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Nitric oxide reduces hypophagia induced by threonine free diet in the rat
Brain Research 808 Ž1998. 129–133
Research report
Nitric oxide reduces hypophagia induced by threonine free diet in the rat M. Monda ) , A. Viggiano, A. Sullo, V. De Luca Dipartimento di Fisiologia Umana e Funzioni Biologiche Integrate ‘Filippo Bottazzi’, Seconda UniÕersita` di Napoli, Via Costantinopoli 16, 80138 Naples, Italy Accepted 14 July 1998
Abstract Food intake and concentrations of glutamic ŽGLU. and aspartic ŽASP. acids in the nucleus accumbens were monitored in male Sprague–Dawley rats fed a threonine free diet. These variables were measured before and after an intracerebroventricular injection of 20 nmole nitroprusside ŽNP., a non-enzymatic nitric oxide donor. The same variables were also monitored in: Ža. rats fed a threonine free diet and injected with saline; Žb. animals fed a standard diet and injected with nitroprusside; Žc. rats fed a standard diet and injected with saline. The results showed that the threonine-free diet reduced food intake and GLU and ASP concentrations in the accumbens. NP reduced the hypophagia, but it did not change GLU and ASP levels in rats fed the threonine-free diet. In animals fed the standard diet, NP increased GLU and ASP concentration, and food intake. No change was found in the animals with saline injection. These findings suggest that nitric oxide reduces the hypophagia in the rats fed a threonine-free diet. The lack of increase in GLU and ASP concentration in the nucleus accumbens of the hypophagic rats indicates that NP acts on hypophagia independently by GLU and ASP. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Aspartic and glutamic acid; Eating behavior; Nitroprusside; Nucleus accumbens; Rat
1. Introduction Rats reduce their food intake when fed an amino-acid imbalanced diet in which a single essential amino acid is growth limiting and the other essential amino acids are replete w4,17x. The decrease in intake of imbalanced diets occurs after a decrease in the concentration of the dietary limiting amino acid in the brain w5x. The reduced food intake appears to be a response to the lower concentration of the dietary limiting amino acid in the brain, since food intake is not reduced when a small amount of the dietary limiting amino acid is infused into the carotid artery w20x. The mechanism responsible for the reduction in food intake seems be located in the prepiriform cortex ŽPPC.. Animals with bilateral lesions of the PPC do not reduce their food intake when fed an imbalanced diet w9x. Because intake of imbalanced diet increases after the dietary limiting amino acid is injected into the PPC w14x, it appears that
) Corresponding author: Tel. q39-81-566-5833-5828; Fax: q39-81566-5820; E-mail: [email protected]
the mechanism responsible for part of the anorectic response to imbalanced diets is initiated by a decrease in the concentration of the dietary limiting amino acid in the PPC w1x. Many other cerebral areas are involved in the control of eating behavior w2,6,10x. The nucleus accumbens, located within the ventromedial striatum, is a critical neural substrate for appetite behavior w8x. The accumbens receives afferent input from PPC and limbic structures, which is primarily coded by excitatory amino acids w16x. In a remarkably brief period of time, nitric oxide ŽNO. has been recognized as a putative neurotransmitter w19x. NO is produced by the following enzymatic reaction: NO-synthase converts L-arginine into citrulline and NO, that in turn acts through stimulation of soluble guanylate cyclase w7x. NO is also produced by nitroprusside, a nonenzymatic NO donor. It has been demonstrated that NO is involved in the control of autonomic nervous system w11– 13x and food intake w3x. The aim of this experiment was to test the effects of NO on: Ž1. eating behavior and Ž2. GLU and ASP concentrations of the accumbens in rats fed with a threonine free diet.
0006-8993r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 7 3 3 - 1
M. Monda et al.r Brain Research 808 (1998) 129–133
130
2. Materials and methods
copolymeric polycarbonate–polyether membranes with a molecular cut-off of 20,000 Da.
2.1. Animals We used male Sprague–Dawley rats Ž N s 72., weighing 280–320 g. These were housed at controlled temperature Ž22 " 18C. and humidity Ž70%. with a 12:12 h light– dark cycle with lights on from 0700 to 1900 h. The animals were divided in 12 groups consisting of 6 each. 2.2. Diet A threonine-free diet was used which was identical to the standard diet except that it lacked threonine. Compositions of standard pellet diet and threonine free diet is reported in Table 1. Food was purchased from Piccioni, Gessate ŽMI., Italy. This diet is high osmotic, as well as the sucrose rich diet. However, the special diets are well tolerated by rats. 2.3. Apparatus Chromatographic separation was performed with a Jasco HPLC system with a reverse phase microsphere C 18 3 mm column from Chrompack eluted with a binary gradient. Collected samples were monitored with a fluorescence detector and evaluated with a Chromjet integrator, using o-phthalaldehyde precolumn derivatization method w21x. Microdialysis probes ŽCMAr11, CMA, Acton, USA. had Table 1 Compositions Žgrkg. of standard pellet diet Žcolumn 1. and threonine free diet Žcolumn 2. L-Arginine
HCl HCl=H 2 O L-Isoleucine L-Leucine L-Lysine HCl L-Methionine L-Phenylalanine L-Threonine L-Tryptophan L-Valine L-Alanine L-Apartic acid L-Glutamic acid Glycine L-Proline L-Cystine L-Serine L-Tyrosine L-Asparagine Glucose Sucrose Maize oil Mineral mix Vitamin mix Inert fiber Total L-Histidine
13.5 4.5 8.2 11.1 18.0 8.2 11.6 8.2 1.7 8.2 3.5 3.5 35.0 23.3 3.5 3.5 3.5 3.5 6.0 443.0 221.5 100.0 50.0 7.0 0.0 1000.0
13.5 4.5 8.2 11.1 18.0 0.0 11.6 8.2 1.7 8.2 3.5 3.5 35.0 23.3 3.5 3.5 3.5 3.5 6.0 443.0 221.5 100.0 50.0 7.0 8.2 1000.0
2.4. Surgery All animals were anaesthetized with i.p. pentobarbital Ž50 mgrkg b.w.. and a 20-gauge stainless guide cannula was positioned stereotaxically w15x above a lateral cerebral ventricle at the following coordinates: 1.7 mm lateral to the midline, 0.4 mm posterior to the bregma, 3.0 mm from the cranial theca. Rats were given 7–10 days to recover from surgery as judged by recovery of preoperative body weight. 2.5. Procedure Food intake was monitored in the first part of the experiment. Twelve animals Žsix in group 1 and six in group 2. were fed a threonine-free diet for 5 days and food intake was measured daily. Then, the animals of group 1 were kept without food for 24 h. Food intake was monitored over a 90 min period after presentation of threonine free food. After an additional 22.5 h of food deprivation, food intake was measured over a 90 min period after food presentation. An intracerebroventricular Ži.c.v.. injection of NP Ž20 nmole dissolved in 5 ml of saline. was made immediately before the second presentation of food. The same procedure was carried out in the animals of group 2, but saline injection replaced NP administration. Twelve rats Žsix in group 3 and six in group 4. fed a standard laboratory diet were used as control. Food intake was monitored after the NP or saline injection, according to the procedure used for the rats fed a threonine-free diet. The same procedure was carried out in rats of another four groups Žgroups 5–8., but an i.c.v. injection of Larginine replaced NP administration. In the second part of the experiment, the microdialysis was performed. Six animals Žgroup 9. were fed a threonine free diet for 5 days. After a 24 h period without food, these rats were anesthetized with urethane Ž1.2 grkg b.w. i.p.. and mounted in a stereotaxic instrument ŽStoelting.. The level of anesthesia was kept constant as evaluated by skeletal muscle relaxation, eye and palpebral responses to stimuli w18x. Heat loss was prevented in the animals by placing them on a heating pad. The skull was exposed and a microdialysis probe was inserted into the accumbens at following coordinates: 3.5 mm anterior to bregma, 1.1 mm lateral to midline, 5.9 mm from cortical surface w15x. The accumbens was perfused with artificial cerebrospinal fluid ŽCFS. at flow speed of 0.5 mlrmin for 4 h to stabilize the preparation. CFS consisted of 140 mM NaCl, 3 mM KCl, 2.5 mM CaCl 2 and 1 mM MgCl 2 , pH 7.4. After stabilization, a fraction of 45 mlr90 min of microdialysate was collected before Žbaseline period. and after an i.c.v. injection of NP Ž20 nmole.. The same variables were recorded
M. Monda et al.r Brain Research 808 (1998) 129–133
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3. Results
Fig. 1. Means"S.E. of food intake and body weight in the rats fed a threonine-free diet or laboratory standard diet. The results of additional experiments are not reported.
in the other 6 rats Žgroup 10. fed a threonine-free diet, but NP injection was substituted with saline administration. The same procedure used with group 9 was carried out in the other 6 animals Žgroup 11. fed a standard laboratory food. In the rats of group 12 fed a standard diet, the same procedure used in group 10 was carried out. The microdialysis was not performed in the rats with i.c.v. injection of L-arginine, because this injection did not modify eating behavior Žsee Section 3..
Upper panel of Fig. 1 illustrates the reduction of food intake in the rats fed a threonine free diet. Analysis of variance showed significant effects for diet w F Ž1,22. s 400.778, p - 0.01x, for time w F Ž4,88. s 2.555, p - 0.05x, for interaction diet = time w F Ž4,88. s 3.026, p - 0.05x. Lower panel shows body weight curves. Analysis of variance showed significant effects for diet w F Ž1,22. s 17.235, p - 0.01x, for time w F Ž5,110. s 424.054, p - 0.01x, for interaction diet = time w F Ž5,110. s 690.783, p - 0.01x. Fig. 2 shows the changes in food intake after cerebral injection of NP or saline. Injection of NP increased food intake in the rats fed a threonine-free diet and in the animal fed a standard diet. The injection of saline did not exert an effect on reduced food intake in the animals fed the imbalanced diet. Saline did not induce eating modification in rats fed a standard diet. Analysis of variance showed significant effects for diet w F Ž1,20. s 42.503, p 0.01x, for cerebral injection w F Ž1,20. s 32.632, p - 0.01x, for time w F Ž1,20. s 124.214, p - 0.01x, for interaction diet = cerebral injection w F Ž1,20. s 7.494, p - 0.05x, diet = time w F Ž1,20. s 21.611, p - 0.01x, cerebral injection= time w F Ž1,20. s 119.607, p - 0.01x and diet = cerebral injection= time w F Ž1, 20. s 25.967, p - 0.01x. Results of post hoc test are indicated in Fig. 2. The changes in food intake after cerebral injection of L-arginine or saline can be seen in Fig. 3. No difference was found in any group. Analysis of variance showed only a significant effect for diet w F Ž1,20. s 157.452, p - 0.01x. Results of post hoc test are indicated in Fig. 3. Fig. 4 shows the changes in GLU concentration in the accumbens. Threonine-free diet reduced the basal level of GLU. NP induced an increase in rats fed with standard
2.6. Histology At the end of the experiment, the location of cannula and microdialysis probe was histologically identified. 2.7. Statistical analysis The values are presented as means" S.E. Statistical analysis was performed using analysis of variance. Multiple comparisons were performed by Newman–Keuls post hoc test. This test was done only on data sets that showed a significant overall ANOVA. 2.8. Additional experiments Other rats were injected with photo-inactivated NP, exposed to room light for 24 h, to test the effects unrelated to NO production. Furthermore, microdialysis was done in pair-fed rats to demonstrate that results were due to the threonine deficiency and not just to an energy deficit.
Fig. 2. Means"S.E. of food intake over a 90 min period after i.c.v. injection Žsecond bars. of nitroprusside or saline in rats fed a threonine-free diet or laboratory standard diet. The first bars indicate the amount of food eaten over a 90 min period without cerebral injection. Bars having different superscripts Ža, b, c. represent differences Ž p- 0.05. among groups. The results of additional experiments are not reported.
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M. Monda et al.r Brain Research 808 (1998) 129–133
Fig. 3. Means"S.E. of food intake over a 90 min period after i.c.v. injection Žsecond bars. of L-arginine or saline in rats fed a threonine free-diet or laboratory standard diet. The first bars indicate the amount of food eaten over a 90 min period without cerebral injection. Bars having different superscripts Ža, b. represent differences Ž p- 0.05. among groups. The results of additional experiments are not reported.
Fig. 5. Means"S.E. of changes in concentration of aspartic acid over a 90 min period before Žfirst bars. and after Žsecond bars. i.c.v. injection of nitroprusside or saline in rats fed a threonine-free diet or laboratory standard diet. Bars having different superscripts Ža, b, c. represent differences Ž p- 0.05. among groups. The results of additional experiments are not reported.
diet, while this modification disappeared in rats fed with threonine free diet. Saline did not change the concentration in GLU. Analysis of variance showed significant effects for diet w F Ž1,20. s 15.221, p - 0.01x, for time w F Ž1,20. s 38.873, p - 0.01x, for interaction diet = cerebral injection w F Ž1,20. s 5.148, p - 0.05x, diet = time w F Ž1,20. s 51.255, p - 0.01x, cerebral injection= time w F Ž1,20. s 36.488, p - 0.01x and diet = cerebral injection = time w F Ž1,20. s 51.494, p - 0.01x. Results of post hoc test are indicated in Fig. 4. ASP concentrations are reported in Fig. 5. ASP concentration was reduced by threonine-free diet. An increase was found after NP injection in animals fed the standard diet, while theonine free diet blocked this increase. Saline
did not cause any modification. Analysis of variance showed significant effects for diet w F Ž1,20. s 38.519, p 0.01x, for interaction diet = cerebral injection w F Ž1,20. s 6.600, p - 0.05x, diet = time w F Ž1,20. s 7.856, p - 0.05x, and diet = cerebral injection= time w F Ž1,20. s 8.528, p - 0.01x. Results of post hoc test are indicated in Fig. 5. The additional experiments indicate no significant modification. These results are not reported in the figures.
Fig. 4. Means"S.E. of changes in concentration of glutamic acid over a 90 min period before Žfirst bars. and after Žsecond bars. i.c.v. injection of nitroprusside or saline in rats fed a threonine-free diet or laboratory standard diet. Bars having different superscripts Ža, b, c. represent differences Ž p- 0.05. among groups. The results of additional experiments are not reported.
4. Discussion This experiment shows that a threonine-free diet causes a reduction of food intake and confirms that threonine is the dietary limiting amino acid w14x. The present data are the first to demonstrate that i.c.v. injection of a NO donor, as NP, increases food intake in rats fed a threonine-free diet. This is a further demonstration that NO plays an important role in the control of eating behavior w3x. NP modifies food intake prevalently in rats fed the threoninefree diet, suggesting that hypophagic behavior induced by threonine-free diet may be the results of a reduction of NO synthesis. Probably, the threonine-free diet modifies enzymatic pathways involved in the NO formation, so that L-arginine injection cannot reduce the hypophagic effect. Another interpretation could be that L-arginine is not limiting for NO production. Nevertheless, NP, a non enzymatic NO donor, changes eating behavior especially in rats fed threonine-free diet. L-arginine does not modify food intake in control rats. This suggests that a surplus of L-arginine does not change the cerebral concentration of NO in rats fed with a standard diet. Furthermore, this experiment emphasizes the effects of threonine-free diet on excitatory amino acids in the accumbens. We report direct evidence showing that ASP and
M. Monda et al.r Brain Research 808 (1998) 129–133
GLU concentrations are reduced by threonine-free diet and no change is induced by NP in the rats fed this diet. This demonstrates that threonine is required to maintain a normal level of ASP and GLU in the accumbens. The reduction induced by threonine-free diet is not modifiable by NP. The lack of increase in ASP and GLU in the accumbens may be the cause of the more evident increase in food intake induced by NP in the hypophagic rats. Indeed, other authors have shown that GLU and ASP, injected into the accumbens, reduces food intake w8x. Thus, a threonine-free diet not only induces hypophagia, but also modifies mechanisms responsive to NO. In conclusion, nitric oxide reduces the hypophagia in the rats fed with a threonine free diet. The lack of increase in GLU and ASP concentration in the accumbens appears to be involved in this phenomenon. Acknowledgements Consiglio Nazionale delle Ricerche, Italy. References w1x J.L. Beverly, D.W. Gietzen, Q.R. Rogers, Effect of dietary limiting amino acid in prepiriform cortex on food intake, Am. J. Physiol. 259 Ž1990. R709–R715. w2x K.D. Carr, T.D. Wolinsky, Regulation of feeding by multiple opioid receptors in cingulate cortex; follow-up to an in vivo autoradiographic study, Neuropeptides 26 Ž1994. 207–213. w3x B. De Luca, M. Monda, A. Sullo, Changes in eating behavior and thermogenic activity following inhibition of nitric oxide formation, Am. J. Physiol. 268 Ž1995. R1533–R1538. w4x D.W. Gietzen, P.M.B. Leung, T.W. Castonguay, W.J. Hartman, Q.R. Rogers, Time course of food intake and plasma and brain amino acid concentration in rats fed amino acid-imbalanced or -deficient diets, in: M.R. Brand, J.G. Brand ŽEds.., Interactions of Chemical Senses with Nutrition, Academic Press, New York, NY, 1986, pp. 415–456. w5x D.W. Gietzen, P.M.B. Leung, Q.R. Rogers, Norepinephrine and amino acids in prepiriform cortex of rats fed imbalanced amino acid diets, Physiol. Behav. 36 Ž1986. 1071–1080.
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w6x R.E. Keesey, T.L. Powley, The regulation of body weight, Ann. Rev. Psychol. 37 Ž1986. 109–133. w7x R.G. Knowles, M. Palacios, R.M.J. Palmer, S. Moncada, Formation of nitric oxide from L-arginine in the central nervous system: a transduction mechanism for stimulation of the soluble guanylate cyclase, Proc. Natl. Acad. Sci. USA 86 Ž1989. 5159–5162. w8x C.S. Maldonado-Irizarry, C.J. Swanson, A.E. Kelley, Glutamate receptors in the nucleus accumbens shell control feeding behavior via the lateral hypothalamus, J. Neurosci. 15 Ž1995. 6779–6788. w9x L.L. Meliza, P.M.B. Leung, Q.R. Rogers, Effect of anterior prepiriform and medial amygdaloid lesions on acquisition of taste-avoidance and response to dietary amino acid imbalance, Physiol. Behav. 26 Ž1981. 1031–1035. w10x M. Monda, S. Amaro, B. De Luca, The influence of exercise on energy balance changes induced by ventromedial hypothalamic lesion in the rat, Physiol. Behav. 54 Ž1993. 1057–1061. w11x M. Monda, S. Amaro, A. Sullo, B. De Luca, Nitric oxide reduces the thermogenic changes induced by lateral hypothalamic lesion, J. Physiol. ŽParis. 88 Ž1994. 347–352. w12x M. Monda, S. Amaro, A. Sullo, B. De Luca, Nitric oxide reduces the thermogenic changes induced by prostaglandin E1, Brain Res. Bull. 38 Ž1995. 489–493. w13x M. Monda, A. Sullo, E. De Luca, N G -methyl-L-arginine increases the hyperthermic effects of prostaglandin E 1 , J. Physiol. ŽParis.. 90 Ž1996. 79–83. w14x M. Monda, A. Sullo, V. De Luca, M.P. Pellicano, A. Viggiano, L-threonine injection into prepyriform cortex modifies food intake by change in lateral hypothalamic activity and sympathetic discharge, Am. J. Physiol. 273 Ž1997. R554–R559. w15x L.J. Pellegrino, A.S. Pellegrino, A.J. Cushman ŽEds.., A Stereotaxic Atlas of the Rat Brain, Plenum, New York, NY, 1979. w16x T.G. Robinson, P.M. Beart, Excitant amino acid projections from rat amygdala and thalamus to nucleus accumbens, Brain Res. Bull. 20 Ž1988. 467–471. w17x Q.R. Rogers, P.M.B. Leung, The influence of amino acids on the neuroregulation of food intake, Federation Proc. 32 Ž1973. 1709– 1719. w18x L.R. Soma ŽEd.., Textbook of Veterinary Surgery, Depth of General Anesthesia, Williams and Wilkins, Baltimore, MD, 1971, pp. 178– 187. w19x S.H. Snyder, D.S. Bredt, Nitric oxide as a neural messenger, Trend Pharmacol. Sci. 12 Ž1991. 125–128. w20x G. Tobin, K.N. Boorman, Carotid and jugular amino acid infusions and food intake in the cockerel, Br. J. Nutr. 41 Ž1979. 157–162. w21x B.H.C. Westerink, Brain microdialysis and its application for the study of animal behaviour, Behav. Brain. Res. 70 Ž1995. 103–124.
Research report
Nitric oxide reduces hypophagia induced by threonine free diet in the rat M. Monda ) , A. Viggiano, A. Sullo, V. De Luca Dipartimento di Fisiologia Umana e Funzioni Biologiche Integrate ‘Filippo Bottazzi’, Seconda UniÕersita` di Napoli, Via Costantinopoli 16, 80138 Naples, Italy Accepted 14 July 1998
Abstract Food intake and concentrations of glutamic ŽGLU. and aspartic ŽASP. acids in the nucleus accumbens were monitored in male Sprague–Dawley rats fed a threonine free diet. These variables were measured before and after an intracerebroventricular injection of 20 nmole nitroprusside ŽNP., a non-enzymatic nitric oxide donor. The same variables were also monitored in: Ža. rats fed a threonine free diet and injected with saline; Žb. animals fed a standard diet and injected with nitroprusside; Žc. rats fed a standard diet and injected with saline. The results showed that the threonine-free diet reduced food intake and GLU and ASP concentrations in the accumbens. NP reduced the hypophagia, but it did not change GLU and ASP levels in rats fed the threonine-free diet. In animals fed the standard diet, NP increased GLU and ASP concentration, and food intake. No change was found in the animals with saline injection. These findings suggest that nitric oxide reduces the hypophagia in the rats fed a threonine-free diet. The lack of increase in GLU and ASP concentration in the nucleus accumbens of the hypophagic rats indicates that NP acts on hypophagia independently by GLU and ASP. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Aspartic and glutamic acid; Eating behavior; Nitroprusside; Nucleus accumbens; Rat
1. Introduction Rats reduce their food intake when fed an amino-acid imbalanced diet in which a single essential amino acid is growth limiting and the other essential amino acids are replete w4,17x. The decrease in intake of imbalanced diets occurs after a decrease in the concentration of the dietary limiting amino acid in the brain w5x. The reduced food intake appears to be a response to the lower concentration of the dietary limiting amino acid in the brain, since food intake is not reduced when a small amount of the dietary limiting amino acid is infused into the carotid artery w20x. The mechanism responsible for the reduction in food intake seems be located in the prepiriform cortex ŽPPC.. Animals with bilateral lesions of the PPC do not reduce their food intake when fed an imbalanced diet w9x. Because intake of imbalanced diet increases after the dietary limiting amino acid is injected into the PPC w14x, it appears that
) Corresponding author: Tel. q39-81-566-5833-5828; Fax: q39-81566-5820; E-mail: [email protected]
the mechanism responsible for part of the anorectic response to imbalanced diets is initiated by a decrease in the concentration of the dietary limiting amino acid in the PPC w1x. Many other cerebral areas are involved in the control of eating behavior w2,6,10x. The nucleus accumbens, located within the ventromedial striatum, is a critical neural substrate for appetite behavior w8x. The accumbens receives afferent input from PPC and limbic structures, which is primarily coded by excitatory amino acids w16x. In a remarkably brief period of time, nitric oxide ŽNO. has been recognized as a putative neurotransmitter w19x. NO is produced by the following enzymatic reaction: NO-synthase converts L-arginine into citrulline and NO, that in turn acts through stimulation of soluble guanylate cyclase w7x. NO is also produced by nitroprusside, a nonenzymatic NO donor. It has been demonstrated that NO is involved in the control of autonomic nervous system w11– 13x and food intake w3x. The aim of this experiment was to test the effects of NO on: Ž1. eating behavior and Ž2. GLU and ASP concentrations of the accumbens in rats fed with a threonine free diet.
0006-8993r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 7 3 3 - 1
M. Monda et al.r Brain Research 808 (1998) 129–133
130
2. Materials and methods
copolymeric polycarbonate–polyether membranes with a molecular cut-off of 20,000 Da.
2.1. Animals We used male Sprague–Dawley rats Ž N s 72., weighing 280–320 g. These were housed at controlled temperature Ž22 " 18C. and humidity Ž70%. with a 12:12 h light– dark cycle with lights on from 0700 to 1900 h. The animals were divided in 12 groups consisting of 6 each. 2.2. Diet A threonine-free diet was used which was identical to the standard diet except that it lacked threonine. Compositions of standard pellet diet and threonine free diet is reported in Table 1. Food was purchased from Piccioni, Gessate ŽMI., Italy. This diet is high osmotic, as well as the sucrose rich diet. However, the special diets are well tolerated by rats. 2.3. Apparatus Chromatographic separation was performed with a Jasco HPLC system with a reverse phase microsphere C 18 3 mm column from Chrompack eluted with a binary gradient. Collected samples were monitored with a fluorescence detector and evaluated with a Chromjet integrator, using o-phthalaldehyde precolumn derivatization method w21x. Microdialysis probes ŽCMAr11, CMA, Acton, USA. had Table 1 Compositions Žgrkg. of standard pellet diet Žcolumn 1. and threonine free diet Žcolumn 2. L-Arginine
HCl HCl=H 2 O L-Isoleucine L-Leucine L-Lysine HCl L-Methionine L-Phenylalanine L-Threonine L-Tryptophan L-Valine L-Alanine L-Apartic acid L-Glutamic acid Glycine L-Proline L-Cystine L-Serine L-Tyrosine L-Asparagine Glucose Sucrose Maize oil Mineral mix Vitamin mix Inert fiber Total L-Histidine
13.5 4.5 8.2 11.1 18.0 8.2 11.6 8.2 1.7 8.2 3.5 3.5 35.0 23.3 3.5 3.5 3.5 3.5 6.0 443.0 221.5 100.0 50.0 7.0 0.0 1000.0
13.5 4.5 8.2 11.1 18.0 0.0 11.6 8.2 1.7 8.2 3.5 3.5 35.0 23.3 3.5 3.5 3.5 3.5 6.0 443.0 221.5 100.0 50.0 7.0 8.2 1000.0
2.4. Surgery All animals were anaesthetized with i.p. pentobarbital Ž50 mgrkg b.w.. and a 20-gauge stainless guide cannula was positioned stereotaxically w15x above a lateral cerebral ventricle at the following coordinates: 1.7 mm lateral to the midline, 0.4 mm posterior to the bregma, 3.0 mm from the cranial theca. Rats were given 7–10 days to recover from surgery as judged by recovery of preoperative body weight. 2.5. Procedure Food intake was monitored in the first part of the experiment. Twelve animals Žsix in group 1 and six in group 2. were fed a threonine-free diet for 5 days and food intake was measured daily. Then, the animals of group 1 were kept without food for 24 h. Food intake was monitored over a 90 min period after presentation of threonine free food. After an additional 22.5 h of food deprivation, food intake was measured over a 90 min period after food presentation. An intracerebroventricular Ži.c.v.. injection of NP Ž20 nmole dissolved in 5 ml of saline. was made immediately before the second presentation of food. The same procedure was carried out in the animals of group 2, but saline injection replaced NP administration. Twelve rats Žsix in group 3 and six in group 4. fed a standard laboratory diet were used as control. Food intake was monitored after the NP or saline injection, according to the procedure used for the rats fed a threonine-free diet. The same procedure was carried out in rats of another four groups Žgroups 5–8., but an i.c.v. injection of Larginine replaced NP administration. In the second part of the experiment, the microdialysis was performed. Six animals Žgroup 9. were fed a threonine free diet for 5 days. After a 24 h period without food, these rats were anesthetized with urethane Ž1.2 grkg b.w. i.p.. and mounted in a stereotaxic instrument ŽStoelting.. The level of anesthesia was kept constant as evaluated by skeletal muscle relaxation, eye and palpebral responses to stimuli w18x. Heat loss was prevented in the animals by placing them on a heating pad. The skull was exposed and a microdialysis probe was inserted into the accumbens at following coordinates: 3.5 mm anterior to bregma, 1.1 mm lateral to midline, 5.9 mm from cortical surface w15x. The accumbens was perfused with artificial cerebrospinal fluid ŽCFS. at flow speed of 0.5 mlrmin for 4 h to stabilize the preparation. CFS consisted of 140 mM NaCl, 3 mM KCl, 2.5 mM CaCl 2 and 1 mM MgCl 2 , pH 7.4. After stabilization, a fraction of 45 mlr90 min of microdialysate was collected before Žbaseline period. and after an i.c.v. injection of NP Ž20 nmole.. The same variables were recorded
M. Monda et al.r Brain Research 808 (1998) 129–133
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3. Results
Fig. 1. Means"S.E. of food intake and body weight in the rats fed a threonine-free diet or laboratory standard diet. The results of additional experiments are not reported.
in the other 6 rats Žgroup 10. fed a threonine-free diet, but NP injection was substituted with saline administration. The same procedure used with group 9 was carried out in the other 6 animals Žgroup 11. fed a standard laboratory food. In the rats of group 12 fed a standard diet, the same procedure used in group 10 was carried out. The microdialysis was not performed in the rats with i.c.v. injection of L-arginine, because this injection did not modify eating behavior Žsee Section 3..
Upper panel of Fig. 1 illustrates the reduction of food intake in the rats fed a threonine free diet. Analysis of variance showed significant effects for diet w F Ž1,22. s 400.778, p - 0.01x, for time w F Ž4,88. s 2.555, p - 0.05x, for interaction diet = time w F Ž4,88. s 3.026, p - 0.05x. Lower panel shows body weight curves. Analysis of variance showed significant effects for diet w F Ž1,22. s 17.235, p - 0.01x, for time w F Ž5,110. s 424.054, p - 0.01x, for interaction diet = time w F Ž5,110. s 690.783, p - 0.01x. Fig. 2 shows the changes in food intake after cerebral injection of NP or saline. Injection of NP increased food intake in the rats fed a threonine-free diet and in the animal fed a standard diet. The injection of saline did not exert an effect on reduced food intake in the animals fed the imbalanced diet. Saline did not induce eating modification in rats fed a standard diet. Analysis of variance showed significant effects for diet w F Ž1,20. s 42.503, p 0.01x, for cerebral injection w F Ž1,20. s 32.632, p - 0.01x, for time w F Ž1,20. s 124.214, p - 0.01x, for interaction diet = cerebral injection w F Ž1,20. s 7.494, p - 0.05x, diet = time w F Ž1,20. s 21.611, p - 0.01x, cerebral injection= time w F Ž1,20. s 119.607, p - 0.01x and diet = cerebral injection= time w F Ž1, 20. s 25.967, p - 0.01x. Results of post hoc test are indicated in Fig. 2. The changes in food intake after cerebral injection of L-arginine or saline can be seen in Fig. 3. No difference was found in any group. Analysis of variance showed only a significant effect for diet w F Ž1,20. s 157.452, p - 0.01x. Results of post hoc test are indicated in Fig. 3. Fig. 4 shows the changes in GLU concentration in the accumbens. Threonine-free diet reduced the basal level of GLU. NP induced an increase in rats fed with standard
2.6. Histology At the end of the experiment, the location of cannula and microdialysis probe was histologically identified. 2.7. Statistical analysis The values are presented as means" S.E. Statistical analysis was performed using analysis of variance. Multiple comparisons were performed by Newman–Keuls post hoc test. This test was done only on data sets that showed a significant overall ANOVA. 2.8. Additional experiments Other rats were injected with photo-inactivated NP, exposed to room light for 24 h, to test the effects unrelated to NO production. Furthermore, microdialysis was done in pair-fed rats to demonstrate that results were due to the threonine deficiency and not just to an energy deficit.
Fig. 2. Means"S.E. of food intake over a 90 min period after i.c.v. injection Žsecond bars. of nitroprusside or saline in rats fed a threonine-free diet or laboratory standard diet. The first bars indicate the amount of food eaten over a 90 min period without cerebral injection. Bars having different superscripts Ža, b, c. represent differences Ž p- 0.05. among groups. The results of additional experiments are not reported.
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Fig. 3. Means"S.E. of food intake over a 90 min period after i.c.v. injection Žsecond bars. of L-arginine or saline in rats fed a threonine free-diet or laboratory standard diet. The first bars indicate the amount of food eaten over a 90 min period without cerebral injection. Bars having different superscripts Ža, b. represent differences Ž p- 0.05. among groups. The results of additional experiments are not reported.
Fig. 5. Means"S.E. of changes in concentration of aspartic acid over a 90 min period before Žfirst bars. and after Žsecond bars. i.c.v. injection of nitroprusside or saline in rats fed a threonine-free diet or laboratory standard diet. Bars having different superscripts Ža, b, c. represent differences Ž p- 0.05. among groups. The results of additional experiments are not reported.
diet, while this modification disappeared in rats fed with threonine free diet. Saline did not change the concentration in GLU. Analysis of variance showed significant effects for diet w F Ž1,20. s 15.221, p - 0.01x, for time w F Ž1,20. s 38.873, p - 0.01x, for interaction diet = cerebral injection w F Ž1,20. s 5.148, p - 0.05x, diet = time w F Ž1,20. s 51.255, p - 0.01x, cerebral injection= time w F Ž1,20. s 36.488, p - 0.01x and diet = cerebral injection = time w F Ž1,20. s 51.494, p - 0.01x. Results of post hoc test are indicated in Fig. 4. ASP concentrations are reported in Fig. 5. ASP concentration was reduced by threonine-free diet. An increase was found after NP injection in animals fed the standard diet, while theonine free diet blocked this increase. Saline
did not cause any modification. Analysis of variance showed significant effects for diet w F Ž1,20. s 38.519, p 0.01x, for interaction diet = cerebral injection w F Ž1,20. s 6.600, p - 0.05x, diet = time w F Ž1,20. s 7.856, p - 0.05x, and diet = cerebral injection= time w F Ž1,20. s 8.528, p - 0.01x. Results of post hoc test are indicated in Fig. 5. The additional experiments indicate no significant modification. These results are not reported in the figures.
Fig. 4. Means"S.E. of changes in concentration of glutamic acid over a 90 min period before Žfirst bars. and after Žsecond bars. i.c.v. injection of nitroprusside or saline in rats fed a threonine-free diet or laboratory standard diet. Bars having different superscripts Ža, b, c. represent differences Ž p- 0.05. among groups. The results of additional experiments are not reported.
4. Discussion This experiment shows that a threonine-free diet causes a reduction of food intake and confirms that threonine is the dietary limiting amino acid w14x. The present data are the first to demonstrate that i.c.v. injection of a NO donor, as NP, increases food intake in rats fed a threonine-free diet. This is a further demonstration that NO plays an important role in the control of eating behavior w3x. NP modifies food intake prevalently in rats fed the threoninefree diet, suggesting that hypophagic behavior induced by threonine-free diet may be the results of a reduction of NO synthesis. Probably, the threonine-free diet modifies enzymatic pathways involved in the NO formation, so that L-arginine injection cannot reduce the hypophagic effect. Another interpretation could be that L-arginine is not limiting for NO production. Nevertheless, NP, a non enzymatic NO donor, changes eating behavior especially in rats fed threonine-free diet. L-arginine does not modify food intake in control rats. This suggests that a surplus of L-arginine does not change the cerebral concentration of NO in rats fed with a standard diet. Furthermore, this experiment emphasizes the effects of threonine-free diet on excitatory amino acids in the accumbens. We report direct evidence showing that ASP and
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GLU concentrations are reduced by threonine-free diet and no change is induced by NP in the rats fed this diet. This demonstrates that threonine is required to maintain a normal level of ASP and GLU in the accumbens. The reduction induced by threonine-free diet is not modifiable by NP. The lack of increase in ASP and GLU in the accumbens may be the cause of the more evident increase in food intake induced by NP in the hypophagic rats. Indeed, other authors have shown that GLU and ASP, injected into the accumbens, reduces food intake w8x. Thus, a threonine-free diet not only induces hypophagia, but also modifies mechanisms responsive to NO. In conclusion, nitric oxide reduces the hypophagia in the rats fed with a threonine free diet. The lack of increase in GLU and ASP concentration in the accumbens appears to be involved in this phenomenon. Acknowledgements Consiglio Nazionale delle Ricerche, Italy. References w1x J.L. Beverly, D.W. Gietzen, Q.R. Rogers, Effect of dietary limiting amino acid in prepiriform cortex on food intake, Am. J. Physiol. 259 Ž1990. R709–R715. w2x K.D. Carr, T.D. Wolinsky, Regulation of feeding by multiple opioid receptors in cingulate cortex; follow-up to an in vivo autoradiographic study, Neuropeptides 26 Ž1994. 207–213. w3x B. De Luca, M. Monda, A. Sullo, Changes in eating behavior and thermogenic activity following inhibition of nitric oxide formation, Am. J. Physiol. 268 Ž1995. R1533–R1538. w4x D.W. Gietzen, P.M.B. Leung, T.W. Castonguay, W.J. Hartman, Q.R. Rogers, Time course of food intake and plasma and brain amino acid concentration in rats fed amino acid-imbalanced or -deficient diets, in: M.R. Brand, J.G. Brand ŽEds.., Interactions of Chemical Senses with Nutrition, Academic Press, New York, NY, 1986, pp. 415–456. w5x D.W. Gietzen, P.M.B. Leung, Q.R. Rogers, Norepinephrine and amino acids in prepiriform cortex of rats fed imbalanced amino acid diets, Physiol. Behav. 36 Ž1986. 1071–1080.
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