Vol. 8(1):109-115, 1991

DOMESTIC ANIMAL ENDOCRINOLOGY

EFFECTS OF NALOXONE ON AD LIBITUM INTAKE AND PLASMA INSULIN, GLUCOSE, AND FREE FATTY ACIDS IN MAINTENANCE-FED SHEEP 1 F.K. Alavi*, J.P. McCann*, S. Sangiah*, and A. Mauromoustakos**

*Department of Physiological Sciences College of Veterinary Medicine Oklahoma State University Stillwater, OK 74078-0353 and

**Agricultural Statistics Laboratory University of Arkansas Fayetville, AR 72701 Received April 23, 1990

ABSTRACt The dose-dependent effects of naloxone on feed intake, and plasma chemicals (insulin, glucose, FFA) purportedly involved in feed intake regulation, were determined in 16-hr fasted sheep that were lean and chronically fed maintenance. Dorset ewes (n = 5) were treated with 0 (saline), 0.3, 1 or 3 mg/kg of naloxone in a generalized randomized block experiment with at least 7 d between successive doses. Feed intakes and plasma insulin, glucose and FFA were determined frequently during 24 hr of ad libitum intake after each naloxone treatment. The 0.3, 1 and 3 mg/kg doses of naloxone reduced (P < 0.01) the 4-hr feed intake by 30, 40, and 60% respectively, whereas the initial feed intake (10 min) was decreased (P < 0.05) 45% only by 3 mg/kg naloxone. However, total 24-hr intakes were similar across all doses because intakes between 4 and 24 hr of feeding in sheep treated with 0.3 (839 g), 1.0 (802 g) and 3.0 (1330 g) mg/kg naloxone exceeded (P < 0.01) that in saline-treated sheep (391 g). Feeding-induced changes in plasma insulin, glucose and FFA concentrations were independent of naloxone treatment, suggesting that endorphinergic control of feed intake may not involve coincidental changes in plasma insulin, glucose and FFA levels which are thought to play a role in systemic regulation of appetite in animals. The endorphinergic regulation of appetite in sheep may involve the central nervous system, rather than peripheral opiate mechanisms that utilize blood-borne signals. Further, the ability of naloxone to suppress appetite in sheep appears inversely related to the duration of fasting or severity of negative energy balance. INTRODUCTION Changes in plasma levels of insulin, glucose and free fatty acids (FFA) are thought to play a role in systemic regulation of feed intake in animals (1,2,3). Endogenous opiates and their receptors also play important roles in the regulation of feed intake in p h y s i o l o g i c a l a n d p a t h o l o g i c a l c o n d i t i o n s (4,5,6). O p i a t e antagonists such as naloxone and naltrexone decrease feed intake in fasted or fed animals and humans (3,7). In addition to their effects on appetite, endorphins also are capable of affecting the plasma concentrations of insulin, FFA and glucose directly via peripheral opiate receptors and(or) indirectly via opiate receptors in the central nervous system (3,8,9,10). Naloxone, therefore, may suppress feed intake by direct effects within the CNS alone or in combination with naloxoneinduced c h a n g e s in the concentrations of p l a s m a chemicals such as insulin, glucose and FFA that serve as peripheral signals in appetite regulation. Conversely, plasma glucose itself may influence appetite by regulating the activity of central opiate receptors in rats (11). Copyright © 1991 by Domendo, Inc,

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ALAVI, McCANN, SANGIAH AND MAUROMOUSTAKOS

Endorphinergic regulation of appetite may differ a m o n g species and betwt_'t:n animals of the same species that are in differing physiological states. For example, doses of naloxone that decreased feed intake in satiated chickens were ineflcctive in 12-hr fasted chickens (12). In these and other similar studies in domestic animals (13), the endorphinergic link between feed intake and changes in plasma insulin, glucose and FFA were not examined. Therefore, the obiectives of this study were to determine d o s e - d e p e n d e n t inhibitory effects of naloxone on a d libiturn intake in lean sheep fasted overnight, and whether effects of naloxone on feed intake were dissociated from concomitant effects of naloxone on plasma concentrations of insulin, glucose and FFA. MATERIALS AND METHODS

Dorset ewes (n = 5), 3 to 4 years of age and weighing 36 + 2 kg, were housed individually in a room with constant light and temperature (23-25 C). Except on experimental days, sheep were fed a maintenance intake (540 g) of a pelleted haygrain feed (14% CP; 12% fibre, 63% TDN; 93% DM) in two equal amounts at 0900 hr and 1700 hr and 90 g of hay at 0900 hr. All feed routinely was c o n s u m e d within 30 min of feeding. Previous work showed that sheep fed this ration were in zeroenergy balance (14,15) as indicated in this study by steady-state b o d y weights over p r o l o n g e d time intervals of at least several m o n t h s b e f o r e a n d during data collection. The weight-to-height ratio in these Dorset ewes indicated a lean body condition that has b e e n associated with a stripped carcass lipid content of about 23% and a live b o d y lipid content of 16% to 20% (14,15). Five of twenty four sequences possible for treatments of 0 (saline), 0.3, 1.0 and 3.0 m g / k g naloxone were assigned to sheep in a generalized block design with sheep considered as blocks. At least 7 d elapsed between successive treatments. Naloxone hydrochloride (DuPont, Wilmington, Delaware) was prepared fresh in sterile saline (5 ml) as needed on each experimental day, and was injected iv by jugular vein catheter 5 rain before 16-hr fasted sheep were allowed a d libitum intake of pellets for the ensuing 24 hr. A sham trial consisting of blood sampling and 24 hr of a d libitum feeding was d o n e in all s h e e p to acclimate t h e m to e x p e r i m e n t a l p r o c e d u r e s . Feed intakes w e r e d e t e r m i n e d and b l o o d s a m p l e s obtained at 10, 20, 40 and 60 rain and 2, 4, 8, 12, 16 and 24 hr of a d libitum feeding; additional blood samples were taken 30, 20 and 5 min before a d libitum feeding. Blood samples were collected via jugular catheter that was inserted at least 12 hr before sample collections. Plasma samples were stored (-22 C) after centrifugation (4 C) of whole blood for 20 rain at 1000 x g. Glucose and FFA concentrations in plasma were determined using enzymatic colorimetric assays (15,16). Plasma concentrations of insulin w e r e d e t e r m i n e d in duplicate by a validated solid-phase radioimmunoassay (16). Treatment effects of animal, naloxone dose, sample time and their interactions were tested by repeated measures analysis of variance using the General Linear Model (GLM) procedure in SAS (17). An analysis of covariance model of SAS for split-unit and repeated measures (18) was done to adjust plasma concentrations of insulin, glucose and FFA for differences in feed intakes a m o n g treatment groups; feed intake was considered as the covariate changing with the subunit levels (time). Data are presented as mean _+ SE. RESULTS Saline-treated sheep, that previously had been meal fed a maintenance intake,

APPETITE REGULATION IN SHEEP

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Table 1. MEAN ( ± SE) CUMtILAT~VEFEEl) INTAKE (G) IN LEAN SHEEP (N=5) TREATED WITH NALOXONE AND FED AD ISB/~t.:' D o s e of N a l o x o n e ( m g / k g ) Time 10 20 40 60 2 4 8 12 16 24

Saline

min min min min h h h h h h

630 885 908 947 1,216 1,397 1,515 1,615 1,699 1,788

_+ _+ +_ +_ _+ + +_ -+ _+

0.3

29" 82" 84 ~' 104 ~' 149 b 216" 220 ~' 194 ~' 166 t' 137"

649 718 730 765 822 987 1,225 1,501 1,684 1,826

1.0

-+ 39" + 64 h _+ 58" -+ 64" + 69' -+ 104' -+ 123 ~ -+ 95 ~" -+ 69 t' -+ 56 h

690 718 718 720 769 836 1,066 1,329 1,489 1,638

3.0

_+ 31" + 40" _+ 40" + 40" + 61' -+ 74 ~ + 114 'a -+ 129 ~d +_ 159' -+ 154 '~

344 392 392 475 482 560 849 1,151 1,443 1,890

+ + -+ -+ -+ -+ ++ -+ -+

79' 115 ~ 114' 114' 117 ~' liP' 171 ~ 157 ~j 15Z' 85 h

:'Sheep w e r e a l l o w e d 24-hr a d l i b i t u m intake after a 16-hr fast. '*~JMeans w i t h i n a time p e r i o d w i t h similar superscripts are not different (P > 0.05).

consumed 1.79 kg of a high-energy diet during 24 hr of a d libitum feeding (Table 1). Approximately 50% and 80% of their total 24-hr intake was consumed in the first 60 min and 4 hr of ad libitum intake, respectively. Relative to control intakes in saline-treated sheep, naloxone at doses of 0.3 and 1.0 mg/kg initially had no significant effect on intakes in the first 60 min of a d libitum intake, but both doses decreased (P < 0.01) the cumulative 2-hr and 4-hr intakes by approximately 35% (Table 1; Fig 1). In contrast, the highest dose of n a l o x o n e (3 m g / k g ) had immediate (10 min) and protracted (4 hr) inhibitory effects on feed intake. Doses of naloxone used had no observable effects on the gross motor behaviour or wellbeing of the sheep. Preliminary experiments by us in two sheep showed that 10 mg/kg naloxone clearly affected motor behaviour adversely. Compared with saline-treated sheep, 0.3 and 1.0 mg/kg naloxone decreased (P < 0.01) cumulative intakes during the first 8 to 12 hr of a d libitum intake, whereas

FEED INTAKE

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ALAVI, McCANN, SANGIAH AND MAUROMOUSTAKOS

3 m g / k g n a l o x o n e decreased (P < 0.01) cumulative intake during the first 1" t() l() hr of a d libitum intake. A h h o u g h 16-hr cumulative intakes in rt;d()xt)nc-lrc;l~{'d s h e e p generally w e r e less than those in saline-treated sheep, the mc;m ~ t c ~l intake b e t w e e n q and 16 hr in s h e e p treated with 0,3 m g , k g (2.0 g mint, 1.() m g / k g (2.7 g/rain) or ,~.0 m g / k g (3.7 g/rain) of naloxtme e x c e e d e d (P < ().(I-S) that during saline treatment (1.2 g/rain). These results suggest that pretreatment with n a l o x o n e effectively suppressed intake only in the first 2 to ~ hr of ad IibitHm intake: thereafter the effects ()f n;flox()ne w,'ancd and a r e b o u n d respt)nsc was ol)servcd as evidenced by greater ct)nsun]ptit)n rate in n:doxt)ne-treated than in contr()l sheep. Consequently, tt)tal intakes at 2,} hr were similar i~] all groups ()1 s h e e p (see Table 1 ;in(] Fig. 1). Plasma concentrations ()f insulin (38 _+ 7 z's 95 _+ 22 flnot/ml) and gluct)se (2. ~ _+ 0.2 rs 3.6 _+ 0.2 txm()lJml) first increased (P < 0.01) a b o v e fasting levels at :{0 rain and ~ hr, respectively, of a d lit;itum intake in saline-treated sheep (Fig. 2). Plasma FFA levels decreased (P < 0.01) from 291 ± i8 nmol/ml t() a sustained lo\~ level of 92 _+ 1,i nmol ml after 60 rain ()f ad lilHtHm int,~ke in saline-treated sheep. C o n c e n t r a t i o n s t)f insulin, glucose ;rod FFA in plasma of (td li#itum-fed s h e e p p r e t r e a t e d with three d o s e s ()f n a l o x o n e w e r e statistically similar to t h o s e in control s h e e p despite the fi~ct that feed intakes were affected hy naloxone. "I% determine nalt)xone effects per se ()n plasma varial)les, the plasma concentr~tions

PLASMA VARIABLES IN NALOXONE-TREATED I FFA 600

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t:ig, 2. P l a s m a c o n c e n t r a t i o n s ( m c a n _+ SE) o f frec riley acids, g l u c o s e a n d insulin b e f o r e (fa,';tcd) a n d after (fed) n a l o x o n e t r e a t m e n t in lean s h e e p (n = 5). A r r o w s i n d i c a t e n a l o x o n e injection S rain b c f o r c start o f 2,i hr o f a d libitum f e e d i n g . T h e p o o l e d s t ; m d a r d e r r o r (PSE) for insulin c o n c e n t r a t i o n s a r c s h o w n for e a c h t r e ~ t m e n t g r o u p : s t a n d a r d e r r o r s ( b a r ) at 16 a n d 24 hr ;ire s h o w n b e c a u s e m e a n c o n c e n t r a t i o n s d i f f e r e d t h e m o s t at t h e s e t i m e s . S t a n d a r d e r r o r b a r s l()r g l u c o s e a n d FFA w c r c n u m e r i c a l l y c o n t a i n e d w i t h i n s y m b o l s fl)r m e a n c o n c e n t r a t i o n , i n d i c a t e d .

APPETITE REGULATION IN SHEEP

113

of insulin, glucose and FFA were adjusted statistically for naloxone-induced differences in feed intake (18,19). This analysis showed significant covariate relationship between plasma variables and feed intake across sheep in all treatment groups. The adjusted treatment means for glucose and FFA remained unaffected by naloxone treatment, but the highest dose of naloxone tended (P = 0.09) to increase plasma insulin concentrations independent of feed intake. Collectively, results suggest that the inhibitory effects of naloxone on appetite in sheep likely were independent of peripheral feedback signals in plasma involving insulin, glucose, or FFA. DISCUSSION

This study demonstrated dose-dependent inhibitory effects of naloxone on ad libitum intake in 16-hr fasted lean sheep that routinely had been meal fed a maintenance intake. However, only the highest dose of naloxone (3 mg/kg) had immediate inhibitory effects on feed intake. Lower doses of naloxone did not reduce intake significantly until sheep had consumed feed in the initial hr of a d libitum intake. The behavioral response of feed intake to low doses of naloxone in this study apparently was modified with time as animals ate. One interpretation is that the strong endorphinergic drive for hunger in our sheep could be antagonized immediately only by high dose naloxone, and that low dose naloxone became effective with time because endorphinergic drive for eating diminished as the animals consumed feed. This postulate is strengthened by comparing our results with those of Baile et al (13) who determined dose-dependent effects of naloxone in 4-hr fasted sheep that previously were maintained on a d libitum intake. Baile et al. (13) reported that as little as 0.03 mg/kg naloxone suppressed 2-hr intake in their sheep, whereas much higher doses of 0.3 and 1.0 mg/kg naloxone were ineffective in our study. Fasting an overfed animal for 4 hr (13) should result in less hunger drive than fasting maintenance-fed animals for 16 hr, as was done in our study. The degree of negative energy balance, therefore, would seem to be directly related to the endorphinergic drive for hunger in sheep, as illustrated by responses to naloxone in this study and that of Baile et al. (13). A further consideration is that our sheep purposefully were lean, whilst those of Baile et al. (13) should have been fatter, relatively, because their sheep chronically were overfed. Preliminary data from this laboratory in lean and obese Rambouillet ewes suggest that body condition p e r se alters opiate regulation of appetite in sheep (Alavi F.K., McCann J.P., Sangiah S., unpublished data). Additionally, it should be noted that the supraphysiological dose of 3 mg/kg of naloxone was incapable of completely suppressing intake in these sheep. In broiler chicks, doses of naloxone as high as 10 mg/kg had no effect whatsoever on intake in 12-hr fasted birds (12). That intake was not suppressed completely by high dose naloxone in fasted sheep or chicks suggests that opiate and nonopiate systems (e.g., monaminergic and gaba-receptors) in the CNS are involved to different degrees in initiating feeding in hunger-driven sheep and chicks. Girard and coworkers reported that initiation of feeding in sheep may involve neurons in the medial hypothalamus that are responsive to gamma-aminobutyric acid (20,21). This study also sought to determine if the inhibitory effects of naloxone on feed intake were mediated in part by changes in the plasma levels of insulin, glucose, or FFA. Increases in the concentrations of insulin or glucose are purportedly feedback signals for satiety, whereas increases in plasma FFA may have a role in hunger drive (3,22). Our results showed that dose-dependent effects of naloxone

114

ALAVI, McCANN, SANGIAH AND MAUROMOUSTAKOS

on appetite in sheep were manifested without concomitant naloxonc-induc