Effects of sauvagine, urotensin I and CRF on food intake in rats

p,.i,tid~, Vol. 6. Suppl. 3. pp. 53-57. 1985, ' AnkhoInternationalInc. Printedin the U.S.A.

0196-9781/85 $3.00 + .00

Effects of Sauvagine, Urotensin I and CRF on Food Intake in Rats LUCIA NEGRI, ~LIA NOVIELLO AND VITTORIA NOVIELLO

Institute o f Medical Pharmacology. University "'La S a p i e n z a , " O0185-Rome, Italy

NEGRI, L., L. NOVIELLO AND V. NOVIELLO. EJ)'ec'ts oJ'sam'agine, urotensin I and CRF on fi~od intake in rats. PEPTIDES 6: Suppl. 3.53-57. 1985.--Sauvagine ISV) is a forty amino acid peptide, isolated from the skin of the South American frog Phylhmwdus~t sauvagei and structurally related to fish Urotensin I (UI) and to mammalian corticotropin releasing factor (CRF). intracerebroventricular (ICV) injections of SV (0.3-2.0 p.g/rat), CRF (1.0-15.0 pg/rat) and UI (0.5-2,0/-tg/rat ) inhibited feeding in 18 hr food deprived rats. By subcutaneous (SC) route, only SV (3.0-10.0 ~g/kg) and UI ( 10.0--20.0tzg/kg) exhibited anorexogenic effects, CRF being completely inactive up to a dose of 200 p,g/kg. Vagotomy did not prevent the feeding inhibition by SC SV. In ICV injected rats. CRF increased grooming in comparison with both food deprived and satiated controls, while SV and U I increased grooming only in comparison with fasted controls. Compared to satiated animals, food deprived rats when injected ICV with anorexogenic doses of the peptides showed decreased resting and increased moving. Rats given SC injections of SV and U I significantly decreased grooming in comparison with both food deprived and satiated controls, while increased resting only in comparison with fasted controls. CRF by the SC route did not affect the behaviour of the rat. Sauvagine

Urotensin I

CRF

Food intake

Behavioural changes

SAUVAGINE (SV) is a peptide isolated from the skin of the South American frog Phylhmtedusa sauvagei [4,14] with a sequence of 40 amino acid residues. Its secondary structure xvas predicted to contain ¢~-helix B-sheets and/3-turns [15]. The presence of SV-like peptides was demonstrated in amphibians and fishes, in organs other than the skin [3,18]. SV may be considered a prototype of a new polypeptide family possessing a peculiar spectrum of biological activity on the central nervous system, the anterior pituitary, the cardiovascular system and the gastrointestinal tract [1, 4, 5, 12, 131. SV is structurally related to CRF [19], which was shown to be a possible mediator of stress related suppression of food intake [10], and to Urotensin I (UI), a peptide isolated from fish urophysis [9]. In the experiments reported here, we compared the central and peripheral effects ofSV, U I and CRF on feeding and associated behaviours in food deprived rats.

the rats with a preweighed amount of food and weighing the food, every 30 min for 4-6 hr, to the nearest 0,1 g. Food weights were corrected for spillage at each measurement. Rats were tested with several doses of the peptides at two day intervals. In each experiment 16 rats were randomly divided into two groups of 8 rats: the first group was injected with the established dose of the peptide and the other with saline. Changes of food intake in peptide injected rats were calculated as the difference between the amount of food consumed by each rat and the mean of food eaten by the control group of saline injected rats during the same period, and expressed as the percentage of the latter. Data from each animal group were averaged and represented as m e a n ± S E . Statistical analysis was carried out by two way ANOVA and Student's t-test. Peptides were dissolved in saline and injected by subcutaneous (SC) and intracerebroventricular (ICV) routes. In rats receiving ICV injections, a permanent polyamide cannula was stereotaxically screwed into the skull at least three days prior to the beginning of the experiment. The stereotaxic coordinates were 1.5 mm posterior to bregma and 1.5 mm lateral to the sagittal suture with the tooth bar 5 mm above the interaural line [17]. The ICV injections (5/zl) were made using a Hamilton microliter syringe connected to a needle which extended beyond the tip of the ventricular cannula into the ventricular space (about 3.4 mm from skull surface). The needle was removed after the injection: an obturator was repositioned into the cannula and the rat re-

METHOD Male Wistar rats (200-300 g. b.w.) were housed singly and tested in individual cages (15×21×13 cm) on natural day/night cycles. Water was given ad lib. The animals were placed on a daily feeding schedule in which food pellets (Mill. Morini) were available for 6 hr (10:00--16:00) daily. The rats were then deprived of food for 18 hr (16:00-10:00). The testing began after an adaptation period of one week to the feeding schedule. Food intake was measured by presenting

'Requests for reprints should be addressed to Dr. L. Negri, Institute of Medical Pharmacology, University of Rome, "La Sapienza," P.za A. Moro 5, 00185 Rome, Italy.

53

54

NEGRI, N O V I E L L O AND N O V I E L L O

FIG. 1. Effects of SC injected SV. U I and CRF on deprivation induced feeding in rats. Data are expressed as means±SE (see the Method sectionL

FIG. 2. Effects of ICV injected SV. UI and CRF on deprivation induced feeding in rats. Data are expressed as means+-SE [see the Method section).

turned to its cage where the food was immediately available. The rats injected ICV received SV at the doses of 0. l, 0.3. 0.5, 1.0.2.0 p.g/rat, CRF at doses of 0.1, 1.0, 5.0, 15,0 p.g/rat and UI at doses of 0.5 and 2.0 p.g/rat. A group of 8 food deprived rats was SC injected with naloxone (I mg/kg) 15 min before ICV injection of SV (1.0 p.g/rat) and their food intake compared with that of 8 rats ICV injected with saline and pretreated with the same SC dose of naloxone. The SC injected rats received the peptide dissolved in saline (2 ml of 0.9% NaCi/kg, b.w.) or the same volume of saline alone (control rats). 15 min before presentation of the

preweighed food pellets. These rats were injected with the following doses of the peptides: 3.0, 5.0.7.0 and 10.0/zg/kg of SV; 20.0, 50.0, 100.0 and 200.0 ptg/kg of CRF, 10.0 and 20.0 /.tg/kg of U l . The anorexogenic effect of SC SV (10 /zg/kg) was studied also in rats vagotomized according to Lorenz et al. [1 l]. Briefly, in anaesthetized rats the anterior and posterior vagal trunks were located on the surface of the oesophagus and each trunk severed between the diaphragm and its hepatic branch. During some experiments a variety of behaviours was recorded. Following the injections, a trained observer, un-

SAUVAGINE,

UROTENSIN

I, C R F A N D F O O D I N T A K E

55

TABLE I EFFECT OF THE SC ADMINISTRATION OF SV (10/,tg/kg) IN VAGOTOMIZED AND SHAM-OPERATED RATS, FASTED FROM 18 HOURS "% Decrease of Cumulative Food Intake Time Imin)

15

30

60

90

120

180

240

Sham Operated Rats

- 6 7 ___ 10

-71 ± ll

-78 _ 8

- 8 0 --_ 7

-82 ± 6

-81 ± 5

-71 --- 5

Vagotomized Rats

- 4 8 ± 15

- 6 7 ± 10

-75 ± 7

- 7 7 _+ 6

-78 ± 5

-72 ± 8

- 6 5 ± 15

Data are expressed as percentage decrease of food intake in SV treated rats respecting saline injected rats.

TABLE 2 EFFECT OF THE ADMINISTRATION OF SV ALONE 11 ~g/rat ICV). NALOXONE 11 mg/kg SC) AND SV PLUS NALOXONE ON FOOD INTAKE OF FASTED RATS % Decrease of Cumulative Food Intake Time (min)

15

30

60

90

120

150

180

NX NX ~ SV SV

- 3 9 ~ 8* - 7 6 ± 7* - 6 5 ± 7*

- 4 0 ± 5"$ - 8 2 ± 5"+ - 6 9 ± 5"+

- 3 5 ± 8"~ - 8 3 ± 5"~¢ - 6 9 ± 6 *÷

- 3 2 ± 55 - 8 5 ± 7"+ - 7 0 ± 3"¢

-21 ± 10~ - 7 8 ± 9*t - 6 8 ± 4"+

- 1 7 ± 10¢ -74± 5*t -64± 3*t

- 1 8 ± 155 - 7 2 ± 9*t - 5 8 ± 9*t

Data are expressed as percentage decrease of food intake in treated rats respecting saline injected rats. Student's t-test: *,o<0.05 vs. saline control: -/9<0.05 vs. naloxone treated rats; $p<0.05 vs. SV treated rats.

CRF (15 jig) eating'X'z~~grooming °* o ~ ~ grooming *+ groomi.~l;iyjDg e a , , n ~ eating~ -7='~vin'tJ"x'~m°ving* ~ g . X - o+ -.~:~restingO-x-+ SV(2]jg) Ul(2l g ) moving eating° groomin~ ~ ~-:-~ ~,~groommg ~resting ~Ti~moving orestln~ SATIATED RATS FASTED RATS FIG. 3. Effect of ICV injections of SV. U I and CRF on a variety of behaviours. Data are recorded as described in the Method section. Student's t-test: *p<0.05 vs. fasted controls; °p<0.05 vs. satiated controls: -~p<0.05 vs. CRF treated rats.

a c q u a i n t e d with the t r e a t m e n t , e x a m i n e d e a c h rat e v e r y 5 rain for t h r e e h o u r s a n d listed the b e h a v i o u r in w h i c h t h e rat was engaged in one o f the following c a t e g o r i e s : eating, resting, g r o o m i n g a n d m o v i n g . T h e d a t a w e r e r e c o r d e d as the n u m b e r of b e h a v i o u r s / h r , t h e n t r a n s f o r m e d to p e r c e n t a g e o f the total o b s e r v e d b e h a v i o u r s . D a t a w a s a n a l y s e d u s i n g Stud e n t ' s t-test.

Drugs Sauvagine was p u r c h a s e d b y B a c h e m F e i n c h e m i k a l i e n AG (Bubendorf, Switzerland), CRF from CRB (Cambridge, U . K . ) , n a l o x o n e from S A L A R S ( C o m o , Italy). U r o t e n s i n I was kindly p r o v i d e d b y Dr. Karl L e d e r i s ( U n i v e r s i t y o f Calgaff, C a n a d a ) . RESULTS Figure I s h o w s the effect o f different d o s e s o f SC S V on feeding. T h e dose of 3.0 p.g/kg did not p r o d u c e a n y significant r e d u c t i o n o f food intake. T h e inhibition p r o d u c e d by 5.0. 7.0. a n d 10.0 p.g/kg o f S V was significant a n d r e a c h e d the m a x i m u m after 90 min. U I inhibited food i n t a k e with a t i m e - c o u r s e similar to SV, but it a p p e a r e d to b e 2-3 t i m e s less p o t e n t t h a n S V (Fig. 1). SC injections o f C R F at d o s e s r a n g i n g f r o m 20.0 to 200.0 p,g/kg did not s h o w a n y effect o n food i n t a k e (Fig. I). As T a b l e I indicates, v a g o t o m i z e d rats w e r e as s e n s i t i v e to SV as s h a m o p e r a t e d a n i m a l s w e r e . T h e s e r e s u l t s suggest t h a t SC S V does not r e d u c e food i n t a k e t h r o u g h a vagally m e d i a t e d process.

56

NEGRI, N O V I E L L O A N D N O V I E L L O

moving

grooming *° " ~.~-"o

. . moving

r°°minO eating ~ m o v i n g re s t i n ~ t l ~

SATIATED RATS

"

FOOD DEPRIVED RATS

SV(to =,el

U1 (zo pg)

FIG. 4. Effect of SC administration of SV and U I on a variety of behaviours. Data are recorded as described in the Method section. Student's t-test: *p<0.05 vs. fasted controls; °p<0.05 vs. satiated controls.

ICV injected SV (Fig. 2) significantly reduced food intake at dose levels of 0.3, 0.5, 1.0 and 2.0 p,g/rat, the maximum effect being recorded within 90--120 min. Doses of 1.0 and 2.0 p,g/rat showed the same potency, but the activity of the higher dose lasted up to six hours. UI appeared to be as potent as SV when injected into the lateral ventricles, however, its action was more rapid (maximum effect within 30 min) and of shorter duration. The ICV injection of C R F produced a significant food intake inhibition at doses of 1.0, 5.0 and 15.0 ttg/rat. CRF appeared to be about 5-7 times less potent than SV. The maximum effect of C R F was reached within the first 30 rain period and vanished after 90-240 min, depending on the dose. Pretreatment of the animals with 1.0 mg/kg of SC naloxone (NX) did not antagonise the inhibitory effect on food intake produced by the ICV injection of 1.0 p,g/rat SV (Table 2). Behaviours induced in rats by ICV and SC injections of CRF, SV and U1, at doses which were equiactive in inhibiting food intake, are shown in Fig. 3 and 4, in comparison with those observed in food deprived and satiated controls. During the first hour after ICV injections (Fig. 3), rats given the peptides (CRF, 15 ttg/rat; SV, 2 ttg/rat; U I , 2 p,g/rat) reduced eating and increased grooming, in comparison with fasted controls. Compared to satiated controls, all treated animals showed increased moving, but only CRF injected rats increased grooming. All the animals injected SC with SV (10 ~g/kg) and UI (20 ~g/kg) showed reduced moving and grooming and increased resting, in comparison with fasted saline injected rats, and appeared calm and drowsy as satiated controls. Rats injected SC with SV and U I showed strong peripheral vasodilatation appearing as reddening of ears, scrotum, tail and paw soles. DISCUSSION

It was postulated that CRF represents a possible mediator of stress-related suppression of food intake [ f3] and this action is not a vagally-mediated process [7]. It has been reported also, that C R F and SV are equipotent in reducing deprivation-induced feeding when they are injected ICV in rats. SV produced a larger and longer-lasting suppressive effect than C R F on spontaneous and ethylchetocyclazocineinduced feeding, a stronger conditioned aversion to saccharin taste [8] and a higher increase of plasma cathecolamine and glucose levels [2].

Morley and Levine [16] demonstrated that the mechanism by which C R F decreases ingestive behaviour appears to be associated with grooming. In the present experiments, ICV SV and Ul displayed higher potency than CRF in reducing deprivation-induced feeding, but significant lesser activity in inducing grooming. Thus grooming appears to be largely independent of central feeding-inhibitory mechanisms. Moreover, CRF did not affect feeding when it was injected SC, while SV and U1 reduced food intake also by this route, SV being 2-3 times more potent than U I. The range of SV doses active on food intake was almost the same after SC and ICV administration. This fact is rather surprising. Also if we suppose that the peptide given peripherally has free access to the brain, the SC dose must be diluted in extracellular fluids and only a small fraction of it will enter the brain. Alternatively, we can hypothesise that centrally injected SV and U l diffuse to the periphery where they could inhibit feeding by activating an unknown peripheral mechanism. However, before accepting this hypothesis as a possible explanation, we must consider that centrally active doses of SV and U l are two and ten times lower respectively, than the equiactive subcutaneous doses. Since it appears to be highly improbable that the rate of the SC absorption of these peptides is 2-10 times slower than their brain-blood diffusion, we must discard any suggestion of peripheral action of centrally injected doses. It has been shown that SC and ICV SV reduces the rate of gastric emptying in fasted rats [I], so that one may argue that this action could explain the inhibition of feeding. Cues of satiety can arise from the gastric distension following reduced rate of gastric emptying and vagotomy has been shown to abolish such cues [6]. Yet vagotomy failed to influence the peripheral action of SV on food intake, demonstrating that the feeding inhibition did not depend on vagally mediated signals. In any case. it is not easy to explain why the structurally related CRF is inactive on feeding when given by SC route. Since SC CRF is more potent than SV and U! in evoking ACTH and /3-endorphin secretion in rats [2]. both pharmacokinetic events and the release of the two pituitary hormones must be ruled out as causative factors in a tentative explanation of the observed differences in the peripheral anorexogenic activities of the three peptides. Subcutaneous injections of SV and U l not only reduced eating but also affected rat behaviour by increasing resting and decreasing grooming. Whether the substitution of resting

S A U V A G I N E , U R O T E N S I N 1, C R F A N D F O O D I N T A K E for eating and g r o o m i n g in SV and U l treated rats reflects a non-specific sedative effect, which may affect the last two b e h a v i o u r s , or a c o n s e q u e n c e of satiety is yet unclear. In any case. rats injected SC with SV and U I appeared calm and dro~vsy as s p o n t a n e o u s l y fed rats, but with a significant reduction of the time spent in grooming activity. Finally, it is not possible to exclude the hypotension produced by SC

57 doses of SV and U 1 [2] might contribute, at least in part, in reducing the l o c o m o t o r activity and feeding. H o w e v e r , SC injection of plainly h y p o t e n s i v e doses of C R F (50, 100, 200 /.tg/kg), did not affect rat behaviour. The most o b v i o u s explanation for these results suggests that the behaviourai effects of peripherally injected SV and U1 are mediated by types of r e c e p t o r that are not activated by C R F .

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10. Levin, A. S., B. Rogers. J. Kneip, M. Grace and J. E. Morley. Effect of centrally administered CRF on multiple feeding paradigms. Neuropharmacology 22: 337-339, 1983. I I. Lorenz. D. N. and S. A. Goldman. Vagal mediation oi the cholecystokinin satiety effect in rats. Physiol Behar 29: 599604, 1982. 12. Melchiorri, P., L. Negri and V. Erspamer. Effects of sauvagine on release of prolactin, adrenocorticotropin, /3-endorphin and corticosterone in rats. In: Neuroendocrinology of Vasopressin Corticoliberin and Opiomelanocortins. edited by A. J. Baertschi and J. J. Dreifuss. London: Academic Press, 1982, pp. 331-342. 13. Melchiorri. P. and L. Negri. Action of sauvagine on the mesenteric vascular bed of the dog. Regul Pept 2: 1-13, 1981. 14. Montecucchi, P. C., A. Henschen and V. Erspamer. Structure of Sauvagine, a vasoactive peptide from the skin of a frog. Hoppe Scylers Z Physiol Chem 360:1178, 1979. 15. Montecucchi, P. C. and L. Gozzini. Secondary structure prediction of sauvagine, a novel biologically active polipeptide from a frog. Int J Pept Protein Res 20: 139-143, 1982. 16. Morley, J. E. and A. S. Levin. Corticotropin releasing factor, grooming and ingestive behavior. Lift, Sci 31: 1459-1464, 1982. 17. Pellegrino, L., A. Pellegrino and A. J. Cushman. A Stereotaxic Atlas of the Rat Brain. New York: Plenum Press, 1979. 18. Renda, T., L. D'este, L. Negri and D. Lomanto. Sauvagine-like immunoreactivity in the bony fish urophysis and caudal neurosecretory system. Basic Appl Histochem 26: 89-98, 1982. 19. Vale, W., S. Spiess, C. Rivier and J. Rivier. Characterization of a 41-residue ovine hypothalamic peptide that stimulates secretion of corticotropin and/3-endorphin. Science 213: 1934. 1981.