The role of the endogenous opiates in zinc deficiency anorexia

Physiology &Behavior,Vol. 32, pp. 475--478.Copyright©Pergamon Press Ltd., 1984. Printed in the U.S.A.

0031-9384/84 $3.00 + .00

The Role of the Endogenous Opiates in Zinc Deficiency Anorexia M ' B . E S S A T A R A , J. E. M O R L E Y , A . S. L E V I N E , M. K . E L S O N , R. B. S H A F E R A N D C. J. M ¢ C L A I N 1

Department of Food Science and Nutrition, University of Minnesota Neuroendocrine Research Laboratory and Department of Medicine Minneapolis Veterans Administration Medical Center and University of Minnesota R e c e i v e d 4 O c t o b e r 1982 ESSATARA, M'B., J. E. MORLEY, A. S. LEVINE, M. K. ELSON, R. B. SHAFER AND C. J. McCLAIN. The role of the endogenous opiates in zinc deficiency anorexia. PHYSIOL BEHAV 32(3) 475-478, 1984.mAnorexia is a major symptom of zinc deficiency, but the mechanism(s) for this anorexia are poorly defined. Recent studies have suggested an integral role for endogenous opiate peptides in appetite regulation. Dynorphin, a leucine-enkephalin containing opiate peptide, is a potent inducer of spontaneous feeding. In this study we showed that zinc deficient animals were relatively resistant to dynorphin-induced feeding. Measurement of dynorphin levels using a highly sensitive radioimmunoassay showed that zinc deficient animals had lower levels of dynorphin in the hypothalamus than did ad lib fed animals, with weight restricted animals having intermediate values. ['~H]-naioxone binding was significantly increased to isolated brain membranes from zinc deficic.,at animals using I nM unlabeled naloxone when compared to ad lib fed controls with the weight restricted animals again having intermediate values. These data suggest that abnormalities in endogenous opiate regulation of appetite may well play a role in the anorexia of zinc deficiency. The effects of zinc deficiency on endogenous opiate action appear to include alterations in receptor affinity, a post-receptor defect and alterations in the synthesis and/or release of dynorphin. Dynorphin

Zinc

Opiate receptor

Feeding

Endorphins

A N O R E X I A represents a primary symptom of zinc deficiency and plays an important role in the production of the growth retardation [6, l l, 44]. The mechanism(s) by which zinc deficiency induce anorexia is unknown. In previous studies in this series, we have demonstrated that zinc deficient animals are relatively resistant to food induction produced by central administration of norepinephrine, the dopamine agonist, bromergocryptine, and the G A B A agonist, muscimol [7]. The anorexia of zinc deficiency can be overcome by mild tail pinch [6], a model of stress induced eating which is dependent on dopaminergic and opiate mechanisms [l, 18, 20, 27, 32]. A number of lines of evidence suggest that endogenous opioid peptides may play a primary role in the regulation of appetite [21, 25, 28]. Dynorphi n, an endogenous opioid peptide, which contains leucine-enkephalin at its N-terminal [l 0] is thought to be the endogenous ligand for the kappa opiate receptor [5,36]. Intraventricular (ICV) administration of dynorphin-(l-13) induces spontaneous feeding in rats which is antagonized by concomitant administration of a small dose of naloxone, ICV [26, 29, 45]. It has been suggested that dynorphin may be the primary opioid peptide involved in appetite regulation [24]. Recently, Stengaard-Pedersen and his colleagues [43] have suggested that zinc may play a physiological role in the regulation of the opiate receptor. In this study, we report the effects of central administration of dynorphin-(l-13) on food ingestion in zinc deficient animals;

Appetite

Opiates

the effect of zinc deficiency on immunoreactive (ir)dynorphin levels in the hypothalamus and cortex and on opiate receptor activity. METHOD

Animals and Diet Seventy-two male weanling Sprague-Dawley rats (Biolab, St. Paul, MN) weighing from 40-46 g were randomly divided into three groups. All animals were fed a commercially prepared zinc deficient pellet diet (Ziegler Bros., Inc., Gardners, PA) which contained 0.7 ppm zinc and had no added phytate. The diet was fed with the following between group variations: zinc deficient and ad lib controls received the diet ad lib and weight restricted animals were weight paired with zinc deficient animals and fed an amount of diet so as to keep the body weights equal. The rats were maintained in an environment especially designed for trace metal studies described by us previously [6]. Rats were housed individually in stainless steel wire-bottom cages, stainless steel feeders were used and plastic water bottles were equipped with silicone stoppers. Distilled and deionized water with 30 ppm replacement of zinc as zinc acetate was supplied ad lib to weight restricted and ad lib control groups, while there was no zinc replacement in the water of zinc deficient animals. After five weeks on their respective diets, 24 of the animals were placed under light ether anesthesia

~Requests for reprints should be addressed to C. J. McClain, Gastroenterology Section, MN 650, Department of Medicine, University of Kentucky Medical Center, 800 Rose Street. Lexington, KY 40536.

475

476

E S S A I A R A /- 1 ,~1

1 CONTROL •

and chronic indwelling cannulas were inserted into their right ventricles as previously described by us [28]. The animals were allowed a 5-7 day post operative recovery period after which testing procedures were undertaken in a randomized order.

6 E 0

All animals had free access to food and water until the experiments were performed. All testing was carried out between 1300 to 1500 hours. Dynorphin-(I-13) (Sigma Chemical Co., St. Louis, MO) was dissolved in methanol/0.1 N hydrochloric acid (1: I vol/vol) and administered |CV in a 5 I volume. Controls received the vehicle only. Immediately after administration of dynorphin or vehicle the animals were returned to their home cage together with 2 pellets of preweighed zinc deficient diet (7-10 g). Food intake is expressed as grams eaten (to the nearest 0.1 g)/60 minutes.

5

v O)

o

3

t--

"o 0

ii0

2 I

l)ynorphin Radioimmunoassay Dynorphin was measured by radioimmunoassay using a modification of the method described by Ghazarossian et al. [9] as previously described by us [22]. Anti-serum (Lucia 9/14) was kindly provided by Avram Goldstein of the Addiction Research Foundation, Palo Alto, CA. Dynorphin-(1-13) was commercially purchased from Sigma Chemical Co. (St. Louis, MO) for use as a standard and also for radioiodination.

Our iodination procedure differed from that of Ghazarossian el a/. [9] in that we used the chloramine T technique of Hunter and Greenwood [15] and the iodination took place in a 15 cc polypropylene centrifuge tube. At the completion of the iodination period we aspirated the fluid from the vial. Due to the significant adherence of dynorphin to the plastic surface, the iodinated dynorphin-(l-13) remains in the vial while the free ~":'Iwas aspirated with the reaction buffer. The [te:'l] dynorphin-(l-13) was then released from the plastic surface by the addition of 0.5 cc of MEOH/0.1 NHC1 (1:1, vol/vol). The 300 pJ RIA incubation mixture consisted of 100 ~tl of a dilution of dynorphin-(l-13) or tissue extract diluted in assay buffer (150 mM) phosphate buffer (pH 7.5) containing 0.1% bovine serum albumin, 0.1% Triton XI00 and 10 mM EDTA; rather than in methanol/HCl as 100 txl of the latter decreases binding by 50%; 100 pJ of a 1:10,000 dilution of anti-serum assay buffer in 100 gl of ['e~I] dynorphin-(I-13) (approximately 5000 cpm) in the assay buffer. All components were mixed in polypropylene tubes and incubated 14-18 hours at 4°C, At the completion of the incubation period bound and free [~:'I] dynorphin-(1-13) were separated with the addition of a 1.0 ml suspension of ice-cold dextran and serum coated charcoal. The tubes were centrifuged and radioactivity was determined in the supernatant. The mean intraassay coefficient of variation was 7.2% and interassay coefficient was .92%. Rats were killed by decapitation and brains were rapidly removed. Cortex and hypothalamus were dissected as previously described [3]. Tissue was kept frozen on dry ice with an acetone slurry until it has homogenized in 1 ml of acidified methanol (consisting of equal parts of methanol and 0.1 M HCI v/v: MEOH-HCI). The homogenate was then centrifuged at 15,000 × g at 4°C for 20 minutes and the supernatant transferred to polypropylene tubes and stored at 0°C until assayed for dynorphin. The protein content of the precipitate was measured by the standard Lowry method [ 19]. The cor-

DYNORPHIN t/Jg

1 ~ DYNORPHIN lO/.Ig

C

Pharmacological Testing

I

f

AL

ZD

WR

FIG. 1. Effect of ICV administration of dynorphin on food intake in zinc deficient (ZD), weight restricted (WR) and ad lib fed (AI3 rats. F=21.62, p<0.005, tp<0.05, *p<0.01 compared to comrol.

tical and hypothalamic extracts demonstrated parallelism with synthetic dynorphin-(l-13) in the radioimmunoassay,

["H/-Nah~xone Binding Fresh whole brain tissue (minus cerebellum) was pooled from two brains obtained from zinc deficient and control animals (n=4 brain preparations/group). Brain tissue was suspended in 100 mM Tris-HCI (pH 7.41 buffer and homogenized with a Brinkman Polytron. The homogenate was centrifuged at 20,000 x g for 15 minutes and the pellet was suspended in tris buffer and rehomogenized. A second 20,000 x g spin for 15 minutes was undertaken and the pellet resuspended in tris buffer and 100 mM NaC1. The opiate receptor assay was carried out using our previously published method [17]. [:~H] naloxone was purchased from New England Nuclear for use as the ligand (specific activity 51) ~Ci/mmol) and nonradiolabelled naloxone was kindly supplied by Endo Laboratory (Menlo Park, N J). The concentration of pH] naloxone was varied over a range of 0.1-3 nM with a constant incubation time of 20 minutes at 37°C to determine the K~ and B ...... .

Measttrement of Zinc ('oncentralions Zinc concentrations were determined applying atomic absorption techniques using a Varian AA375 Atomic Absorption Spectrophotomete'r. All specimens for zinc analysis were stored in polyethylene containers that had been previously soaked in a 1% solution of EDTA and deionized water, and then rinsed with deionized water in order to avoid zinc contamination. The tissues for zinc analysis were dry ashed as described previously [37]. The presence of zinc deficiency was confirmed by tissue analysis in all animals used in this study (data not shown).

Statistics All results are expressed as the mean±S.E.M. Results were compared using urinalysis of variance and the two tailed unpaired Student's t-test.

ZINC A N D OPIATES

477 TABLE 1 THE EFFECT OF ZINC DEFICIENCYON IR-DYNORPHINLEVELS IN THE CORTEX AND HYPOTHALAMUS

RESULTS Feeding Studies (Fig. I) Dynorphin (1 /zg) induced spontaneous feeding in ad lib control rats (p<0.025) and enhanced the feeding seen in weight restricted animals (p<0.05) but was ineffective at inducing feeding in zinc deficient animals. A higher concentration of dynorphin (10/~g) induced feeding in zinc deficient animals (p<0.05) and to a greater extent in ad lib controls (p<0.0t). Dynorphin Radioimmunoassay (Table 1)

Zinc Deficient Ad Lib Controls Weight Restricted

n

Hypothalamus

Cortex

7 8 8

384 _+ 20* 480 _+ 18 426 _+ 24 F=5.19, p<0.025

252 -+ 36 166 _+ 16 222 _+ 26 F=2.71, NS

*p<0.01 vs. ad lib controls.

Zinc deficient animals had lower levels of ir-dynorphin in the hypothalamus than did the ad lib controls (p<0.01) with weight restricted animals having intermediate values. Cortical levels of ir-dynorphin tended to be increased in the zinc deficient animals compared to ad lib controls with weight restricted animals again having intermediate values. [3H]-Naloxone Binding [3H]-naloxone binding was significantly increased by isolated brain membranes from zinc deficient animals (74.3_+2.8× 1~~ cpm/mg protein) compared with the ad lib group ( 5 3 . 5+- 10.0x lff~ cpm/mg protein, p<0.05) when incubated with 1 nM unlabeled naloxone. Brain membranes from the weight restricted rats bound an intermediate amount of ['~H]-naloxone (63.3-+4.1x1~ ~ cpm/mg protein) incubated with 1 mM unlabled naloxone. Based on Scatchard analysis the KD for the zinc deficient, weight restricted and ad lib control groups were 2.2, 2.1 and 3.4 riM, respectively and the B,~x 268.0x lff~, 190.9× 10a and 211.1 x 103 cpm/mg protein, respectively. DISCUSSION A number of lines of evidence have suggested that endogenous opioid peptides may play a primary role in the regulation of appetite. Systemic administration of the opiate antagonist, naloxone, decreases feeding in food deprived animals [4,14] during stress-induced eating [20,27], after muscimol-induced [30], norepinephrine-induced [31] or diazepam-induced [41,42] eating, and in responses on operant schedules for food reinforcement [8]. A number of studies have suggested that the classical opiate, morphine, facilitates feeding [39]. Intra-hypothalamic injection of /3-endorphin initiates feeding [12,16]. The long acting methionine-enkephalin analog, D-alaZ-met-enkephalinamide, has also been shown to induce feeding in sated rats [2] and to reverse the satiety effect of a number of putative satiety factors such as cholecystokinin, bombesin, thryotropinreleasing hormone and its metabolite, histidyl-proline diketopiperazine and prostaglandins [28, 32, 33]. Zinc inhibits the stereospecific binding of [:~H]-

enkephalinamide to opiate receptor thiols and zinc ions. In addition, zinc ions and enkephalins have been shown to be topographically located in similar areas in the brain [43]. This is particularly true in the hippocampus, an area which has been suggested to be important in the induction of opiate induced feeding [24]. Dynorphin is a recently isolated basic opioid peptide [10]. After ICV injection, dynorphin has been shown to produce analgesia [13], catalepsy [13], excessive grooming [46], alterations in centrally stimulated gastric acid secretion [34] and in pituitary hormone secretion [40]. In addition, dynorphin-(l-13) has been demonstrated to be a potent inducer of spontaneous feeding via a mechanism that includes activation of the dopaminergic system [24,45]. Ir-dynorphin levels have been shown to be altered in a number of situations associated with alterations in the feeding drive [22,23]. In the studies reported here we found that zinc deficient animals are resistant to the food-inducing effects of dynorphin-(l-13), just as they are resistant to the foodinducing effects of a number of other substances [7]. The mechanism by which zinc deficiency produces the inhibition of feeding due to dynorphin is uncertain but appears to involve a post-receptor mechanism as zinc deficiency enhanced stereospecific [:3H]-naloxone binding. The enhancement of opiate receptor binding in zinc deficient animals would be in accord with the previously reported inhibition of opiate binding by zinc ions [43]. The alteration in irdynorphin levels in the cortex and hypothalamus would suggest that in turn zinc deficiency leads to changes in the synthesis and/or release of this endogenous opioid peptide. The difficulties in interpreting which of these mechanisms is involved based on immunoassay data have previously been discussed by us [35]. We conclude that these data suggest that abnormalities in endogenous opiate regulation of appetite may well play a role in the anorexia of zinc deficiency. The effects of zinc deficiency on endogenous opiate action appear to include alterations in receptor affinity, a post-receptor defect and alterations in the synthesis and/or release of the dynorphin.

REFERENCES I. Antelman, S. M., H. Szechtman, P. Chin and A. E. Fisher. Tail pinch induced eating, gnawing and licking behavior in rats: Dependence on nigrostriatal dopamine system. Brain Res 99:319337, 1975. 2. Belluzi, J. D. and L. Stein. Do enkephalin systems mediate drive reduction? Soc Neurosci Abstr 8: 405, 1978.

3. Brammer, G. L., J. E. Morley, E. Geller, A. Yuwiler and J. M. Hershman. Hypothalamic-pituitary-thyroid axis interactions with the pineal gland in the rat. Am J Physiol 236: E416-E420, 1979. 4. Brown, D. R. and S. G. Holtzman. Suppression of deprivation induced food and water intake in rats and mice by naloxone. Pharmacol Biochem Behav 11: 567-573, 1979.

478 5. Chavkin, C. and A. Goldstein. Specific receptor for the opioid peptide dynorphin: Structure activity relationships. Proe Natl Aead Sci USA 78: 6543-6547, 1981. 6. Essatara, M'B., A. S. Levine, J. E. Morley and C. J. McClain. Zinc deficiency and anorexia in rats: Normal feeding patterns and stress induced feeding. Physiol Behav 32: 46%474, 1984. 7. Essatara, M'B., C. J. McClain, A. S. Levine and J. E. Morley. Zinc deficiency and anorexia in rats: The effect of central administration of norepinephrine, muscimol and bromergocryptine. Physiol Behav 32: 47%482, 1984. 8. Gellert, V. F. and S. B. Sparber. A comparison of the effects of naloxone upon body weight loss and suppression of fixed-ratio operant behavior in morphine-dependent rats. J Pharmaeol Exp Ther 201: 44-54, 1977. 9. Ghazarossian, V. E., C. Chavkin and A. Goldstein. A specific radioimmunoassay for the novel opioid peptide, dynorphin. Lift, Sei 27: 75-86, 1980. 10. Goldstein, A., W. Fischil, L. I. Lowney, M. Hunkapiller and L. Hood. Porcine pituitary dynorphin: Complete amino acid sequence of the biologically active heptadecapeptide. Proc Natl Aead Sei USA 78: 7219-7223, 1981. 11. Gordon, E. F.. J. T. B o n d , R. C. Gordon and M. R. Denny. Zinc deficiency and behavior: A developmental perspective. Physiol Behav 28: 893-897, 1982. 12. Grandison, S. and A, Guidotti. Stimulation of food intake by muscimol and beta-endorphin. Neuropharmaeology 16: 533536, 1977. 13. Herman, B. H., F. Leslie and A. Goldstein. Behavioral effects and in vivo degradation of intraventricular administration of dynorphin-( I-I 3) and D-Ala2-Dynorphin-(I-I1) in rats. Lift, Sci 27: 883-892, 1980. 14. Holtzman, S. G. Behavioral effects of separate and combined administration of naloxone and d-amphetamine. J Pharmacol I~:~p 7her 189: 51-60, 1974. 15. Hunter, W. M. and F. C. Greenwood. Preparation of Iodine-131 labeled human growth hormone of high specific activity. Nature 194: 495-496, 1962. 16. Lebiowitz, S. F. and L. Hor. Endorphinergic and alphanoradrenergic systems in the paraventricular nucleus: Effects on eating behavior. Peptides 3: 421-428, 1982. 17. Levine, A. S., J. E. Morley and S. Hess. Alcohol and the opiate receptor. Alcoholism 7: 83-89, 1983. 18. Levine, A. S., J. E. Morley, G. Wilcox, D, M. Brown and B. S. Handwerger. Tail pinch behavior and analgesia in diabetic mice. Physiol Behav 28: 39-43, 1982. 19. Lowry, O. H., N. J. Rosebrough, A. J. Farr and R. J. Randall. Protein measurement with the folin phenol reagent. J Biol Chem 193: 265-275. 1951. 20. Lowy, M. T., R. R. Maickel and G. K. W. Yim. Naloxone reduction of stress related feeding. Lift" Sei 26:2113-2118, 1980. 21. Morley, J. E. The neuroendocrine control of appetite: The role of the endogenous opiates, cholecystokinin, TRH, gammaamino butyric acid and the diazepam receptor. Lift~ Sei 27: 355-368, 1980, 22. Morley, J. E., M. K. Elson, A. S. Levine and R. B. Shafer. The effects of stress on central nervous system concentrations of the opioid peptide, dynorphin. Peptides 3: 901-906, 1982. 23. Morley, J. E., M. K. Elson, A. S. Levine and R. B. Shafer. Levels of ir-dynorphin in brain and pituitary of hyperthyroid and hypothyroid rats. Eur J Pharmaeol 78: 125-127, 1982. 24. Morley. J. E. and A. S. Levine. Opiates, dopamine and feeding. In: l h e Neural Basis ~]'Feeding and Reward. Brunswick, ME: The Haer Institute, in press, 1982.

E S S A T A R A P,1 A L .

25. Morley, J. E. and A. S. Levine. The role of the endogenous opiates as regulators of appetite. A m J ('lin Nutr 35: 757-761, 1982. 26. Morley, J. E. and A. S. Levine. Dynorphin-(l-13) induces spontaneous feeding in rats. Lift, Sci 29: 1901-1903, 1981. 27. Morley, J. E. and A. S. Levine. Stress induced eating is mediated through endogenous opiates. Science 209: 1259-1261, 1980. 28. Morley, J. E. and A. S. Levine. Thyrotropin releasing hormone (TRH) suppresses stress induced eating. Life Sci 27: 269-274, 1980. 29. Morley, J. E., A. S. Levine, M. Grace and J. Kneip. Dynorphin-(l-13), dopamine and feeding in rats. Pharmacol Bioehem Behav 16: 701-705, 1982. 30. Morley, J. E., A. S. Levine and J. Kneip. Muscimol induced feeding: A model to study the hypothalamic regulation of appetite. Lift, Sci 29: 1213-1218, 1981. 31. Morley, J. E., A. S. Levine, S. S. Murray and J. Kneip. Peptidergic regulation of norepinephrine induced feeding. Pharmacol Biochem Behav 16: 225-228, 1982. 32. Morley, J. E., A. S. Levine, S. S. Murray, J. Kneip and M. Grace. Peptidergic regulation of stress-induced eating. Am ,I Physiol 243: RI59-R163, 1982. 33. Morley, J. E., A. S. Levine and C. Prasad. Histidyl-proline diketopiperazine decreases food intake in rats. Brain Res 210: 475-478, 1981. 34. Morley, J. E., A. S. Levine and S. E. Silvis. Endogenous opiates inhibit gastric acid secretion induced by central administration of thyrotropin releasing hormone (TRH). Lift' Sei 29: 293-297, 1981. 35. Morley, J. E., Y. Yamada, J. H. Walsh, C. B. Lamers, H. Wong, A. Shulkes, D. A. Damassa, J. Gordon, H. E. Carlson and J. M. Hershman. Morphine addiction and withdrawal alters brain peptide concentrations. Life Sci 26: 2239-2244, 1980. 36. Oka, T., K. Negishi, M. Suda, A. Sarva, M. Fujino and M. Wakimasu. Evidence that dynorphin-(l-13) acts as an agonist on opioid K-receptors. Ear J Pharmaeol 77: 137-141, 1982. 37. O'Leary, M. J., C. J. McClain and P. V. J. Hegarty. Effect of zinc deficiency on the weight, cellularity and zinc concentration of different skeletal muscles in the post-weanling rat. Br ,I Nutr 42: 487-495. 1979. 38. Sanger, D. J. Endorphinergic mechanisms in the control of food and water intake. Appetite 2: 193-208, 1981. 39. Sanger, D. J. and P. S. McCarthy. Differential effects of morphine on food and water intake in food deprived and freshlyfeeding rats. Psyehopharmaeology (Berlin) 72: 103-106, 1980. 40. Schulz, R., A. Wilhelm, K. M. Perke, C. Gramsch and A. Herz. /3-endorphin and dynorphin control serum luteinizing hormone level in immature female rats. Nature 294: 757-759, 1981. 41. Soubrie, P., A. Jobert and H. M. Thiebot. Differential effects of naloxone aversive situations. Psychopharmacology (Berlin 69: 101-105, 1980. 42. Stapleton, J., M. D. Lind, V. J. Merriman and L. D. Reid. Naloxone inhibits diazepam-induced feeding in rats, L(/~, Sei 24: 2421-2426, 1979. 43. Stengaard-Pederson, K., K. Fredens and L.-I. Larsson. Enkephalin and zinc in the hippocampal mossy fiber system. Brain Res 212: 230-233, 1981. 44. Underwood, E. S. Trace Elements in Haman and Animal Nutrition. New York: Academic Press, 1977, pp. 196-262. 45. Walker. J. M., R. J. Katz and H. Akil. Behavioral effects of dynorphin-(I-13) in the mouse and rat: Initial observations. Peptide,s l: 341-345, 1980. 46. Zwiers, H,, V. J. Aloyo and W. H. Gispen. Behavioral and neurochemical effects of the new opioid peptide, dynorphin-tl-13)--comparison with other neuropeptides. Li/~~ Sci 28: 2545-2551, 1981.