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Inhibition of deprivation-induced feeding by naloxone and cholecystokinin in rats: Effects of central alloxan
0361-9230/90 $3.00 + .oO
Brain Research Bulletin, Vol. 24, pp. 375379. +QPergamon Press plc, 1990. printed in the U.S.A.
Inhibition of Deprivation-Induced Feeding by Naloxone and Cholecystokinin in Rats: Effects of Central Alloxan DULMANIE
ARJUNE AND RICHARD
J. BODNAR’
Depa~en~ of P~ychoiogy and Neuropsy~hology Doctoral cub-Program Queens College, City University of New York, Flushing, NY 11361 Received
20 July 1989
ARJUNE, D. AND R. J. BODNAR. ln~ib~gionof depri~afian-induced feeding by naloxone and c~o~e~sto~injn in rats: E&cfs of central alfoxun. BRAIN RES BULL 24(3) 375-379, 1990.-Central administration of alloxan reduces the hyperphagic, but not the
hyperglycemic response to glucoprivation by presumably acting upon brain glucoreceptors or a glucoprivic control mechanism. The present stndy evaluated whether central alloxan pretreatment respectively altered the dose-dependent suppressant effects upon deprivation (24~hr)-induced feeding of naloxone (0.01-10 mg/kg, IP) and cholecystokinin octapeptide (CCK-8: l-8 pgikg, IP) in rats. Central alloxan (200 p,g, ICV) failed to alter body weight, free-feeding and deprivation-induced feeding. Both naloxone and CCK-8 produced significant dose-dependent inhibitions of deprivation-induced feeding in control rats. Central alloxan treatment significantly ionized peak nafoxone hy~phagia induced by 2.5 and 10 mgikg doses, and CCK-8 hy~phagia induced by the 1 and 4 pgkg doses. Coadministration of 3 M D-glucose, which acts as a cytopmtectant against alloxan-induced diabetes, blocked the attenuating actions of alloxan upon naloxone and CCK-8 hypophagia. These data indicate the effectiveness of central alloxan in restricting the ability of pharmacological agents to either stimulate or inhibit food intake in rats without altering basal intake or body weight maintenance. Alloxan
Naloxone
Cholecystokinin
Deprivation-induced feeding
3 M D-Glucose
Rats
responses. Both the opiate receptor antagonist, naloxone (6, 7, 15) and the gut peptide, cholecystokinin octapeptide (CCK-8) (1, 16, 35) decrease deprivation-induced feeding. The relationship between glucose availability and opioid control of food intake has been established in that either genetically diabetic or streptozototin-treated mice are more sensitive to naloxone (21), and that streptozotocin-treated rats display less hyperphagia following morphine (20) or Tyr-D-ala-gly-(Me)-gly-ol-enkephalin (DAGO) (17). 2DG hyperphagia is also altered by opioid manipulations (5,23). The hyperphagic actions of glucoprivic agents (3, 9, 29) and hypophagic actions of CCK-8 (10, 11, 14, 38) depend in part upon the projection systems of the area postrema (AP), nucleus tractus solitarius (NTS) and sending dorsal vagal complex. The present studies examined whether central alloxan altered the suppression of deprivation-induced feeding by naloxone and CCK-8, and whether coadministration of 3 M D-glucose, a cytoprotectant against alloxan-induced diabetes (33,41), would reverse any alloxan-induced alterations upon hypophagia induced by naloxone or CCK-8.
WHILE peripheral administration of high doses of alloxan produces diabetes and hyperglycemia by its toxic effects upon pancreatic beta cells (8, 13, 19, 28), central administration of far lower doses of alloxan alters feeding behavior without producing hyperglyce~a (3 1,32). While central alloxan at low doses (10-20 Fg) stimulates feeding (32), higher central doses of alloxan (40-200 p.g) significantly attenuate hyperphagia induced by 2deoxy-D-glucose (2DG) without altering body weight or food intake (27, 31, 40). The laboratories of Ritter (27, 30, 31) and Woods (40) have postulated that central alloxan may impair glucoprivic feeding by inducing a toxic effect upon brain glucoreceptors in a similar manner as peripheral alloxan alters lingual glucoreception (4 1). If central alloxan is acting through such a mechanism, then it should alter other pharmacological manipulations that depend in part upon glucoprivic controls. Further, nonspecific actions of alloxan could account for the reduction in glucoprivic hyperphagia; a more convincing demonstration of alloxan’s specificity would be to diminish ph~cologic~ly induced hy~phagic
‘Requests for reprints should be addressed to Dr. Richard J. Bodnar.
375
ARJUNE AND BODNAR
METHOD
Seventy-one naive male albino Sprague-Dawley rats (400-550 g) were maintained individually in wire mesh cages on a 12-hr light: 12-hr dark schedule at ambient temperatures between 22” and 25°C with pelleted rat chow and water available ad lib. In all studies, rats were monitored daily for body weight and food intake over four consecutive days, and were divided into treatment groups by matching these variables. Each rat was administered chlorpromazine HCl (3 mg/ml normal saline/kg body weight, IP) 20 min before anesthetization with ketamine HCl (100 mg/ml sterile water/kg body weight, IM). A stainless steel 22-gauge guide cannula (Plastic Products) was stereotaxically (Kopf) aimed so that its tip was positioned 0.3 mm above the left lateral ventricle. With the incisor bar set at +5 mm, coordinates were 0.5 mm anterior to the bregma suture, 1.3 mm lateral to the sagittal suture and 3.6 mm from the top of the skull. With the guide cannula in place, a stainless steel 28-gauge internal cannula (Plastic Products) was inserted so that it protruded 0.5 mm beyond the tip of the guide cannula. Based upon group assignment, rats received either a control (5 ~1 normal saline), alloxan (200 kg/5 (*I normal saline, Sigma) or alloxan/3 M D-glucose injection by infusing the injectate using a Hamilton microsyringe and polyethylene tubing at a rate of 1 ~1 every 20 sec. The alloxan dose was chosen for its previously established efficacy in reducing 2DG hyperphagia (27,31). Following injection, the internal cannula and the guide cannula were subsequently removed, and the rat was then sutured. Two weeks were allowed for recovery from surgery and anesthetic clearance. Each rat was then again monitored daily for postoperative body weight and food intake over four consecutive days. In the first experiment, control (n=9) and alloxan-treated (n = 9) rats received five injection conditions at weekly intervals according to an incompletely counterbalanced design: a) vehicle (1 ml normal saline/kg body weight, IP), and naloxone (1 ml/kg body weight, IP, Sigma) at doses of b) 0.01 mg/kg, c) 0.1 mglkg, d) 1 .O mg/kg and e) 10.0 mg/kg. All rats were deprived of food, but were allowed ad lib access to water for 24 hr prior to each injection; preand postdeprivation body weights were also determined. Following each injection condition which occurred 1-2 hr into the light cycle, deprivation-induced intake was determined 2 and 24 hr after injection. The naloxone regimen was chosen for its previously determined dose-dependent efficacy in inhibiting deprivationinduced intake (6, 7, 15). Intake in this and all subsequent protocols was determined by weighing food pellets prior to and after each condition and adjusting for spillage. The weekly interval between conditions in this and subsequent experiments allowed full recovery of body weight. In a second experiment, control (n= 9) and alloxan-treated (n =9) rats received vehicle and naloxone (2.5 mg/kg) as described previously. Time-dependent actions of naloxone hypophagia were assessed 30, 60 and 120 min at a dose considered to act specifically as an opiate receptor antagonist (34). In a third experiment, control (n = 8) and alloxan-treated (n= 9) rats received five injection conditions of CCK-8 (1 ml/kg body weight, Peninsula) at doses of 0, 1, 2, 4 and 8 (*g/kg as described previously. Deprivation-induced intake was determined 0.5, 2 and 24 hr after injection as per its previously determined efficacy in inhibiting deprivation-induced intake (1, 16, 35). In the fourth experiment, control rats (n = 9) or rats coadministered alloxan with a 3 M D-glucose solution (n=9) received three injection conditions as described previously: a) vehicle, b) naloxone (10 mg/kg) and c) CCK-8 (4 kg/kg). Deprivation-induced intake was determined at 0.5, 2 and 24 hr after injection. Histological confirmation of correct cannula placement was conducted in all experiments; only animals with verifiable place-
-
Ii
’
M
’
0.1
’
10
’
104
NALOXONE DOSE (mg/kg) 1. Alterations in deprivation (24 hr)-induced intake (2 hr: Mean, SEM) by systemic naloxone (0.01-10 mgkg, IP) in control (5 (~1normal
FIG.
saline, ICV, open circles) and alloxan-treated (200 kg, ICV, closed circles) rats. The open stars denote significant differences in intake between naloxone and corresponding vehicle injections. The enclosed star denotes significant differences between control and alloxan treatments for that condition.
ments in the lateral ventricle were used in the data analysis. Split-plot analyses of variance assessed significant effects upon individual intake points and body weights; Dunnett and Dunn comparisons were used to discern differences between vehicle and naloxone, and control and alloxan treatments, respectively. RESULTS
In all experiments, treatment with alloxan either alone or coadministered with 3 M D-glucose failed to alter postoperative body weights relative to control treatment two weeks after injection. Alloxan treatments also failed to alter food intake following food deprivation paired with a vehicle injection. All treatments failed to alter food intake 24 hr after reintroduction of food. Finally, alloxan treatments failed to alter body weight loss induced by deprivation, or subsequent body weight gain. First, naloxone significantly and dose-dependently decreased deprivation-induced intake 2 hr after reintroduction of food, F(4,64) = 17.43, p
ALLOXAN ALTERS NALOXONE
s Y
‘;: $G
s 8 P
*
differ in rats receiving control injections or alloxan coadministered with 3 M D-glucose (Fig. 4, left panel). The significant decreases induced by CCK-8 (4 &kg) in deprivation-induced intake 0.5 hr after reintroduction of food, F(1,16)=38.87, p
*O *
*
2-
0 0.0
0.5
DISCUSSION
+
CONTROUVEHICLE
*
_-II
ALLOXANNEHICLE CONTROUNALOXONE
+
ALLOXANINALOXONE
1.0
1.5
1 2.5
2.0
TIME (h) FIG. 2 Time-dependent alterations in deprivation-induced intake by systemic naloxone (2.5 mg/kg) in control and alloxan-treated rats. The open and enclosed stars respectively denote significant differences in intake between naloxone and corresponding vehicle injections, and between control and alloxan treatments for that condition.
illustrates the significant inhibition of food intake in controls by CCK-8 doses of 1 (32%), 2 (41%), 4 (58%) and 8 (64%) *g/kg. Alloxan-treated rats only displayed significant inhibition of deprivation-induced intake following CCK-8 doses of 2 (34%), 4 (33%) and 8 (54%) pg/kg. Alloxan significantly diminished CCK-8 hypophagia induced by the 4 p&kg dose, and eliminated CCK-8 hypophagia induced by the 1 l&kg dose. Fourth, coadministration of alloxan with 3 M D-glucose reversed alloxan-induced deficits. The significant decreases induced by naloxone (10 mg/kg) in deprivation-induced intake 2 hr after reintroduction of food, F( 1,16) = 10.00, p
& +
;
’
1
371
AND CCK-8 HYPOPHAGIA
-
2
CONTROL ALLOXAN
-
4
e
CCK-6 DOSE (w/kg) FIG. 3. Alterations in deprivation-induced intake (0.5 hr: Mean, SEM) by systemic cholecystokinin octapeptide (CCK-8: l-8 p&kg, IP) in control and alloxan-treated rats. The open and enclosed stars respectively denote significant differences in intake between CCK-8 and corresponding vehicle injections, and between control and alloxan treatments for that condition.
Central alloxan pretreatment significantly diminished the suppressant effects of both naloxone and CCK-8 upon deprivationinduced intake in rats. Naloxone hypophagia induced by the 10 mg/kg (62% inhibition) and 2.5 mg/kg (33% inhibition) doses in control rats was significantly reduced in alloxan-treated rats to 41% and 17%, respectively. However, naloxone hypophagia at lower doses or at earlier time points than 2 hr appeared unaffected by alloxan pretreatment. While alloxan failed to alter the peak (8 pgkg) inhibitory effects of CCK-8 upon deprivation-induced intake, it eliminated CCK-8 hypophagia at a 1 *g/kg dose, and suppressed CCK-8 hypophagia at a 4 pg/kg dose (alloxan: 33%, control: 58%). Alloxan-induced reductions in naloxone and CCK-8 hypophagia appeared selective for the following reasons. Coadministration with 3 M D-glucose reversed the alloxan-induced deficits upon naloxone and CCK-8 hypophagia, just as it acts as a cytoprotectant against alloxan-induced diabetes (33,41). Thus, alloxan appears to be working through gluco-sensitive mechanisms, although periventricular damage following alloxan has been noted using silver stain degeneration techniques (S. Ritter, personal communication). Alloxan failed to affect body weight and food intake under baseline and deprivation conditions, indicating that the animals were not debilitated, and could respond normally to a physiological challenge. Therefore, alloxan does not appear to alter either normal free feeding or deprivation-induced intake in and of itself, but appears to restrict the ability of pharmacological and physiological agents to either stimulate (e.g., 2-deoxy-D-glucose) (27, 31, 32, 40) or inhibit (e.g., naloxone and CCK-8) intake. That central alloxan treatment decreases the hypophagic properties of naloxone provides further support for a selective interaction with opioid systems. Alloxan decreased both 2DG analgesia (24) and hyperphagia (27,31) which are opioid-mediated (4,5,23, 36). Further, central alloxan treatment decreases morphine, but not nonopioid swim analgesia (25). The decreased sensitivity to naloxone hypophagia in alloxan-treated rats contrasts with the increased sensitivity to naloxone hypophagia in streptozotocintreated mice (21). This difference may be attributed to the novel test environment since streptozotocin-treated rats show increased sensitivity to naloxone hypophagia when tested in a novel environment, but are insensitive to naloxone hypophagia when tested in their home cage (22). This latter effect parallels the present findings and testing conditions, but our effects occurred in the absence of hyperglycemia, increased body weights and increased food intakes induced by streptozotocin (20). The present study cannot address whether central alloxan effects upon naloxone and CCK-8 hypophagia are working through similar sites and/or mechanisms of action. The medial hypothalamus, and particularly the paraventricular nucleus appears most sensitive to stimulation of intake by opioid agonists (18, 26, 37, 39). CCK-8 hypophagia can be induced following peripheral (1, 16, 35) or central (12) administration. The satiating actions of peripheral CCK-8 can be blocked by lesions placed in the AP (38), NTS (14), NTS-hypothalamic pathway (1 l), and the paraventricular and dorso-medial hypothalamic nuclei (2,lO). The above hindbrain structures also appear to modulate 2DG hyperphagia (3, 9, 29). Thus, medial hypothalamic and hindbrain structures may
ARJUNE AND BODNAR
378
q CONTROL n ALLoxANl 3M O-GLUCOSE
0
NALOXONE DOSE (mglkg)
4
CCK-8 DOSE (pg!kg)
FIG. 4. Alterations in deprivation-induced intake (Mean, SEM) by systemic naloxone (2 hr: 10 mgikg, IP, left panel) and CCK-8 (0.5 hr: 4 kg/kg, IP, right panel) in control rats and rats coadministered alloxan in 3 M D-glucose. The stars denote significant differences in intake between either naloxone or CCK-8 and corresponding vehicle injections.
modulate inhibition of food intake by naloxone and CCK-8, and may also contain those gluco-sensitive mechanisms sensitive to central alloxan effects.
ACKNOWLEDGEMENTS This research was supported by NIH DA04 194-O 1A2 and PSCiCUNY Grants 668244 and 669213 to R.J.B.
REFERENCES 1. Antin, J.; Gibbs, J.; Holt, J.; Young, R. C.; Smith, G. P. Cholecystokinin elicits the complete behavioral sequence of satiety in rats. J. Comp. Physiol. Psychol. 89:784-790; 1975. 2. Bellinger, L. L.; Bemardis, L. L. Suppression of feeding by cholecystokinin but not bombesin is attenuated in dorsomedial hypothalamic nuclei lesioned rats. Peptides 5:547-552; 1984. 3. Bird, E.; Cardone, C. C.; Conteras, R. J. Area postrema lesions disrupt food intake induced by cerebra-ventricular infusions of 5thioglucose in the rat. Brain Res. 270:193-196; 1983. 4. Bodnar, R. J.; Kelly, D. D.; Glusman, M. 2-Deoxy-D-glucose analgesia: Influences of opiate and non-opiate factors. Pharmacol. Biochem. Behav. 11:297-301; 1979. A. L.; 5. Bodnar, R. J.; Kramer, E.; Simone, D. A.; Kirchgessner, Scalisi, J. Dissociation of analgesic and hyperphagic responses following 2-deoxy-D-glucose. Int. J. Neurosci. 21:225-236; 1983. 6. Brands, B. J.; Thomhill, J. A.; Hirst, M.; Gowdey, C. W. Suppression of food intake and body weight gain by naloxone in rats. Life Sci. 24:1773-1778; 1979. 7. Brown, D. R.; Holtzman, S. G. Suppression of deprivation induced food and water intake in rats and mice by naloxone. Pharmacol. Biochem. Behav. 11:567-583; 1979. 8. Chang, A. Y.; Diani, A. R. Chemically and hormonally-induced diabetes. In: Volk, B. W.; Arquilla, E. R., eds. The diabetic pancreas, 2nd ed. New York: Plenum; 1985:415-438. 9. Conteras, R. J.; Fox, E.; Drugovich, M. L. Area postrema lesions produce feeding deficits in the rat: Effects of preoperative dieting and 2-deoxy-D-glucose. Physiol. Behav. 29:875-884; 1982. 10. Crawley, J. N.; Kiss, J. Z. Paraventricular nucleus lesions abolish the inhibition of feeding induced by systemic cholecystokinin. Peptides 6:133-137; 1985. 11. Crawley, J. N.; Kiss, J. Z.; Mezey, E. Bilateral midbrain transections block the behavioral effects of cholecystokinin on feeding and exploration in rats. Brain Res. 232:316-321; 1984. 12. Della-Fera, M. A.; Baile, C. A. Cholecystokinin octapeptide: continuous picomole injections into the cerebral ventricles of sheep suppress feeding. Science 206:471473; 1979. 13. Dunn, J. S.; Sheehan, H. L.; McLetchie, N. G. Necrosis of islets of Langerhans produced experimentally. Lancet 244:484-+87; 1943. 14. Edwards, G. L.; Ladenheim, E. E.; Ritter, R. C. Dorsal hindbrain participation in cholecystokinin-induced satiety. Am. J. Physiol. 251:R971-R977; 1986.
15. Frenk, H.; Rogers, G. H. The suppressant effects of naloxone on food and water intake in the rat. Behav. Neural Biol. 26:23-40; 1979. 16. Gibbs, J.; Young, R. C.; Smith, G. P. Cholecystokinin decreases food intake in rats. J. Comp. Physiol. Psychol. 84:488495; 1973. 17. Gosnell, B. A.; Grace, M.; Billington, C. J.; Levine, A. S. Effects of streptozotocin-induced diabetes on feeding induced by centrallyadministered opioid agonists. Life Sci. 45:31-40; 1989. 18. Gosnell, B. A.; Morely, J. E.; Levine, A. S. Opioid-induced feeding: localization of sensitive brain sites. Brain Res. 369: 177-184; 1986. 19. Gunnarsson, R.; Hellerstrom, C. Acute effects of alloxan on the metabolism and insulin secretion of the pancreatic beta cells. Horm. Metab. Res. 5:404-409; 1973. 20. Levine, A. S.; Grace, M.; Billington, C. J.; Gosnell, B. A.; Krahn, D. D.; Brown, D. M.; Morley, J. E. Effect of morphine and nalmefene on energy balance in diabetic and non-diabetic rats. Pharmacol. Biochem. Behav. 29:495-500; 1988. 21. Levine, A. S.; Morley, J. E.; Brown, D. M.; Handwerger, B. S. Extreme sensitivity of diabetic mice to naloxone-induced suppression of food intake. Physiol. Behav. 28:987-989; 1982. 22. Levine, A. S.; Morley, J. E.; Kneip, J.; Grace, M.; Brown, D. M. Environmental modulates naloxone’s suppressive effect on feeding in diabetic and non-diabetic rats. Physiol. Behav. 34:391-393; 1985. 23. Lowy, M. T.; Maickel, R. P.; Yim, G. K. W. Naloxone reduction of stress-related feeding. Life Sci. 26:2113-2118; 1980. 24. Lubin, E.; Bodnar, R. J. Intracerebroventricular alloxan reduces 2-deoxy-D-glucose analgesia. Physiol. Behav. 42:465-470; 1988. 25. Lubin, E.; Bodnar, R. J. Differential actions of central alloxan upon opioid and nonopioid antinociception in rats. Pharmacol. Biochem. Behav. 34:511-516; 1989. 26. McLean, S.; Hoebel, B. G. Feeding induced by opiates injected into the paraventricular hypothalamus. Peptides 4:287-292; 1983. 27. Murnane, J. M.; Ritter, S. Intraventricular alloxan impairs feeding to both central and systemic glucoprivation. Physiol. Behav. 34:609613; 1985. 28. Rerup, C. C. Drugs producing diabetes through damage to insulin secreting cells. Pharmacol. Rev. 22:485-518; 1970. 29. Ritter, R. C.; Slusser, P. G.; Stone, S. Brain glucoreceptor controlling feeding and blood glucose: location in the hindbrain. Science 213: 451453; 1981. 30. Ritter, S. Glucoprivation and the glucoprivic control of food intake. In: Ritter, R. C.; Ritter, S.; Barnes, C. D., eds. Feeding behavior.
ALLOXAN ALTERS NALOXONE AND CCK-8 HYPOPHAGIA
379
Orlando, FL: Academic Press; 1986:271-313. 31. Ritte.r, S.; Mumane, J. M.; Ladenheim, E. E. Glucoprivic feeding is impaired by lateral or fourth ventricular alloxan injection. Am. J. Physiol. 243:R312-R317; 1982. 32. Ritter, S.; Strang, M. Fourth ventricular alloxan injection causes feeding, but not hy~rglycemia in rats. Brain Res. 249:198-211; 1982. 33. Rossini, A. A.; Berger, M.; Shadden, J.; Cahill, G. F. Beta cell protection to alloxan necrosis by anomers of D-glucose. Science 183:424; 1974. 34. Sawynok, J.; Pinsky, C.; LaBella, F. S. On the specificity of naloione as an opiate antagonist. Life Sci. 2531621-1632; 1979. 35. Smith. G. P.: Gibbs. J.: Young. R. C. Cholecvsto~nin and intestinal satiety in the rat. Fed. Proc. 33:1146-l 149; 1!?74. 36. Spiaggia, A.; Bodnar, R. J.; Kelly, D. D.; Glusman, M. Opiate and
non-opiate mechanisms of stress-induced analgesia: Cross-tolerance between stressors. Phatmacol. B&hem. Behav. 10:761-765; 1979. Stanley, B. G.; Lanthier, D.; Leibowitz, S. F. Multiple brain sites sensitive to feeding stimulation by opioid agonists: A cannulamapping study. Pharmacol. Biochem. Behav. 31:825-832; 1989. Van der Kooy, D. Area postrema: site where CCK acts to decrease food intake. Brain Res. 295:345-347; 1984. Woods, J. S.; Leibowitz, S. F. Hypothalamic sites sensitive to morphine and naloxone: Effects on feeding behavior. Pharmacol. Biochem. Behav. 23:431-438; 1985. Woods, S. C.; McKay, L. D. Intraventricular alloxan eliminates feeding elicited by 2-deoxy-D-glucose. Science 202: 1209-1211; 1978. Zawalich, W. S.; Biedler, L. M. Glucose and alloxan interactions in the pancreatic islets. Am. J. Physiol. 224:%3-966; 1973.
37.
38. 39.
40. 41.
Brain Research Bulletin, Vol. 24, pp. 375379. +QPergamon Press plc, 1990. printed in the U.S.A.
Inhibition of Deprivation-Induced Feeding by Naloxone and Cholecystokinin in Rats: Effects of Central Alloxan DULMANIE
ARJUNE AND RICHARD
J. BODNAR’
Depa~en~ of P~ychoiogy and Neuropsy~hology Doctoral cub-Program Queens College, City University of New York, Flushing, NY 11361 Received
20 July 1989
ARJUNE, D. AND R. J. BODNAR. ln~ib~gionof depri~afian-induced feeding by naloxone and c~o~e~sto~injn in rats: E&cfs of central alfoxun. BRAIN RES BULL 24(3) 375-379, 1990.-Central administration of alloxan reduces the hyperphagic, but not the
hyperglycemic response to glucoprivation by presumably acting upon brain glucoreceptors or a glucoprivic control mechanism. The present stndy evaluated whether central alloxan pretreatment respectively altered the dose-dependent suppressant effects upon deprivation (24~hr)-induced feeding of naloxone (0.01-10 mg/kg, IP) and cholecystokinin octapeptide (CCK-8: l-8 pgikg, IP) in rats. Central alloxan (200 p,g, ICV) failed to alter body weight, free-feeding and deprivation-induced feeding. Both naloxone and CCK-8 produced significant dose-dependent inhibitions of deprivation-induced feeding in control rats. Central alloxan treatment significantly ionized peak nafoxone hy~phagia induced by 2.5 and 10 mgikg doses, and CCK-8 hy~phagia induced by the 1 and 4 pgkg doses. Coadministration of 3 M D-glucose, which acts as a cytopmtectant against alloxan-induced diabetes, blocked the attenuating actions of alloxan upon naloxone and CCK-8 hypophagia. These data indicate the effectiveness of central alloxan in restricting the ability of pharmacological agents to either stimulate or inhibit food intake in rats without altering basal intake or body weight maintenance. Alloxan
Naloxone
Cholecystokinin
Deprivation-induced feeding
3 M D-Glucose
Rats
responses. Both the opiate receptor antagonist, naloxone (6, 7, 15) and the gut peptide, cholecystokinin octapeptide (CCK-8) (1, 16, 35) decrease deprivation-induced feeding. The relationship between glucose availability and opioid control of food intake has been established in that either genetically diabetic or streptozototin-treated mice are more sensitive to naloxone (21), and that streptozotocin-treated rats display less hyperphagia following morphine (20) or Tyr-D-ala-gly-(Me)-gly-ol-enkephalin (DAGO) (17). 2DG hyperphagia is also altered by opioid manipulations (5,23). The hyperphagic actions of glucoprivic agents (3, 9, 29) and hypophagic actions of CCK-8 (10, 11, 14, 38) depend in part upon the projection systems of the area postrema (AP), nucleus tractus solitarius (NTS) and sending dorsal vagal complex. The present studies examined whether central alloxan altered the suppression of deprivation-induced feeding by naloxone and CCK-8, and whether coadministration of 3 M D-glucose, a cytoprotectant against alloxan-induced diabetes (33,41), would reverse any alloxan-induced alterations upon hypophagia induced by naloxone or CCK-8.
WHILE peripheral administration of high doses of alloxan produces diabetes and hyperglycemia by its toxic effects upon pancreatic beta cells (8, 13, 19, 28), central administration of far lower doses of alloxan alters feeding behavior without producing hyperglyce~a (3 1,32). While central alloxan at low doses (10-20 Fg) stimulates feeding (32), higher central doses of alloxan (40-200 p.g) significantly attenuate hyperphagia induced by 2deoxy-D-glucose (2DG) without altering body weight or food intake (27, 31, 40). The laboratories of Ritter (27, 30, 31) and Woods (40) have postulated that central alloxan may impair glucoprivic feeding by inducing a toxic effect upon brain glucoreceptors in a similar manner as peripheral alloxan alters lingual glucoreception (4 1). If central alloxan is acting through such a mechanism, then it should alter other pharmacological manipulations that depend in part upon glucoprivic controls. Further, nonspecific actions of alloxan could account for the reduction in glucoprivic hyperphagia; a more convincing demonstration of alloxan’s specificity would be to diminish ph~cologic~ly induced hy~phagic
‘Requests for reprints should be addressed to Dr. Richard J. Bodnar.
375
ARJUNE AND BODNAR
METHOD
Seventy-one naive male albino Sprague-Dawley rats (400-550 g) were maintained individually in wire mesh cages on a 12-hr light: 12-hr dark schedule at ambient temperatures between 22” and 25°C with pelleted rat chow and water available ad lib. In all studies, rats were monitored daily for body weight and food intake over four consecutive days, and were divided into treatment groups by matching these variables. Each rat was administered chlorpromazine HCl (3 mg/ml normal saline/kg body weight, IP) 20 min before anesthetization with ketamine HCl (100 mg/ml sterile water/kg body weight, IM). A stainless steel 22-gauge guide cannula (Plastic Products) was stereotaxically (Kopf) aimed so that its tip was positioned 0.3 mm above the left lateral ventricle. With the incisor bar set at +5 mm, coordinates were 0.5 mm anterior to the bregma suture, 1.3 mm lateral to the sagittal suture and 3.6 mm from the top of the skull. With the guide cannula in place, a stainless steel 28-gauge internal cannula (Plastic Products) was inserted so that it protruded 0.5 mm beyond the tip of the guide cannula. Based upon group assignment, rats received either a control (5 ~1 normal saline), alloxan (200 kg/5 (*I normal saline, Sigma) or alloxan/3 M D-glucose injection by infusing the injectate using a Hamilton microsyringe and polyethylene tubing at a rate of 1 ~1 every 20 sec. The alloxan dose was chosen for its previously established efficacy in reducing 2DG hyperphagia (27,31). Following injection, the internal cannula and the guide cannula were subsequently removed, and the rat was then sutured. Two weeks were allowed for recovery from surgery and anesthetic clearance. Each rat was then again monitored daily for postoperative body weight and food intake over four consecutive days. In the first experiment, control (n=9) and alloxan-treated (n = 9) rats received five injection conditions at weekly intervals according to an incompletely counterbalanced design: a) vehicle (1 ml normal saline/kg body weight, IP), and naloxone (1 ml/kg body weight, IP, Sigma) at doses of b) 0.01 mg/kg, c) 0.1 mglkg, d) 1 .O mg/kg and e) 10.0 mg/kg. All rats were deprived of food, but were allowed ad lib access to water for 24 hr prior to each injection; preand postdeprivation body weights were also determined. Following each injection condition which occurred 1-2 hr into the light cycle, deprivation-induced intake was determined 2 and 24 hr after injection. The naloxone regimen was chosen for its previously determined dose-dependent efficacy in inhibiting deprivationinduced intake (6, 7, 15). Intake in this and all subsequent protocols was determined by weighing food pellets prior to and after each condition and adjusting for spillage. The weekly interval between conditions in this and subsequent experiments allowed full recovery of body weight. In a second experiment, control (n= 9) and alloxan-treated (n =9) rats received vehicle and naloxone (2.5 mg/kg) as described previously. Time-dependent actions of naloxone hypophagia were assessed 30, 60 and 120 min at a dose considered to act specifically as an opiate receptor antagonist (34). In a third experiment, control (n = 8) and alloxan-treated (n= 9) rats received five injection conditions of CCK-8 (1 ml/kg body weight, Peninsula) at doses of 0, 1, 2, 4 and 8 (*g/kg as described previously. Deprivation-induced intake was determined 0.5, 2 and 24 hr after injection as per its previously determined efficacy in inhibiting deprivation-induced intake (1, 16, 35). In the fourth experiment, control rats (n = 9) or rats coadministered alloxan with a 3 M D-glucose solution (n=9) received three injection conditions as described previously: a) vehicle, b) naloxone (10 mg/kg) and c) CCK-8 (4 kg/kg). Deprivation-induced intake was determined at 0.5, 2 and 24 hr after injection. Histological confirmation of correct cannula placement was conducted in all experiments; only animals with verifiable place-
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NALOXONE DOSE (mg/kg) 1. Alterations in deprivation (24 hr)-induced intake (2 hr: Mean, SEM) by systemic naloxone (0.01-10 mgkg, IP) in control (5 (~1normal
FIG.
saline, ICV, open circles) and alloxan-treated (200 kg, ICV, closed circles) rats. The open stars denote significant differences in intake between naloxone and corresponding vehicle injections. The enclosed star denotes significant differences between control and alloxan treatments for that condition.
ments in the lateral ventricle were used in the data analysis. Split-plot analyses of variance assessed significant effects upon individual intake points and body weights; Dunnett and Dunn comparisons were used to discern differences between vehicle and naloxone, and control and alloxan treatments, respectively. RESULTS
In all experiments, treatment with alloxan either alone or coadministered with 3 M D-glucose failed to alter postoperative body weights relative to control treatment two weeks after injection. Alloxan treatments also failed to alter food intake following food deprivation paired with a vehicle injection. All treatments failed to alter food intake 24 hr after reintroduction of food. Finally, alloxan treatments failed to alter body weight loss induced by deprivation, or subsequent body weight gain. First, naloxone significantly and dose-dependently decreased deprivation-induced intake 2 hr after reintroduction of food, F(4,64) = 17.43, p
ALLOXAN ALTERS NALOXONE
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differ in rats receiving control injections or alloxan coadministered with 3 M D-glucose (Fig. 4, left panel). The significant decreases induced by CCK-8 (4 &kg) in deprivation-induced intake 0.5 hr after reintroduction of food, F(1,16)=38.87, p
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DISCUSSION
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ALLOXANNEHICLE CONTROUNALOXONE
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ALLOXANINALOXONE
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TIME (h) FIG. 2 Time-dependent alterations in deprivation-induced intake by systemic naloxone (2.5 mg/kg) in control and alloxan-treated rats. The open and enclosed stars respectively denote significant differences in intake between naloxone and corresponding vehicle injections, and between control and alloxan treatments for that condition.
illustrates the significant inhibition of food intake in controls by CCK-8 doses of 1 (32%), 2 (41%), 4 (58%) and 8 (64%) *g/kg. Alloxan-treated rats only displayed significant inhibition of deprivation-induced intake following CCK-8 doses of 2 (34%), 4 (33%) and 8 (54%) pg/kg. Alloxan significantly diminished CCK-8 hypophagia induced by the 4 p&kg dose, and eliminated CCK-8 hypophagia induced by the 1 l&kg dose. Fourth, coadministration of alloxan with 3 M D-glucose reversed alloxan-induced deficits. The significant decreases induced by naloxone (10 mg/kg) in deprivation-induced intake 2 hr after reintroduction of food, F( 1,16) = 10.00, p
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AND CCK-8 HYPOPHAGIA
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CONTROL ALLOXAN
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CCK-6 DOSE (w/kg) FIG. 3. Alterations in deprivation-induced intake (0.5 hr: Mean, SEM) by systemic cholecystokinin octapeptide (CCK-8: l-8 p&kg, IP) in control and alloxan-treated rats. The open and enclosed stars respectively denote significant differences in intake between CCK-8 and corresponding vehicle injections, and between control and alloxan treatments for that condition.
Central alloxan pretreatment significantly diminished the suppressant effects of both naloxone and CCK-8 upon deprivationinduced intake in rats. Naloxone hypophagia induced by the 10 mg/kg (62% inhibition) and 2.5 mg/kg (33% inhibition) doses in control rats was significantly reduced in alloxan-treated rats to 41% and 17%, respectively. However, naloxone hypophagia at lower doses or at earlier time points than 2 hr appeared unaffected by alloxan pretreatment. While alloxan failed to alter the peak (8 pgkg) inhibitory effects of CCK-8 upon deprivation-induced intake, it eliminated CCK-8 hypophagia at a 1 *g/kg dose, and suppressed CCK-8 hypophagia at a 4 pg/kg dose (alloxan: 33%, control: 58%). Alloxan-induced reductions in naloxone and CCK-8 hypophagia appeared selective for the following reasons. Coadministration with 3 M D-glucose reversed the alloxan-induced deficits upon naloxone and CCK-8 hypophagia, just as it acts as a cytoprotectant against alloxan-induced diabetes (33,41). Thus, alloxan appears to be working through gluco-sensitive mechanisms, although periventricular damage following alloxan has been noted using silver stain degeneration techniques (S. Ritter, personal communication). Alloxan failed to affect body weight and food intake under baseline and deprivation conditions, indicating that the animals were not debilitated, and could respond normally to a physiological challenge. Therefore, alloxan does not appear to alter either normal free feeding or deprivation-induced intake in and of itself, but appears to restrict the ability of pharmacological and physiological agents to either stimulate (e.g., 2-deoxy-D-glucose) (27, 31, 32, 40) or inhibit (e.g., naloxone and CCK-8) intake. That central alloxan treatment decreases the hypophagic properties of naloxone provides further support for a selective interaction with opioid systems. Alloxan decreased both 2DG analgesia (24) and hyperphagia (27,31) which are opioid-mediated (4,5,23, 36). Further, central alloxan treatment decreases morphine, but not nonopioid swim analgesia (25). The decreased sensitivity to naloxone hypophagia in alloxan-treated rats contrasts with the increased sensitivity to naloxone hypophagia in streptozotocintreated mice (21). This difference may be attributed to the novel test environment since streptozotocin-treated rats show increased sensitivity to naloxone hypophagia when tested in a novel environment, but are insensitive to naloxone hypophagia when tested in their home cage (22). This latter effect parallels the present findings and testing conditions, but our effects occurred in the absence of hyperglycemia, increased body weights and increased food intakes induced by streptozotocin (20). The present study cannot address whether central alloxan effects upon naloxone and CCK-8 hypophagia are working through similar sites and/or mechanisms of action. The medial hypothalamus, and particularly the paraventricular nucleus appears most sensitive to stimulation of intake by opioid agonists (18, 26, 37, 39). CCK-8 hypophagia can be induced following peripheral (1, 16, 35) or central (12) administration. The satiating actions of peripheral CCK-8 can be blocked by lesions placed in the AP (38), NTS (14), NTS-hypothalamic pathway (1 l), and the paraventricular and dorso-medial hypothalamic nuclei (2,lO). The above hindbrain structures also appear to modulate 2DG hyperphagia (3, 9, 29). Thus, medial hypothalamic and hindbrain structures may
ARJUNE AND BODNAR
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q CONTROL n ALLoxANl 3M O-GLUCOSE
0
NALOXONE DOSE (mglkg)
4
CCK-8 DOSE (pg!kg)
FIG. 4. Alterations in deprivation-induced intake (Mean, SEM) by systemic naloxone (2 hr: 10 mgikg, IP, left panel) and CCK-8 (0.5 hr: 4 kg/kg, IP, right panel) in control rats and rats coadministered alloxan in 3 M D-glucose. The stars denote significant differences in intake between either naloxone or CCK-8 and corresponding vehicle injections.
modulate inhibition of food intake by naloxone and CCK-8, and may also contain those gluco-sensitive mechanisms sensitive to central alloxan effects.
ACKNOWLEDGEMENTS This research was supported by NIH DA04 194-O 1A2 and PSCiCUNY Grants 668244 and 669213 to R.J.B.
REFERENCES 1. Antin, J.; Gibbs, J.; Holt, J.; Young, R. C.; Smith, G. P. Cholecystokinin elicits the complete behavioral sequence of satiety in rats. J. Comp. Physiol. Psychol. 89:784-790; 1975. 2. Bellinger, L. L.; Bemardis, L. L. Suppression of feeding by cholecystokinin but not bombesin is attenuated in dorsomedial hypothalamic nuclei lesioned rats. Peptides 5:547-552; 1984. 3. Bird, E.; Cardone, C. C.; Conteras, R. J. Area postrema lesions disrupt food intake induced by cerebra-ventricular infusions of 5thioglucose in the rat. Brain Res. 270:193-196; 1983. 4. Bodnar, R. J.; Kelly, D. D.; Glusman, M. 2-Deoxy-D-glucose analgesia: Influences of opiate and non-opiate factors. Pharmacol. Biochem. Behav. 11:297-301; 1979. A. L.; 5. Bodnar, R. J.; Kramer, E.; Simone, D. A.; Kirchgessner, Scalisi, J. Dissociation of analgesic and hyperphagic responses following 2-deoxy-D-glucose. Int. J. Neurosci. 21:225-236; 1983. 6. Brands, B. J.; Thomhill, J. A.; Hirst, M.; Gowdey, C. W. Suppression of food intake and body weight gain by naloxone in rats. Life Sci. 24:1773-1778; 1979. 7. Brown, D. R.; Holtzman, S. G. Suppression of deprivation induced food and water intake in rats and mice by naloxone. Pharmacol. Biochem. Behav. 11:567-583; 1979. 8. Chang, A. Y.; Diani, A. R. Chemically and hormonally-induced diabetes. In: Volk, B. W.; Arquilla, E. R., eds. The diabetic pancreas, 2nd ed. New York: Plenum; 1985:415-438. 9. Conteras, R. J.; Fox, E.; Drugovich, M. L. Area postrema lesions produce feeding deficits in the rat: Effects of preoperative dieting and 2-deoxy-D-glucose. Physiol. Behav. 29:875-884; 1982. 10. Crawley, J. N.; Kiss, J. Z. Paraventricular nucleus lesions abolish the inhibition of feeding induced by systemic cholecystokinin. Peptides 6:133-137; 1985. 11. Crawley, J. N.; Kiss, J. Z.; Mezey, E. Bilateral midbrain transections block the behavioral effects of cholecystokinin on feeding and exploration in rats. Brain Res. 232:316-321; 1984. 12. Della-Fera, M. A.; Baile, C. A. Cholecystokinin octapeptide: continuous picomole injections into the cerebral ventricles of sheep suppress feeding. Science 206:471473; 1979. 13. Dunn, J. S.; Sheehan, H. L.; McLetchie, N. G. Necrosis of islets of Langerhans produced experimentally. Lancet 244:484-+87; 1943. 14. Edwards, G. L.; Ladenheim, E. E.; Ritter, R. C. Dorsal hindbrain participation in cholecystokinin-induced satiety. Am. J. Physiol. 251:R971-R977; 1986.
15. Frenk, H.; Rogers, G. H. The suppressant effects of naloxone on food and water intake in the rat. Behav. Neural Biol. 26:23-40; 1979. 16. Gibbs, J.; Young, R. C.; Smith, G. P. Cholecystokinin decreases food intake in rats. J. Comp. Physiol. Psychol. 84:488495; 1973. 17. Gosnell, B. A.; Grace, M.; Billington, C. J.; Levine, A. S. Effects of streptozotocin-induced diabetes on feeding induced by centrallyadministered opioid agonists. Life Sci. 45:31-40; 1989. 18. Gosnell, B. A.; Morely, J. E.; Levine, A. S. Opioid-induced feeding: localization of sensitive brain sites. Brain Res. 369: 177-184; 1986. 19. Gunnarsson, R.; Hellerstrom, C. Acute effects of alloxan on the metabolism and insulin secretion of the pancreatic beta cells. Horm. Metab. Res. 5:404-409; 1973. 20. Levine, A. S.; Grace, M.; Billington, C. J.; Gosnell, B. A.; Krahn, D. D.; Brown, D. M.; Morley, J. E. Effect of morphine and nalmefene on energy balance in diabetic and non-diabetic rats. Pharmacol. Biochem. Behav. 29:495-500; 1988. 21. Levine, A. S.; Morley, J. E.; Brown, D. M.; Handwerger, B. S. Extreme sensitivity of diabetic mice to naloxone-induced suppression of food intake. Physiol. Behav. 28:987-989; 1982. 22. Levine, A. S.; Morley, J. E.; Kneip, J.; Grace, M.; Brown, D. M. Environmental modulates naloxone’s suppressive effect on feeding in diabetic and non-diabetic rats. Physiol. Behav. 34:391-393; 1985. 23. Lowy, M. T.; Maickel, R. P.; Yim, G. K. W. Naloxone reduction of stress-related feeding. Life Sci. 26:2113-2118; 1980. 24. Lubin, E.; Bodnar, R. J. Intracerebroventricular alloxan reduces 2-deoxy-D-glucose analgesia. Physiol. Behav. 42:465-470; 1988. 25. Lubin, E.; Bodnar, R. J. Differential actions of central alloxan upon opioid and nonopioid antinociception in rats. Pharmacol. Biochem. Behav. 34:511-516; 1989. 26. McLean, S.; Hoebel, B. G. Feeding induced by opiates injected into the paraventricular hypothalamus. Peptides 4:287-292; 1983. 27. Murnane, J. M.; Ritter, S. Intraventricular alloxan impairs feeding to both central and systemic glucoprivation. Physiol. Behav. 34:609613; 1985. 28. Rerup, C. C. Drugs producing diabetes through damage to insulin secreting cells. Pharmacol. Rev. 22:485-518; 1970. 29. Ritter, R. C.; Slusser, P. G.; Stone, S. Brain glucoreceptor controlling feeding and blood glucose: location in the hindbrain. Science 213: 451453; 1981. 30. Ritter, S. Glucoprivation and the glucoprivic control of food intake. In: Ritter, R. C.; Ritter, S.; Barnes, C. D., eds. Feeding behavior.
ALLOXAN ALTERS NALOXONE AND CCK-8 HYPOPHAGIA
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Orlando, FL: Academic Press; 1986:271-313. 31. Ritte.r, S.; Mumane, J. M.; Ladenheim, E. E. Glucoprivic feeding is impaired by lateral or fourth ventricular alloxan injection. Am. J. Physiol. 243:R312-R317; 1982. 32. Ritter, S.; Strang, M. Fourth ventricular alloxan injection causes feeding, but not hy~rglycemia in rats. Brain Res. 249:198-211; 1982. 33. Rossini, A. A.; Berger, M.; Shadden, J.; Cahill, G. F. Beta cell protection to alloxan necrosis by anomers of D-glucose. Science 183:424; 1974. 34. Sawynok, J.; Pinsky, C.; LaBella, F. S. On the specificity of naloione as an opiate antagonist. Life Sci. 2531621-1632; 1979. 35. Smith. G. P.: Gibbs. J.: Young. R. C. Cholecvsto~nin and intestinal satiety in the rat. Fed. Proc. 33:1146-l 149; 1!?74. 36. Spiaggia, A.; Bodnar, R. J.; Kelly, D. D.; Glusman, M. Opiate and
non-opiate mechanisms of stress-induced analgesia: Cross-tolerance between stressors. Phatmacol. B&hem. Behav. 10:761-765; 1979. Stanley, B. G.; Lanthier, D.; Leibowitz, S. F. Multiple brain sites sensitive to feeding stimulation by opioid agonists: A cannulamapping study. Pharmacol. Biochem. Behav. 31:825-832; 1989. Van der Kooy, D. Area postrema: site where CCK acts to decrease food intake. Brain Res. 295:345-347; 1984. Woods, J. S.; Leibowitz, S. F. Hypothalamic sites sensitive to morphine and naloxone: Effects on feeding behavior. Pharmacol. Biochem. Behav. 23:431-438; 1985. Woods, S. C.; McKay, L. D. Intraventricular alloxan eliminates feeding elicited by 2-deoxy-D-glucose. Science 202: 1209-1211; 1978. Zawalich, W. S.; Biedler, L. M. Glucose and alloxan interactions in the pancreatic islets. Am. J. Physiol. 224:%3-966; 1973.
37.
38. 39.
40. 41.