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Reduced Food Intake Following Intracerebroventricular Administration of a Low Molecular Weight Fraction of Plasma from Free-Feeding Fowl
PHYSIOLOGY AND REPRODUCTION Reduced Food Intake Following Intracerebroventricular Administration of a Low Molecular Weight Fraction of Plasma from Free-Feeding Fowl P. A. SKEWES, D. M. DENBOW1, M. P. LACY, and H. P. VAN KREY Department of Poultry Science, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061 (Received for publication February 25, 1985)
1986 Poultry Science 6 5:172-176 INTRODUCTION Regulation of food intake has generally been ascribed t o well-defined areas within t h e h y p o t h a l a m u s . Lesioning (Brobeck et al, 1943) and anesthetizing (Epstein, 1960) t h e ventromedial h y p o t h a l a m u s (VMH), or electrically stimulating t h e lateral h y p o t h a l a m u s (LH) (Miller, 1 9 6 0 ) , increased food c o n s u m p t i o n in t h e rat. On t h e o t h e r hand, food intake was reduced b y stimulating t h e VMH (Krasne, 1962) or b y lesioning t h e LH (Anand and Brobeck, 1 9 5 1 ) . Similarly, hyperphagia and obesity occurred in chickens (Lepkovsky and Yasuda, 1966) and White-throated sparrows (Zonotrichia albicollis) (Kuenzel and Helms, 1970) following lesioning of t h e VMH, whereas hypophagia and weight loss occurred in t h e chicken (Smith, 1 9 6 9 ) and t h e White-throated sparrow (Kuenzel, 1972) following lesioning of t h e LH.
broventricular (ICV) injection of a narrowspectrum molecular weight fraction of h u m a n serum into 96-hr fasted rats reduced food intake (Knoll, 1979). In t h e domestic fowl, food intake was reduced following ICV inj e c t i o n of plasma collected from free-feeding birds and c o n c e n t r a t e d t o t w o or four times n o r m a l c o n c e n t r a t i o n , while injection of c o n c e n t r a t e d plasma collected from 24-hr fasted birds did n o t affect food i n t a k e (Skewes et al, 1 9 8 4 ) . It appears, therefore, t h a t t h e h y p o t h a l a m u s m a y control food intake by m o n i t o r i n g t h e levels of various plasma comp o n e n t s . Based on these reports, t h e objective of this study was t o d e t e r m i n e t h e molecular weight range of a factor present in t h e plasma of t h e free-feeding domestic fowl t h a t inhibits food intake.
The n a t u r e of t h e factor acting u p o n t h e h y p o t h a l a m u s in vivo has n o t been d e t e r m i n e d , although current evidence suggests t h a t t h e factor m a y be present in t h e blood. Intracere-
1
To whom correspondence should be addressed. 172
MATERIALS AND METHODS Animal Preparation. Single C o m b White Leghorn cockerels were raised in electrically heated b a t t e r y b r o o d e r s with food and water available ad libitum. At 8 weeks of age, t h e birds were transferred t o individual cages. Following sodium p e n t o b a r b i t a l anesthesia (25 mg/kg), a 23-gauge thin-walled stainless steel quide cannula was stereotaxically i m p l a n t e d i n t o t h e right lateral ventricle as described b y
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ABSTRACT Plasma was collected from free-feeding Leghorn cockerels and partitioned by gel filtration into fractions of different molecular weight ranges. The individual fractions were then lyophilized and reconstituted to four times the original concentration. The plasma was administered to 10-week-old Leghorn cockerels via a stainless steel guide cannula, stereotaxically implanted into the lateral cerebral ventricle. Sated cockerels received 10 nl intracerebroventricular (ICV) injections of one of the concentrated plasma fractions or of a control injection of artificial cerebrospinal fluid. Food and water intake were monitored following injection. Food intake was significantly decreased by the ICV injection of plasma fractions less than 1500 mol wt, whereas water consumption was not significantly different from that of the controls. The 1500 to 5000 mol wt fraction and the fraction above 5000 did not alter either food or water intake. These results suggest that the plasma of free-feeding domestic fowl contains a low molecular weight factor that is involved in the regulation of food intake. (Key words.- food intake, satiety factor, intracerebroventricular, anorexigenic factor, chicken, plasma)
REDUCED FOOD INTAKE TABLE 1. Experimental
173
treatments
Experiment 1
2
Control 1 Whole plasma2 <5000 mol wt plasma2 >5000 mol wt plasma2
3 Control 1 <5000 mol wt plasma2 5000-1500 mol wt plasma2 <1500moi wt plasma2
Artificial cerebrospinal fluid.
2
Plasma collected from free-feeding birds.
3
Plasma collected from 24-hr fasted birds.
Denbow et al. (1981). The guide cannula was occluded with a 27-gauge stylet between injections. Following a 3-day recovery period, validation of cannula location was verified by monitoring the colonic temperature response to an injection of 67 fig of norepinephrine in 10 /A of artificial cerebrospinal fluid (aCSF) administered via the guide cannula. Only those birds exhibiting a decrease in body temperature of 1.0 C or greater were used in the experiments. Plasma Preparation. Prior to each experiment, blood was collected via cardiac puncture from free-feeding and 24-hr fasted cockerels. Samples from each group were pooled and centrifuged at 3000 g for 20 min. The plasma was separated into fractions using gel filtration. A PD-10 column (Pharmacia Fine Chemicals) containing 5 cm of Sephadex G-25 or G-15 gel (Pharmacia Fine Chemicals) was used to divide the plasma at 5000 mol and 1500 mol wt cutoff points, respectively, yielding four fractions (<1500 mol wt, 1500 to 5000 mol wt, <5000 mol wt, and >5000 mol wt). Following elution with distilled water, the fractions were lyophilized and reconstituted to four times the original concentration with distilled water and stored at —20 C. Injection Procedure. Injections were made via a 27-gauge stainless steel injection cannula connected to a 10 jul Hamilton syringe. In Experiments 1 and 2, the cockerels received either 10 jul of aCSF or a plasma fraction collected from free-feeding birds (Table 1). In Experiment 3, they also received fractions of plasma collected from 24-hr fasted birds.
2 The aCSF consisted of 155 m/W Na+, 2.5 mAf Ca++, 3.7 mM K+, 2.1 m/W Mg++, 140 mM CI - and 23 mM H C 0 3 - (Anderson and Hazelwood, 1969).
Food and water intakes were monitored (by weight and volume, respectively) at 15-min intervals for the first hr, 30-min intervals for the second hour, and hourly thereafter. Statistical Design and Analysis. A repeated Latin Square design (Snedecor and Cochran, 1980) was utilized. Nonorthogonal contrasts were used to make comparisons of cumulative food and water intake between the control and treated birds at each time period. Bonferroni F values (P<.05) were used in determining statistical significance (Games, 1972). RESULTS Experiment 1. Injection of whole plasma collected from free-feeding birds and concentrated to four times normal significantly reduced cumulative food intake at 90 min postinjection (Table 2). The fraction of plasma with a molecular weight below 5000 reduced cumulative food intake at 60, 90, and 120 min postinjection; however, the fraction above 5000 mol wt did not alter food intake. Water intake was not significantly altered by any of the treatments. Experiment 2. Cumulative food intake of the birds receiving the fraction of plasma below 1500 mol wt was reduced below that of the control birds by 45, 60, 90, and 180 min postinjection (Table 3). No reduction in food intake occurred following injection with the 1500 to 5000 mol wt or the <5000 mol wt fraction. Water intake again was not altered by any treatment. Experiment 3. Birds receiving the below 1500 mol wt fraction of plasma collected from free-feeding birds consumed less food by 120 min postinjection than did the control birds, but food intake was not reduced in the birds
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1
Control 1 <1500 mol wt plasma2 <1500 mol wt plasma3
174
SKEWES ET AL.
TABLE 2. Mean cumulative food and water intake of free-feeding Single Comb White Leghorn cockerels following a 10-p.l intracerebroventricular injection of four times normal concentration of whole, <5000 molecular weight, or >5000 molecular weight fractions of plasma collected from free-feeding birds T i m e postinfection (mini) 15
30
45
60
90
120
180
240
Control 1 Whole plasma > 5 0 0 0 m o l w t plasma <5O0O m o l wt plasma SEM 2
3.7 2.5 3.2 2.4 .5
4.9 3.3 4.8 3.6 .6
6.2 4.3 5.9 4.6 .6
7.7 5.8 6.9 5.3* .5
10.5 8.0* 9.6 7.5* .7
11.5 10.4 11.4 9.0* .7
16.2 14.3 15.4 12.9 .9
18.8 18.1 18.3 15.0 1.1
Control1 Whole plasma > 5 0 0 0 m o l w t plasma < 5 0 0 0 m o l w t plasma SEM 2
1.7 1.7 1.3 .0 1.0
2.5 2.9 1.7 .4 1.1
3.3 2.9 2.5 .8 1.4
5.0 4.6 3.8 2.1 1.9
7.1 8.8 6.3 5.4 2.7
11.7 11.7 9.2 7.9 2.7
19.2 19.2 15.4 10.4 2.9
24.6 23.8 19.2 15.4 3.1
Water
1
Artificial cerebrospinal fluid.
'Standard error of the mean (n = 12). •Significantly different from control (P<.05).
TABLE 3. Mean cumulative food and water intake of free-feeding Single Comb White Leghorn cockerels following a 10-fJ.l intracerebroventricular injection of four times normal concentration of <5000 molecular weight, 1500 to 5000 molecular weight,or <1500 molecular weight fractions of plasma collected from free-feeding birds T i m e postinjection (min ) Treatment
15
30
45
90
60 Food
1
120
180
240
11.9 11.7 10.7
17.1 15.6 14.4
19.8 18.7 18.9
9.6 .7
13.4* 1.0
16.4 1.1
•
Control <5O0O m o l wt plasma 1 5 0 0 - 5 0 0 0 m o l wt plasma < 1 5 0 0 m o l wt plasma SEM 2
3.4 3.8 2.8
5.3 5.5 4.6
6.5 6.7 5.3
7.2 7.2 6.3
10.5 9.8 8.8
2.6 .4
3.8 .5
4.3* 5
5.0* .6
Control1 < 5 0 0 0 m o l wt plasma 1 5 0 0 - 5 0 0 0 m o l wt plasma < 1 5 0 0 m o l w t plasma SEM 2
1.7 .4 .4
2.9 1.3 1.7
5.0 1.7 3.8
5.4 2.9 5.4
7.5 6.7 7.5
11.3 9.6 10.8
18.3 17.9 17.9
24.2 21.7 24.6
.8 .5
1.3 .8
2.5 1.1
4.2 1.1
5.0 1.5
8.3 1.9
12.9 2.6
16.3 2.7
7.0* .6 Water i
1
Artificial cerebrospinal fluid.
'Standard error of the mean (n = 12). 'Significantly different from control (P<.05).
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Treatment
REDUCED FOOD INTAKE
receiving the same fraction collected from 24-hr fasted birds (Table 4). Water consumption was not significantly altered by either treatment.
DISCUSSION
(Knoll, 1979). Satietin was proposed to contain a limited number of peptides with molecular weights in the range of 40,000 to 60,000. It was reported that the proposed molecular weight range of satietin conflicted with the separation procedures employed, which included filtering all samples through an Amicon UM-10 membrane (known to withhold compounds with molecular weight above 10,000). The lack of a response following ICV injection of plasma collected from 24-hr fasted birds was probably the result of a reduction in the level of the factor due to the nutritional status of the donors. The concentration of the factor in the plasma may decrease in proportion to the length of time the animal has gone without feeding. This type of response has been demonstrated in the rat where the amount of food consumed by a sated recipient rat, following transfusion with blood from a fasted donor rat, was inversely proportional to the length of deprivation of the donor rat (Davis etal, 1971). The nature of the low molecular weight factor(s) present in plasma that reduces food intake when injected ICV is unknown. Numerous endogenous substances with molecular weights below 1500 have been shown to
TABLE 4. Mean cumulative food intake of free-feedingSingle Comb White Leghorn cockerels following a 10-y.l intracerebroventricular injection of four times normal concentration of <1500 molecular weight fractions of plasma collected from free-feeding or 24-hr fasted birds T i m e postinjection (min) Treatment
15
30
45
60
Control1 < 1 5 0 0 mol wt plasma 2 < 1 5 0 0 mol wt plasma 3 SEM4
1.9 1.9 1.8 .4
2.8 2.6 2.9 .6
3.9 3.6 4.0 .7
4.8 4.1 4.9 .7
Control1 < 1 5 0 0 mol wt plasma 2 < 1 5 0 0 mol w t p l a s m a 3 SEM4
1.1 1.1 .0 .5
1.7 1.7 .6 .9
2.8 3.3 2.2 1.0
3.3 4.4 2.8 1.1
120
180
240
7.4 5.8 7.8 .7
9.6 6.3* 9.4 .7
11.3 9.7 11.9 1.1
14.8 12.1 14.1 1.3
5.6 5.0 2.8 1.2
5.6 6.1 3.9 1.2
7.8 8.9 7.8 2.3
11.7 13.3 11.1 2.1
90 F o o d intake
1
Artificial cerebrospinal fluid.
2
Plasma collected from free-feeding birds.
3
Plasma collected from 24-hr fasted birds.
"Standard error of the mean (n = 9). 'Significantly different from control (P<.05).
ig;
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Concentrated plasma fractions below 5000 mol wt and below 1500 mol wt reduced food intake if the plasma had been collected from free-feeding birds, but it did not reduce food intake if the plasma had been collected from 24-hr fasted birds. The absence of a response following injection with the below 5000 mol wt fraction in Experiment 2 is to be questioned in light of the reduction in food intake by the <5000 mol wt fraction Experiment 1 and the <1500 mol wt fraction in Experiments 2 and 3. It is possible that this fraction was overdiluted when reconstituted, thereby reducing the concentration of the active factor in the fraction. It appears, therefore, that the "satiety" factor previously shown to exist in the plasma of the free-feeding domestic fowl (Skewes et al, 1984), has a molecular weight below 1500. In a similar study, a compound called "satietin", which reduced food intake when injected into the lateral ventricle of 96-hr fasted rats, was shown to exist in human serum
175
SKEWES ET AL.
176
The results of these experiments suggest that a satiety factor having a molecular weight of less than 1500 exists in the plasma of the free-feeding domestic fowl. Further, the plasma concentration of this factor appeared to be related to the nutritional status of the bird. Because water intake was not reduced by any of the treatments, it can be assumed that the effect of the treatments was specific for food intake. Whether this factor originates from the gastrointestinal tract as a secretion, e.g., cholecystokinin or bombesin, or from the absorption of nutrients, e.g., glucose, remains to be elucidated. ACKNOWLEDGMENT This study was supported, in part, by a grant from the John Lee Pratt Animal Nutrition Program. The authors would also like to thank K. E. Webb, Jr., for his technical advice.
REFERENCES Anand, B. K., and J. R. Brobeck, 1951. Hypothalamic control of food intake in rats and cats. Yale J. Biol. Med. 24:123-140. Anderson, D. K., and R. L. Hazelwood, 1969. Chicken cerebrospinal fluid: normal composition and response to insulin administration. J. Physiol. 202:83-95. Avery, D. D., and S. B. Calisher, 1982. The effects of injections of bombesin into the cerebral ventricles on food intake and body temperature in fooddeprived rats. Neuropharmacology 21:1059— 1063. Brobeck, J. R., J. Tepperman, and C. N. Long, 1943. Experimental hypothalamic hyperphagia in the albino rat. Yale J. Biol. Med. 15:831-853.
Davis, J. D., C. S. Campbell, R. J. Gallagher, and M. A. Zurakov, 1971. Disappearance of a humoral satiety factor during food deprivation. J. Comp. Physiol. Psychol. 75:476-482. Denbow, D. M., J. A. Cherry, P. B. Siegel, and H. P. Van Krey, 1981. Eating, drinking and temperature response of chicks to brain catecholamine injections. Physiol. & Behav. 27:265-269. Denbow, D. M., and R. D. Myers, 1982. Eating, drinking and temperature responses to intraventricular cholecystokinin in the chick. Peptides 3:739-743. Epstein, A. N., 1960. Reciprocal changes in feeding behavior produced by intrahypothalamic chemical injections. Am. J. Physiol. 199:969-974. Games, P. A., 1972. An improved t table for simultaneous control on g contrasts. J. Am. Statist. Assoc. 72:531. Knoll, J., 1979. Satietin: A highly potent anorexogenic substance in human serum. Physiol. & Behav. 23:497-502. Krasne, F. B., 1962. General disruption resulting from electrical stimulus of the ventromedial hypothalamus. Science 138:822-823. Kuenzel, W. J., 1972. Dual hypothalamic feeding system in a migratory bird, Zonotrichia albicollis. Am. J. Physiol. 223:1138-1142. Kuenzel, W. J., and C. W. Helms, 1970. Hyperphagic, polydipsia and other effects of hypothalamic lesions in the White-throated sparrow, Zonotrichia albicollis. Condor 72:66—75. Lepkovsky, S., and M. Yasuda, 1966. Hypothalamic lesions, growth, and body composition of male chickens. Poultry Sci. 45:582-588. Maddison, S., 1977. Intraperitoneal and intracranial cholecystokinin depress operant responding for food. Physiol. & Behav. 19:819-824. Matei-Vladescu, C , G. Apostol, and V. Popescu, 1977. Reduced food intake following cerebral intraventricular infusion of glucose in Gallus domesticus. Physiol. & Behav. 19:7 — 10. Miller, N. E., 1960. Motivational effects of brain stimulation and drugs. Fed. Proc. 19:846—853. Parrot, R. F., and B. A. Baldwin, 1982. Centrallyadministered bombesin produces effects unlike short-term satiety in operant feeding pigs. Physiol. & Behav. 28:521-524. Skewes, P. A., D. M. Denbow, M. P. Lacy, and H. P. Van Krey, 1984. Alteration of food intake following lateral cerebral ventricular injection of plasma from a fasted fowl. Poultry Sci. 67(suppl. 1): 3 7 - 3 8 . (Abstr.) Smith, C.J.V., 1969. Alterations in the food intake of chickens as a result of hypothalamic lesions. Poultry Sci. 48:475-477. Snedecor, G. W., and W. G. Cochran, 1980. Statistical Methods. Iowa State University Press, Ames, Iowa. Stuckey, J. A., and J. Gibbs, 1982. Lateral hypothalamic injection of bombesin decreases food intake in rats. Brain Res. Bull. 8:617—621.
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decrease food intake when injected ICV. A hypophagic condition was reported in the rat (Avery and Calisher, 1982; Stuckey and Gibbs, 1982) and pig (Parrot and Baldwin, 1982) following ICV administration of bombesin. Intracerebroventricular injection of cholecystokinin reduced food intake in the rat (Maddison, 1977) and chicken (Denbow and Myers, 1982). Nutrient-related blood components have also been shown to be involved in regulating food intake. Infusion of 6% glucose into the lateral ventricle of domestic fowl reduced food intake for up to 3 hr postinjection (MateiVladescuef a/., 1977).
1986 Poultry Science 6 5:172-176 INTRODUCTION Regulation of food intake has generally been ascribed t o well-defined areas within t h e h y p o t h a l a m u s . Lesioning (Brobeck et al, 1943) and anesthetizing (Epstein, 1960) t h e ventromedial h y p o t h a l a m u s (VMH), or electrically stimulating t h e lateral h y p o t h a l a m u s (LH) (Miller, 1 9 6 0 ) , increased food c o n s u m p t i o n in t h e rat. On t h e o t h e r hand, food intake was reduced b y stimulating t h e VMH (Krasne, 1962) or b y lesioning t h e LH (Anand and Brobeck, 1 9 5 1 ) . Similarly, hyperphagia and obesity occurred in chickens (Lepkovsky and Yasuda, 1966) and White-throated sparrows (Zonotrichia albicollis) (Kuenzel and Helms, 1970) following lesioning of t h e VMH, whereas hypophagia and weight loss occurred in t h e chicken (Smith, 1 9 6 9 ) and t h e White-throated sparrow (Kuenzel, 1972) following lesioning of t h e LH.
broventricular (ICV) injection of a narrowspectrum molecular weight fraction of h u m a n serum into 96-hr fasted rats reduced food intake (Knoll, 1979). In t h e domestic fowl, food intake was reduced following ICV inj e c t i o n of plasma collected from free-feeding birds and c o n c e n t r a t e d t o t w o or four times n o r m a l c o n c e n t r a t i o n , while injection of c o n c e n t r a t e d plasma collected from 24-hr fasted birds did n o t affect food i n t a k e (Skewes et al, 1 9 8 4 ) . It appears, therefore, t h a t t h e h y p o t h a l a m u s m a y control food intake by m o n i t o r i n g t h e levels of various plasma comp o n e n t s . Based on these reports, t h e objective of this study was t o d e t e r m i n e t h e molecular weight range of a factor present in t h e plasma of t h e free-feeding domestic fowl t h a t inhibits food intake.
The n a t u r e of t h e factor acting u p o n t h e h y p o t h a l a m u s in vivo has n o t been d e t e r m i n e d , although current evidence suggests t h a t t h e factor m a y be present in t h e blood. Intracere-
1
To whom correspondence should be addressed. 172
MATERIALS AND METHODS Animal Preparation. Single C o m b White Leghorn cockerels were raised in electrically heated b a t t e r y b r o o d e r s with food and water available ad libitum. At 8 weeks of age, t h e birds were transferred t o individual cages. Following sodium p e n t o b a r b i t a l anesthesia (25 mg/kg), a 23-gauge thin-walled stainless steel quide cannula was stereotaxically i m p l a n t e d i n t o t h e right lateral ventricle as described b y
Downloaded from http://ps.oxfordjournals.org/ at Florida International University on May 30, 2015
ABSTRACT Plasma was collected from free-feeding Leghorn cockerels and partitioned by gel filtration into fractions of different molecular weight ranges. The individual fractions were then lyophilized and reconstituted to four times the original concentration. The plasma was administered to 10-week-old Leghorn cockerels via a stainless steel guide cannula, stereotaxically implanted into the lateral cerebral ventricle. Sated cockerels received 10 nl intracerebroventricular (ICV) injections of one of the concentrated plasma fractions or of a control injection of artificial cerebrospinal fluid. Food and water intake were monitored following injection. Food intake was significantly decreased by the ICV injection of plasma fractions less than 1500 mol wt, whereas water consumption was not significantly different from that of the controls. The 1500 to 5000 mol wt fraction and the fraction above 5000 did not alter either food or water intake. These results suggest that the plasma of free-feeding domestic fowl contains a low molecular weight factor that is involved in the regulation of food intake. (Key words.- food intake, satiety factor, intracerebroventricular, anorexigenic factor, chicken, plasma)
REDUCED FOOD INTAKE TABLE 1. Experimental
173
treatments
Experiment 1
2
Control 1 Whole plasma2 <5000 mol wt plasma2 >5000 mol wt plasma2
3 Control 1 <5000 mol wt plasma2 5000-1500 mol wt plasma2 <1500moi wt plasma2
Artificial cerebrospinal fluid.
2
Plasma collected from free-feeding birds.
3
Plasma collected from 24-hr fasted birds.
Denbow et al. (1981). The guide cannula was occluded with a 27-gauge stylet between injections. Following a 3-day recovery period, validation of cannula location was verified by monitoring the colonic temperature response to an injection of 67 fig of norepinephrine in 10 /A of artificial cerebrospinal fluid (aCSF) administered via the guide cannula. Only those birds exhibiting a decrease in body temperature of 1.0 C or greater were used in the experiments. Plasma Preparation. Prior to each experiment, blood was collected via cardiac puncture from free-feeding and 24-hr fasted cockerels. Samples from each group were pooled and centrifuged at 3000 g for 20 min. The plasma was separated into fractions using gel filtration. A PD-10 column (Pharmacia Fine Chemicals) containing 5 cm of Sephadex G-25 or G-15 gel (Pharmacia Fine Chemicals) was used to divide the plasma at 5000 mol and 1500 mol wt cutoff points, respectively, yielding four fractions (<1500 mol wt, 1500 to 5000 mol wt, <5000 mol wt, and >5000 mol wt). Following elution with distilled water, the fractions were lyophilized and reconstituted to four times the original concentration with distilled water and stored at —20 C. Injection Procedure. Injections were made via a 27-gauge stainless steel injection cannula connected to a 10 jul Hamilton syringe. In Experiments 1 and 2, the cockerels received either 10 jul of aCSF or a plasma fraction collected from free-feeding birds (Table 1). In Experiment 3, they also received fractions of plasma collected from 24-hr fasted birds.
2 The aCSF consisted of 155 m/W Na+, 2.5 mAf Ca++, 3.7 mM K+, 2.1 m/W Mg++, 140 mM CI - and 23 mM H C 0 3 - (Anderson and Hazelwood, 1969).
Food and water intakes were monitored (by weight and volume, respectively) at 15-min intervals for the first hr, 30-min intervals for the second hour, and hourly thereafter. Statistical Design and Analysis. A repeated Latin Square design (Snedecor and Cochran, 1980) was utilized. Nonorthogonal contrasts were used to make comparisons of cumulative food and water intake between the control and treated birds at each time period. Bonferroni F values (P<.05) were used in determining statistical significance (Games, 1972). RESULTS Experiment 1. Injection of whole plasma collected from free-feeding birds and concentrated to four times normal significantly reduced cumulative food intake at 90 min postinjection (Table 2). The fraction of plasma with a molecular weight below 5000 reduced cumulative food intake at 60, 90, and 120 min postinjection; however, the fraction above 5000 mol wt did not alter food intake. Water intake was not significantly altered by any of the treatments. Experiment 2. Cumulative food intake of the birds receiving the fraction of plasma below 1500 mol wt was reduced below that of the control birds by 45, 60, 90, and 180 min postinjection (Table 3). No reduction in food intake occurred following injection with the 1500 to 5000 mol wt or the <5000 mol wt fraction. Water intake again was not altered by any treatment. Experiment 3. Birds receiving the below 1500 mol wt fraction of plasma collected from free-feeding birds consumed less food by 120 min postinjection than did the control birds, but food intake was not reduced in the birds
Downloaded from http://ps.oxfordjournals.org/ at Florida International University on May 30, 2015
1
Control 1 <1500 mol wt plasma2 <1500 mol wt plasma3
174
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TABLE 2. Mean cumulative food and water intake of free-feeding Single Comb White Leghorn cockerels following a 10-p.l intracerebroventricular injection of four times normal concentration of whole, <5000 molecular weight, or >5000 molecular weight fractions of plasma collected from free-feeding birds T i m e postinfection (mini) 15
30
45
60
90
120
180
240
Control 1 Whole plasma > 5 0 0 0 m o l w t plasma <5O0O m o l wt plasma SEM 2
3.7 2.5 3.2 2.4 .5
4.9 3.3 4.8 3.6 .6
6.2 4.3 5.9 4.6 .6
7.7 5.8 6.9 5.3* .5
10.5 8.0* 9.6 7.5* .7
11.5 10.4 11.4 9.0* .7
16.2 14.3 15.4 12.9 .9
18.8 18.1 18.3 15.0 1.1
Control1 Whole plasma > 5 0 0 0 m o l w t plasma < 5 0 0 0 m o l w t plasma SEM 2
1.7 1.7 1.3 .0 1.0
2.5 2.9 1.7 .4 1.1
3.3 2.9 2.5 .8 1.4
5.0 4.6 3.8 2.1 1.9
7.1 8.8 6.3 5.4 2.7
11.7 11.7 9.2 7.9 2.7
19.2 19.2 15.4 10.4 2.9
24.6 23.8 19.2 15.4 3.1
Water
1
Artificial cerebrospinal fluid.
'Standard error of the mean (n = 12). •Significantly different from control (P<.05).
TABLE 3. Mean cumulative food and water intake of free-feeding Single Comb White Leghorn cockerels following a 10-fJ.l intracerebroventricular injection of four times normal concentration of <5000 molecular weight, 1500 to 5000 molecular weight,or <1500 molecular weight fractions of plasma collected from free-feeding birds T i m e postinjection (min ) Treatment
15
30
45
90
60 Food
1
120
180
240
11.9 11.7 10.7
17.1 15.6 14.4
19.8 18.7 18.9
9.6 .7
13.4* 1.0
16.4 1.1
•
Control <5O0O m o l wt plasma 1 5 0 0 - 5 0 0 0 m o l wt plasma < 1 5 0 0 m o l wt plasma SEM 2
3.4 3.8 2.8
5.3 5.5 4.6
6.5 6.7 5.3
7.2 7.2 6.3
10.5 9.8 8.8
2.6 .4
3.8 .5
4.3* 5
5.0* .6
Control1 < 5 0 0 0 m o l wt plasma 1 5 0 0 - 5 0 0 0 m o l wt plasma < 1 5 0 0 m o l w t plasma SEM 2
1.7 .4 .4
2.9 1.3 1.7
5.0 1.7 3.8
5.4 2.9 5.4
7.5 6.7 7.5
11.3 9.6 10.8
18.3 17.9 17.9
24.2 21.7 24.6
.8 .5
1.3 .8
2.5 1.1
4.2 1.1
5.0 1.5
8.3 1.9
12.9 2.6
16.3 2.7
7.0* .6 Water i
1
Artificial cerebrospinal fluid.
'Standard error of the mean (n = 12). 'Significantly different from control (P<.05).
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Treatment
REDUCED FOOD INTAKE
receiving the same fraction collected from 24-hr fasted birds (Table 4). Water consumption was not significantly altered by either treatment.
DISCUSSION
(Knoll, 1979). Satietin was proposed to contain a limited number of peptides with molecular weights in the range of 40,000 to 60,000. It was reported that the proposed molecular weight range of satietin conflicted with the separation procedures employed, which included filtering all samples through an Amicon UM-10 membrane (known to withhold compounds with molecular weight above 10,000). The lack of a response following ICV injection of plasma collected from 24-hr fasted birds was probably the result of a reduction in the level of the factor due to the nutritional status of the donors. The concentration of the factor in the plasma may decrease in proportion to the length of time the animal has gone without feeding. This type of response has been demonstrated in the rat where the amount of food consumed by a sated recipient rat, following transfusion with blood from a fasted donor rat, was inversely proportional to the length of deprivation of the donor rat (Davis etal, 1971). The nature of the low molecular weight factor(s) present in plasma that reduces food intake when injected ICV is unknown. Numerous endogenous substances with molecular weights below 1500 have been shown to
TABLE 4. Mean cumulative food intake of free-feedingSingle Comb White Leghorn cockerels following a 10-y.l intracerebroventricular injection of four times normal concentration of <1500 molecular weight fractions of plasma collected from free-feeding or 24-hr fasted birds T i m e postinjection (min) Treatment
15
30
45
60
Control1 < 1 5 0 0 mol wt plasma 2 < 1 5 0 0 mol wt plasma 3 SEM4
1.9 1.9 1.8 .4
2.8 2.6 2.9 .6
3.9 3.6 4.0 .7
4.8 4.1 4.9 .7
Control1 < 1 5 0 0 mol wt plasma 2 < 1 5 0 0 mol w t p l a s m a 3 SEM4
1.1 1.1 .0 .5
1.7 1.7 .6 .9
2.8 3.3 2.2 1.0
3.3 4.4 2.8 1.1
120
180
240
7.4 5.8 7.8 .7
9.6 6.3* 9.4 .7
11.3 9.7 11.9 1.1
14.8 12.1 14.1 1.3
5.6 5.0 2.8 1.2
5.6 6.1 3.9 1.2
7.8 8.9 7.8 2.3
11.7 13.3 11.1 2.1
90 F o o d intake
1
Artificial cerebrospinal fluid.
2
Plasma collected from free-feeding birds.
3
Plasma collected from 24-hr fasted birds.
"Standard error of the mean (n = 9). 'Significantly different from control (P<.05).
ig;
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Concentrated plasma fractions below 5000 mol wt and below 1500 mol wt reduced food intake if the plasma had been collected from free-feeding birds, but it did not reduce food intake if the plasma had been collected from 24-hr fasted birds. The absence of a response following injection with the below 5000 mol wt fraction in Experiment 2 is to be questioned in light of the reduction in food intake by the <5000 mol wt fraction Experiment 1 and the <1500 mol wt fraction in Experiments 2 and 3. It is possible that this fraction was overdiluted when reconstituted, thereby reducing the concentration of the active factor in the fraction. It appears, therefore, that the "satiety" factor previously shown to exist in the plasma of the free-feeding domestic fowl (Skewes et al, 1984), has a molecular weight below 1500. In a similar study, a compound called "satietin", which reduced food intake when injected into the lateral ventricle of 96-hr fasted rats, was shown to exist in human serum
175
SKEWES ET AL.
176
The results of these experiments suggest that a satiety factor having a molecular weight of less than 1500 exists in the plasma of the free-feeding domestic fowl. Further, the plasma concentration of this factor appeared to be related to the nutritional status of the bird. Because water intake was not reduced by any of the treatments, it can be assumed that the effect of the treatments was specific for food intake. Whether this factor originates from the gastrointestinal tract as a secretion, e.g., cholecystokinin or bombesin, or from the absorption of nutrients, e.g., glucose, remains to be elucidated. ACKNOWLEDGMENT This study was supported, in part, by a grant from the John Lee Pratt Animal Nutrition Program. The authors would also like to thank K. E. Webb, Jr., for his technical advice.
REFERENCES Anand, B. K., and J. R. Brobeck, 1951. Hypothalamic control of food intake in rats and cats. Yale J. Biol. Med. 24:123-140. Anderson, D. K., and R. L. Hazelwood, 1969. Chicken cerebrospinal fluid: normal composition and response to insulin administration. J. Physiol. 202:83-95. Avery, D. D., and S. B. Calisher, 1982. The effects of injections of bombesin into the cerebral ventricles on food intake and body temperature in fooddeprived rats. Neuropharmacology 21:1059— 1063. Brobeck, J. R., J. Tepperman, and C. N. Long, 1943. Experimental hypothalamic hyperphagia in the albino rat. Yale J. Biol. Med. 15:831-853.
Davis, J. D., C. S. Campbell, R. J. Gallagher, and M. A. Zurakov, 1971. Disappearance of a humoral satiety factor during food deprivation. J. Comp. Physiol. Psychol. 75:476-482. Denbow, D. M., J. A. Cherry, P. B. Siegel, and H. P. Van Krey, 1981. Eating, drinking and temperature response of chicks to brain catecholamine injections. Physiol. & Behav. 27:265-269. Denbow, D. M., and R. D. Myers, 1982. Eating, drinking and temperature responses to intraventricular cholecystokinin in the chick. Peptides 3:739-743. Epstein, A. N., 1960. Reciprocal changes in feeding behavior produced by intrahypothalamic chemical injections. Am. J. Physiol. 199:969-974. Games, P. A., 1972. An improved t table for simultaneous control on g contrasts. J. Am. Statist. Assoc. 72:531. Knoll, J., 1979. Satietin: A highly potent anorexogenic substance in human serum. Physiol. & Behav. 23:497-502. Krasne, F. B., 1962. General disruption resulting from electrical stimulus of the ventromedial hypothalamus. Science 138:822-823. Kuenzel, W. J., 1972. Dual hypothalamic feeding system in a migratory bird, Zonotrichia albicollis. Am. J. Physiol. 223:1138-1142. Kuenzel, W. J., and C. W. Helms, 1970. Hyperphagic, polydipsia and other effects of hypothalamic lesions in the White-throated sparrow, Zonotrichia albicollis. Condor 72:66—75. Lepkovsky, S., and M. Yasuda, 1966. Hypothalamic lesions, growth, and body composition of male chickens. Poultry Sci. 45:582-588. Maddison, S., 1977. Intraperitoneal and intracranial cholecystokinin depress operant responding for food. Physiol. & Behav. 19:819-824. Matei-Vladescu, C , G. Apostol, and V. Popescu, 1977. Reduced food intake following cerebral intraventricular infusion of glucose in Gallus domesticus. Physiol. & Behav. 19:7 — 10. Miller, N. E., 1960. Motivational effects of brain stimulation and drugs. Fed. Proc. 19:846—853. Parrot, R. F., and B. A. Baldwin, 1982. Centrallyadministered bombesin produces effects unlike short-term satiety in operant feeding pigs. Physiol. & Behav. 28:521-524. Skewes, P. A., D. M. Denbow, M. P. Lacy, and H. P. Van Krey, 1984. Alteration of food intake following lateral cerebral ventricular injection of plasma from a fasted fowl. Poultry Sci. 67(suppl. 1): 3 7 - 3 8 . (Abstr.) Smith, C.J.V., 1969. Alterations in the food intake of chickens as a result of hypothalamic lesions. Poultry Sci. 48:475-477. Snedecor, G. W., and W. G. Cochran, 1980. Statistical Methods. Iowa State University Press, Ames, Iowa. Stuckey, J. A., and J. Gibbs, 1982. Lateral hypothalamic injection of bombesin decreases food intake in rats. Brain Res. Bull. 8:617—621.
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decrease food intake when injected ICV. A hypophagic condition was reported in the rat (Avery and Calisher, 1982; Stuckey and Gibbs, 1982) and pig (Parrot and Baldwin, 1982) following ICV administration of bombesin. Intracerebroventricular injection of cholecystokinin reduced food intake in the rat (Maddison, 1977) and chicken (Denbow and Myers, 1982). Nutrient-related blood components have also been shown to be involved in regulating food intake. Infusion of 6% glucose into the lateral ventricle of domestic fowl reduced food intake for up to 3 hr postinjection (MateiVladescuef a/., 1977).