peptides, 5 (1984) 1071-1075. 21 Morley, J.E., The neuroendocrine control of appetite: the role of the endogenous opiates, cholecystokinin, TRH, y-aminobutyric acid and the diazepam receptor, Life Sci., 27 (1980) 355368.
279 22 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, New York, 1982. 23 Riviere, P. and Bueno, L., Influence of regimen and insulinemia on orexigenic effects of GRF (1-44) in sheep, Physiol. Behay., 39 (1987) 347-350. 24 Sawchenko, EE., Swanson, L.W., Rivier, J. and Vale, W.W., The distribution of growth-hormone-releasing factor (GRF) immumoreactivity in the central nervous system of the rat: an immunohistochemical study using antisera directed against rat hypothalamic GRF, J. Comp. Neurol., 237 (1985) 100-115. 25 Stanley, B.G., Chin, A.S. and Leibowitz, S.E, Feeding and drinking elicited by central injection of neuropeptide Y: evidence for a hypothalamic site(s) of action, Brain Res. Bull., 14 (1985) 521-524. 26 Tannenbaum, G.S. and Patel, Y.C., On the fate of centrally
administered somatostatin in the rat: massive hypersomatostatinemia resulting from leakage into the peripheral circulation has effects on growth hormone secretion and giucoregulation, Endocrinology, 118 (1986) 2137-2143. 27 Tsujii, S. and Bray, G.A., Acetylation alters the feeding response to MSH and fl-endorphin, Brain Res. Bull., 23 (1989) 165-169. 28 Vaccarino, EJ., Bloom, EE., Rivier, J., Vale, W. and Koob, G.E, Stimulation of food intake in rats by centrally administered hypothalamic growth hormone-releasing factor, Nature, 314 (1985) 167-168. 29 Vaccarino, EJ. and Hayward, M., Microinjections of growth hormone-releasing factor into the medial preoptic area/suprachiasmatic nucleus region of the hypothalamus stimulate food intake in rats, Reg. Peptides, 21 (1988) 21-28.
273
BRES 16949
Effect of hypothalamic administration of growth hormone-releasing factor (GRF) on feeding behavior in rats Yasuo Tanaka, Masato Egawa, Shuji Inoue and Yutaro Takamura Third Department of Internal Medicine, Yokohama City University, Yokohama (Japan) (Accepted 23 April 1991)
Key words: Ventromedial hypothalamic nucleus; Lateral hypothalamic area; Paraventricular nucleus; Medial preoptic area; Microinjection; Insulin; Food intake; Glucose; Fatty acid
To examine the role and working site of growth hormone-releasing factor (GRF) in feeding behavior, we first tested the effect of the intracerebroventficular (i.c.v.) injection of GRF on food intake after 24 h of food deprivation. Cumulative food intake was measured 1, 3 and 6 h after injection. A lower dose of GRF stimulated food intake in a dose dependent manner (3 h; GRF 100 pmol 8.64 ± 1.06 g vs saline 5.50 ± 0.60 g, P < 0.05), while a higher dose (1 nmol, 500 pmol) suppressed food intake (3 h; GRF 1 nmol 2.65 ± 0.70 g vs saline 5.50 ± 0.60 g, P < 0.01). Second, the effect of i.c.v, injection of 100 pmol of GRF on peripheral metabolites was examined. The subsequent levels of plasma insulin, glucagon, glucose and non-esterified fatty acid showed no significant difference from those of saline administration. Third, the effect of microinjection of GRF (5 pmol) into several hypothalamic areas on food intake was examined. Injection into the ventromedial hypothalamic nucleus (VMN) stimulated food intake (3 h; GRF 5 pmol 10.32 ± 1.04 g vs saline 6.92 ± 0.32 g, P < 0.05), but no significant effect was observed following injection either into the lateral hypothalamie area (LHA), paraventricular nucleus (PVN) or medial preoptic area (MPOA). Finally, we tested the stimulatory effect of GRF on food intake in bilateral VMN lesioned rats. I.c.v. injection in these animals had no more significant effect on food intake than did saline injection in VMN lesioned rats (3 h; GRF 100 pmol 6.27 ± 0.87 g vs saline 5.34 ± 0.44 g). These results suggest that administration of a low dose of GRF centrally enhances feeding behavior and that the VMN is one of the working sites of this effect. INTRODUCTION Growth hormone-releasing factor ( G R F ) is k n o w n to be a physiological stimulant of growth h o r m o n e s'12. Recently, it has also b e e n reported to be a stimulator 23'28 or suppressor 1° of food intake when administered centrally. Vaccarino et al. 28 reported that intracerebroventricular (i.c.v.) injection of a low dose of G R F stimulated food intake in fasted rats, whereas Imaki et al. 10 reported that i.c.v, injection of a high dose of G R F suppressed food intake u n d e r the same conditions. The cause of this difference in results is u n k n o w n , however, there is a possibility that the effect of G R F is different according to the dose administered. Many brain-gut peptides are also k n o w n as food intake regulators. Some are stimulators 4"5, others are suppressors 14, and most of them have b e e n well discussed with regard ulates food lar nucleus (VMN), or
to their working sites. N e u r o p e p t i d e Y stimintake when injected into the paraventricu(PVN), ventromedial hypothalamic nucleus the lateral hypothalamic area ( L H A ) 16'25
Corticotropin-releasing h o r m o n e suppresses food intake
when injected into the PVN 14. Little is known, however, regarding the working site of GRF. Vaccarino's group reported that this site was within the medial preoptic area ( M P O A ) and that there was no effect within the PVN 7"29, but we found no reports of microinjection studies of feeding behavior into other hypothalamic areas. In the present study, we examined the effect of G R F on food intake with regard to dose d e p e n d e n c y and time course after its injection into the 3rd ventricle or hypothalamic nuclei, which are k n o w n to be feeding regulat o r y areas, for the purpose of investigating its working m a n n e r and sites. We also examined peripheral metabolites which may be related to feeding behavior to learn whether or not changes in their levels mediate the effect of GRF. MATERIALS AND METHODS
Animals Female Sprague-Dawley rats weighing 200-250 g were used in this study. Rats were housed in inidividual stainless steel cages under a controlled temperature (24 - 2 °C) and a 12-h diurnal light cycle (lights on at 08.00 h/lights off at 20.00 h) with free access to powdered chow which was placed in a stainless steel food box with
Correspondence: S. Inoue, Third Department of Internal Medicine, Yokohama City University, 3-9 Fukuura, Kanazawa-ku, Yokohama, Japan.
274 an acrylic holder to prevent spillage. Each rat was subjected to only one administration test at 10.00 h.
Experimental Procedures Experiment 1: effect of i.c.v, administration of GRF on food intake. Rats were anesthetized with 100 mg/kg i.p. hexobarbital, then stereotaxically implanted with a 23-gauge 15 mm stainless steel guide cannula aimed at the 3rd cerebroventriele. Each rat was handled for a minimum of 5 mirdday. After 7 days of recovery, rats were deprived of food for 24 h, and then each animal was injected over a 5-min period with 5/~1 saline or GRF dissolved in 5/A saline through a cannula using a 28-gauge stainless steel injector connected to a silastic tube and microinjeetor. To avoid backflow through the tract, the injector was left in place for 10 min following this procedure. It was then pulled out and rats were returned to their cages with pre-weighed powdered chow. At 1, 3, 6 h after injection, the cumulative amount of food intake was calculated.
Experiment 2: effect of i. c.v. administration of GRF on peripheral metabolites. Rats were implanted with a guide cannula aimed at the 3rd cerebroventricle as described in Exp. 1 under hexobarbital anesthesia. At the same time, a blood sampling catheter (silastic) was installed at the depth of the right atrium through the jugular vein and filled with heparinized saline (50 U/ml) to avoid occlusion. After 7 days of recovery, saline or GRF was administered i.c.v, under a freely moving condition. A sample of 1 ml of blood was taken through the catheter 0, 15, 30, 60 and 120 min after saline or GRF (100 pmol) injection. Immediately after each blood sampling, the same dose of heparinized blood was supplemented to avoid blood depletion 2. Determination of concentrations of plasma glucose, insulin, glucagon and non-esterified fatty acid (NEFA) was made from the samples.
Experiment 3: effect of hypothalamic nuclei administration of GRF on food intake. Under hexobarbital anesthesia (100 mg/kg i.p.), rats were implanted with a guide cannula aimed into the unilateral hypothalamic nucleus, VMN, LHA, PVN or MPOA using stereotaxic coordinates according to the brain map 22. An incisor bar was set 3.3 mm below the interauricular line (VMN: 2.8 mm AP (posterior to the bregma), 0.7 mm L (lateral to sagittal sinus) and 9.0 mm H (below the surface of the skull). LHA: 2.8 mm AP, 1.9 mm L and 9.0 mm H. PVN: 1.8 mm AP, 0.3 mm L and 8.0 mm H. MPOA: 0.5 mm AP, 0.5 mm L and 9.0 mm H). After 7 days of recovery, rats were deprived of food for 24 h, and 0.5 gl of saline for control or 5 pmol of GRF dissolved in 0.5/~1 saline was administered at each site. This dose was about 10% that of the i.c.v. administration of GRF, thus we considered it appropriate for microinjection into hypothalamic nuclei. Cumulative food intake was measured 1, 3 and 6 h after the injection.
Experiment 4: effect of i. c.v. administration of GRF on food in-
**
take in VMN lesioned rats. Under hexobarbital anesthesia, the rat's head was fixed using a stereotaxic instrument. An electrode with just the tip exposed was inserted into the VMN, and the bilateral VMN was destroyed by passing a direct cathodal current through it (2 mA, 20 s) as previously described 11. In sham-operated rats, the electrode was inserted into the VMN but no current was passed through. After 7 days, VMN-lesioned rats remarkably increased in body weight, and those with an increase of more than 40 g a week were used in this study. A guide cannula was implanted into the 3rd ventricle by the method described in Exp. 1. After 7 more days of recovery, saline or GRF was injected into the 3rd ventricle and cumulative food intake was measured 1, 3 and 6 h thereafter.
Histology After the experiment, all rats were injected with dye (5 Hi Methylene blue for i.c.v, and 0.2 Hl for intra hypothalamic nucleus) through the same injectors. One hundred ml of a 1(~ formalin solution was then perfused through the animals' hearts under an overdose of anesthesia, and the brains were removed and placed in 10% formalin solution. They were sectioned to confirm that the VMN had been lesioned and to determine the location of the injection (Fig. 3). Rats which had not been injected in the target nucleus (VMN, n = 11; LHA, n = 10; PVN, n = 8; MPOA, n = 5) were excluded from the statistical analysis.
Statistical Analysis All results were expressed as mean -+ S.E.M. Data were analyzed by one-way analysis of variance (ANOVA). Comparisons between the two groups were made by post-hoe test (P < 0.05 was taken as the criterion for significance).
RESULTS
E x p e r i m e n t 1: effect o f i. c.v. administration o f G R F on
food intake. Fig. la shows the dose dependent increase of cumulative food intake after i.c.v, injection of GRF at a lower dose (50 pmol, 100 pmol). The group injected with 100 pmol of GRF (n = 4) increased its food intake significantly compared to the saline-injected group (n = 5) 1, 3 and 6 h after GRF injection (P < 0.05, P < 0.05 and P < 0.01). Although the group injected with 50 pmol of GRF (n = 5) revealed an increasing tendency compared to the saline-injected group at each of the 3
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Fig. 1. Cumulative food intake 1, 3 and 6 h after 50, 100, 500 pmol and 1 nmol of GRF i.c.v, administration. **P < 0,01, *P < 0,05 compared to the saline-injected rats. a: 0, 50, 100 pmol. b: 0, 500 pmol, 1 nmol.
275 time points, there was no significant difference. Fig. lb shows the dose dependent decrease of cumulative food intake with administration of higher doses of G R F (500 pmol, 1 nmol). The group injected with 500 pmol (n = 5) significantly decreased food intake at 1 and 3 h compared to the saline-injected group (P < 0.01, P < 0.01), but no significant difference was observed at 6 h. Groups injected with 1 nmol of G R F (n = 5) significantly decreased food intake at 1, 3 h (P < 0.01, P < 0.01) and showed a lower tendency at 6 h.
Experiment 2: effect of i.c.v, administration of GRF on peripheral metabolites. To examine the effect of i.c.v. G R F injection on peripheral metabolites which may affect feeding behavior, several metabolites in plasma were measured after G R F administration. Fig. 2 shows plasma glucose, insulin, glucagon and N E F A levels after i.c.v. injection of G R F (100 pmol, n = 5) compared to saline injection (n = 5). There was a slight decreasing tendency of G R F on plasma glucose and NEFA, although no significant difference was observed between the GRF-injected groups and the saline-injected group. There was also no significant difference in plasma insulin or glucagon at any time point between the two groups following G R F injection.
Experiment 3: effect of the hypothalamic nuclei administration of GRF on food intake. To examine the stimulatory effect of G R F on food intake in the hypothalamus, 5 pmol of G R F was injected into several hypothalamic areas. Fig. 3 illustrates microinjection sites in each nucleus for rats which were successfully injected in the target nuclei. Fig. 4a shows the effect of administration into the VMN. Compared to the saline-injected group (n = 5), food intake was increased at 1, 3 and 6 h in the group injected with 5 pmol of G R F (n = 5). The difference in values between the two groups was statistically significant at 1 and 3 h (P < 0.01 and P < 0.05). Fig. 4b shows the effect of 5 pmol of G R F administered to the L H A . The GRF-injected group (n = 5) revealed a tendency to decrease food intake compared to the saline-injected group (n = 5) at the 3 time points, but the differences were not significant. Fig. 4c, d shows the effect of 5 pmol of G R F injected into the PVN and the MPOA. The GRF-injected group (PVN, n = 5; MPOA, n = 5) revealed a tendency to increase food intake compared to the saline-injected group (PVN, n = 5; MPOA, n = 5) 3, 6 h after the injection in the PVN, and 1, 3, 6 h after injection in the MPOA, but no significant difference was observed between the GRF-injected groups
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276
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and saline-injected groups in either hypothalamic area. Experiment 4: effect of i. c.v. administration of GRF on food intake in V M N lesioned rats. To confirm whether or not the VMN is a working site of GRF, VMN-lesioned rats were used for this experiment. The increase in their body weight one week after the VMN lesions was significantly greater than sham-operated rats (sham 3.5 --- 2.6 g vs VMN-lesioned 49.4 - 4.3 g, P < 0.01). Fig. 5 illustrates the destroyed areas of VMN lesions which included the entire VMN. Fig. 6 indicates that the food intake of VMN-lesioned rats injected with 100 pmol of GRF (n = 5) was not significantly different from that of VMN-lesioned rats injected with saline (n = 5), although there was an increasing tendency at the 3 time points.
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i
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GRF was reported to be a feeding stimulator when administered through the lateral cerebroventricle in sheep (100 ng/kg) z3 and in male rats in a low dose (0.2-20 pmol) 28, while i.c.v, administration of GRF in a high dose (1 nmol) reportedly decreased food intake 1°. The contrasting results of these previous studies may have been due to the difference in dosages. The same result was reported regarding fl-endorphin 27. Therefore, we examined the effect of GRF using low and high doses in this study, and confirmed the factor's stimulatory effect on feeding behavior at low dose and suppressive effect at high dose in female rats. We also found that GRF has a feeding stimulating and suppressing effect when administered through the 3rd ventricle as well as through the lateral ventricle. We speculate, however, that the dose eliciting the suppressive effect is high considering the physiological state. Therefore, there is a possibility of there being a non-specific toxic effect with high dosage, The VMN, PVN and L H A are reported to be the working sites involved in the feeding behavior of other peptides. Vaccarino and Hayward 29 reported that the working site of GRF was within the MPOA and that GRF had no effect on food intake when injected into the PVN 7. We examined the GRF working site by injecting it into the VMN, PVN, LHA, and MPOA, and found that it stimulated feeding behavior only when injected into the VMN. The injections into the L H A had a tendency to decrease food intake and injections into the PVN had a tendency to increase it, although there was no statistical difference. We also found that the stimulatory effect of GRF into the MPOA was not significant, although injections showed a slight tendency to increase food intake. The reason for the difference between our results and those of Vaccarino and Hayward is not known at present; however, our reference search revealed that the MPOA has not previously been known to be an important area of feeding behavior 16'21. On the other hand, the VMN has been established as a feeding regulatory area, and has responded to microinjection with norepinephrine, neuropeptide Y, fl-endorphin and GABA, resuiting in increased food intake in rats 9'13A5'25. Furthermore, immunohistochemical studies of GRF showed that neurons containing this factor exist around the VMN 3'6' 19,20,24. However, we used a single dose of 5 pmol of GRF, and thus the possibility remains that another dose, 0.1 or 1 pmol is effective when administered into the MPOA 7. To further confirm that the VMN is a working site of the stimulatory effect of GRF, we injected GRF i.c.v, in VMN-lesioned rats and found that these animals showed
278 no significant response in feeding behavior. This result supports that the V M N is a major working site of the stimulating action of G R F . The possibility remains, however, that h y p e r p h a g i a already caused by the V M N lesions m a y affect this result. H o w e v e r , we are inclined to deny this possibility, since the effect of G R F was greater at 1 h in s h a m - o p e r a t e d rats, and the food intake at 1 h surpassed the levels of V M N - l e s i o n e d rats. The mechanism of stimulating action within the V M N by the administration of G R F is not well u n d e r s t o o d , but it is reasonable to consider that G R F may inhibit the satiety role o f the V M N . This speculation is compatible with the stimulatory effect of microinjections of p e n t o b a r b i t a l into the V M N on feeding b e h a v i o r TM. Cholecystokinin ( C C K ) is known to be a feeding suppressor, and its action is said to be due to the hyperglycemia it produces 17. Therefore, a n o t h e r possibility is that the effect of i.c.v, a d m i n i s t e r e d G R F is m e d i a t e d by changes of p e r i p h e r a l metabolites such as plasma glucose, insulin or glucagon. T h e r e was, however, no significant effect on these 3 metabolites or on N E F A level by i.c.v, administration of G R F at an equivalent dose to that used in the feeding experiment. Thus, the stimulatory effect of G R F on feeding b e h a v i o r is not due to the changes of these metabolites or h o r m o n e levels that
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might affect this behavior. We did not measure plasma growth h o r m o n e , but it is r e p o r t e d that the peripheral injection of growth h o r m o n e which p r o d u c e d an equivalent level of plasma growth h o r m o n e to that released by e n d o g e n o u s G R F secretion showed no significant effect on feeding behavior 2~. W h a t role does this stimulatory effect of G R F have on feeding behavior under physiological conditions? G R F is well known to be a physiological stimulator of growth h o r m o n e secretion. O n the other hand, somatostatin is a suppressor of the growth h o r m o n e in the hypothalamus 26. I.c.v. injection of somatostatin also decreases feeding behavior 1, although there is no r e p o r t of the effect of hypothalamic injection of somatostatin on food intake. Therefore, it is possible that G R F and somatostatin act on feeding behavior integratively in the same way that they m o d u l a t e growth h o r m o n e secretion. Many o t h e r p e p t i d e s also play a role in appetite regulation. A s M o r l e y suggested, the final integration of food intake m a y be brought about by a delicate balance m a i n t a i n e d in the concentrations of a n u m b e r of interacting peptides and m o n o a m i n e s 21. G R F may be one of these peptides. In summary, the results suggested that G R F has a stimulatory effect on feeding behavior and that one of its working sites in the h y p o t h a l a m u s is the VMN.
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279 22 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, New York, 1982. 23 Riviere, P. and Bueno, L., Influence of regimen and insulinemia on orexigenic effects of GRF (1-44) in sheep, Physiol. Behay., 39 (1987) 347-350. 24 Sawchenko, EE., Swanson, L.W., Rivier, J. and Vale, W.W., The distribution of growth-hormone-releasing factor (GRF) immumoreactivity in the central nervous system of the rat: an immunohistochemical study using antisera directed against rat hypothalamic GRF, J. Comp. Neurol., 237 (1985) 100-115. 25 Stanley, B.G., Chin, A.S. and Leibowitz, S.E, Feeding and drinking elicited by central injection of neuropeptide Y: evidence for a hypothalamic site(s) of action, Brain Res. Bull., 14 (1985) 521-524. 26 Tannenbaum, G.S. and Patel, Y.C., On the fate of centrally
administered somatostatin in the rat: massive hypersomatostatinemia resulting from leakage into the peripheral circulation has effects on growth hormone secretion and giucoregulation, Endocrinology, 118 (1986) 2137-2143. 27 Tsujii, S. and Bray, G.A., Acetylation alters the feeding response to MSH and fl-endorphin, Brain Res. Bull., 23 (1989) 165-169. 28 Vaccarino, EJ., Bloom, EE., Rivier, J., Vale, W. and Koob, G.E, Stimulation of food intake in rats by centrally administered hypothalamic growth hormone-releasing factor, Nature, 314 (1985) 167-168. 29 Vaccarino, EJ. and Hayward, M., Microinjections of growth hormone-releasing factor into the medial preoptic area/suprachiasmatic nucleus region of the hypothalamus stimulate food intake in rats, Reg. Peptides, 21 (1988) 21-28.
