GABA and feeding: Reversal of overeating by central GABA-transaminase inhibition

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GABA

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AND FBEDINGz BE’VEBSAL OF OVERBATING GABA-TBANSAMINASE INHIBITION DONALD

BY CENTRAL

V. COSCINA

Section of Biopsychology, Clarke Institute of Psychiatry University of Toronto, Toronto, Ontario, Canada

(Final form, June, 1983)

Abstract Coscina, Donald V.: GABA and feeding: reversal of overeating by central GABA-transaminase inhibition. Prog. Neuro-Psychopharmacol.Q Biol. Psychiat. 1983, 7 (4-5):463-467. 1. Past literature is reviewed briefly which suggests that variations in brain GABA metabolism may be involved in the control of food intake in rats. 2. Recent experiments from the author's laboratory are summarized in which brain GABA has been elevated in adult female rats by intracisternalinjection of the GABA-transaminase inhibitor, ethanolamine-O-sulfate(EOS). 3. Central EOS pretreatment produced dose-dependent anorexia in normal subjects and prevented acute overeating in response to systemic insulin (12 U/kg) or Z-deoxyglucose (750 mg/kg). 4. Similar EOS pretreatment essentially reversed the chronic overeating induced by diet palatability, bilateral medial hypothalamic lesions or genetic predisposition (in Zucker fatty rats). 5. The ubiquity of these anorexic effects in the absence of clear motor debilitation suggests that drugs which elevate brain GABA deserve further investigation for their potential utility in the clinical treatment of overeating. Keywords: anorexic drugs, dietary obesity, ethanolamine-O-sulfate,gamma-aminobutyricacid, genetic obesity, hypothalamic obesity, insulin, overeating, rat, 2-deoxyglucose Abbreviations: body weight (BW); ethanolamine-O-sulfate(EOS); gala-~inobutyric acid (GABA); glutsmic acid decarboxylase (GAD); intracisternal(i.c.); intraperitoneal(i.p.1; medial hypothalamus (MU); subcutaneous (s.c.)

Introduction GABA appears to be a major inhibitory neurotransmitterwhich is located throughout the mammalian CNS (McGeer et al 1978). Aberrations of GRS GABA metabolism have been implicated in a variety of neuropsychiatric illnesses including Alzheimer's disease, epilepsy, Huntington's disease, Parkinson's disease and schizo-affectivedisorders (see Enna 1980). Contrasting to the intensive research efforts devoted to such disorders, little attention has focused on the potential role of CNS GABA metabolism in the etiology or treatment of overeating. As obesity due to food intake in excessive of bioenergic demands is a major clinical problem, investigations into this area may be fruitful. The purpose of this brief paper is to summarize recent work from my laboratory which has investigated the ability of pharmacologically-induced elevations of brain GABA to inhibit overeating induced in rodents by a variety of experimental procedures.

Background Research over the past ten years has suggested that variations in hypothalamic 9 metabolism may be importantly involved in regulating feeding behavior (see Leibowitc 1980 for

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review). A study from my own laboratory showed that bilateral lesions of the MH, sufficient to induce chronic overeating in rats, was associated with reliable decrements in levels of hypothalamic GAD which correlated significantlywith the degree of obesity (Coscina and Lloyd 19801. As GAD is the synthetic enzyme for GABA production and is generally acknowledged to be a valid index of neuronal GABA integrity, these findings were compatible with suggestions that MH GABA neurons are normally involved in the control of feeding. What specific role those neurons might play in that control could not be addressed by that particular experimental design. However, additional research (summarized in Leibowitz 1980) implied that enhancing GABA function in the MH leads to increased feeding. From a clinical perspective, those suggestions might mean that administering GABA antagonists which act selectively on MH neurons might diminish food intake in some forms of human overeating. Of course, there is presently no means by which to selectively target drugs to specific areas of the brain like the MH. Theoretically such drug specificity would be required as GABA neurons are located ubiquitously in the CNS, hence systemic administrationof GABA antagonistswould have widespread effects, many of which could be undesirable. As one important goal of our work was to determine if there were ways in which GABA metabolism could be altered to selectively diminish overeating, we were greatly interested when additional work appeared by Howard et al (1980) which showed that pharmacologicallyinduced elevations of whole brain GABA could produce dose-dependent anorexia in normal male rats. Of particular importance for potential future clinical use, such anorexia could be obtained by systemic administrationof the drug. The compound used in that work was EOS, whose primary mode of action is believed to be the irreversible inhibition of GABA's catabolic enzyme, GABA-transaminase. The evidence by Howard et al (1980) that such EOS-induced anorexia could be achieved without major motoric effects or other non-specific debilitations prompted my group to investigate the capacity of that drug to reverse overeating in a variety of animal models.

General Methods In the experiments to be summarized, adult female rats were chosen almost exclusively for study. Although most researchers tend to employ male-rats, a variety of evidence suggests that females may be more useful in modeling long-term human feeding disorders (see Coscina and Dixon 1983). Also, the research design used for all of these experiments employed i.c. administration of EOS. Although EOS can produce anorexia when administered systemically,a number of our experiments called for drug treatment of severely obese animals. Had we chosen to give EOS systemically in the extremely high doses required to cross the blood-brain barrier (see Howard et al 19801, we would have been faced with large variations in dose-response relationships between obese and control subjects basing dosages on gross BW. Furthermore, evidence has been accumulating which suggests that GABA in the periphery may exert a variety of physiological effects. To circumvent both of those difficulties and test specifically the possible anti-overeatingeffects of CNS EOS in obesity, we selected the i.c. route of administration. The particulars of how such injections were made has been reported previously (Coscina et al 1973). For the experiments reported here, all injections were made in a constant volume of 20 ul while rats were lightly anesthetized with ether. Vehicle for injections was deionized water. Throughout these studies, animals were housed in separate metal cages in a colony illuminated 0800-2000 hrs daily. Room temperature was maintained at 21 + 1'c.

EOS and acute overeatin Rats can be induced to overeat in response to acute glucoprivation. To test the general ability of EOS to prevent overeating, we (Nobrega and Coscina 1982) conducted two separate experiments in which rats received 400 ug EOS or vehicle i.c. 24 hrs before systemic glucoprivic challenges with insulin (12 U/kg s.c.1 or 2-deoxyglucose (2DG; 750 mg/kg i.p.1. This dose of EOS was chosen as earlier pilot work revealed it to be effective in producing anorexia for several days (see also Howard et al 1980). The decision to administer glucoprivic challenges 24 hrs after EOS treatment was based on observations that maximal anorexia plus maximal elevation of brain GABA occur at this dosage-time combination (Howard et al 1980). For each experiment, additional EOS- or vehicle-injectedrats received equal volumes of deionized water by the same routes to control for the glucoprivic injection

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procedures. In both experiments, all groups (ns = 7-8 per condition) were given ad lib access to weighed amounts of Purina Lab Chow (4% fat) in pellet form for 8 days after i.c. injections. BW, food and water intakes were recorded daily. In addition, on the day of glucoprivic challenges (day 2 after i.c. injections), intakes were recorded 1, 2, 3, 4, and 5 hrs after systemic drug administrations. The results of these experiments were almost identical. First, as expected from previous research on male rats (Howard et al 19801, 400 ug EOS produced profound anorexia. During the first 24 hrs, rats receiving EOS consumed only 10-20X of the food eaten by i.c. vehicle controls. In response to insulin challenge, i.c. vehicle controls consumed an average of 4.5 g food over the 5 hr test, which was significantlymore (p < .02) than the 2.0 g eaten by double vehicle controls. Contrasting to this, rats pretreated with EOS i.c. ate only 1.0 g of food after insulin, which was little more than the 0.5 g eaten by EOS + vehicle controls. Glucoprivic challenge with 2DG in i.c. vehicle controls produced an average intake of 4.0 g food over the 5 hr test, which was significantlymore (p < .02) than the 1.4 g consumed by double vehicle controls. During this same time, however, rats pretreated with EOS ate only 0.8 g food, which was little more than the 0.1 g eaten by EOS + vehicle controls. In both experiments, glucoprivic challenge did not lead to increased 24 hr consumption either the day after testing or for the remaining 2 days of EOS-induced anorexia. These results show that two well-established stimuli for eliciting acute increases in feeding are relatively ineffective in rats made anorexic by central injection of EOS. The exact mechanism(s) responsible for this refractory eating response is unknown (but see Nobrega and Coscina 1982 for discussion of possibilities). However, these general findings suggest that elevating whole brain GABA may be effective in impeding overeating in response to acute physiological stimuli.

EOS and chronic overeating Perhaps a more clinically relevant test of EOS's potential utility as an anorexigen would involve demonstrating the drug's efficacy in attenuating chronic overeating. To determine this, we have performed dose-response studies after i.c. EOS in two such models: dietaryinduced overeating and hypothalamic hyperphagia (Coscina and Muir 1981). In addition, a preliminary one-dose study has been performed on genetically hyperphagic Zucker fatty rats (unpublished observations). To induce chronic overeating in normal rats, 10 animals were given ad lib access to Teklad pellets (4% fat), a high-fat diet (67% pellets in powdered form, 33% Crisco vegetable shortening by weight), and a sweet milk solution (50% Borden's Eagle brand sweetened condensed milk, 50% water by volume) for 75 days prior to study. Compared to 9 age-matched controls fed pellets alone, the "dietary" group was quite obese at the time of testing (y + SE BW = 468 + 17 g vs 329 + 4 g; p < .OOl) and continuing to eat approximately 25% more kilzjoules per day than controls (p < .OOl). The sequence of i.c. drug testing for both groups was: day 1, vehicle; day 5, 100 ug EOS; day 15, 200 ug EOS; day 27, 400 ug EOS. BW plus food and water intakes were measured daily for 40 consecutive days. In the second experiment, 10 rats received bilateral Mlilesions and 10 received sham surgery (see Coscina and Lloyd 1980 for details of methods). All rats were fed Teklad pellets ad lib throughout the drug study, which cormsencedtwo weeks after surgery. At the time of testing, MH rats were already obese (?I+ SE BW = 341 + 6 g vs. 278 + 7 g for controls; p < .Ol) and still eating 60-100X more than-controls (p <-.OOl). The sequence of i.c. drug testing for both groups was: day 1, 400 ug EOS; day 13, 200 ug EOS; day 25, 100 ug EOS; day 36, vehicle. BW plus food and water intakes were measured daily for 42 consecutive days. In the third experiment, 3 female and 4 male homoeygously recessive Zucker "fatty" rats (fafa) were compared to 3 female and 4 male lean litter-mates(Pa?). All rats were fed Purina Lab Chow pellets (4% fat) ad-lib throughout the study. At the time the experiment began, fatties were already obese (XL SE BW = 295 2 6 g vs. 204 + 15 g for lean controls; p < .OOl) and still gaining weight rapidly. During a lo-day baseline period, fatties consumed about 50% more food than lean controls (p < .OOl). After this baseline, both groups received 400 ug BOS l.C., followed 12 days later by i.c. vehicle control. BW and daily intakes were measured for 17 consecutive days.

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Several general results emerged from these investigations. First, EOS was effective in control subjects in eliciting reliable, dose-dependent anorexia which was replicable in duration and magnitude across experiments. Irrespective of dosage order, 100 ug EOS suppressed intake 30-50X the first day after treatment (p < .Ol) with recovery to normal intake occurring by day 3. At the 200 ug dose, intake was suppressed SO-70% for the first two days (ps < .OOl) with recovery occurring by day 5. intake was suppressed 70-90X for the At the 400 ug dose, first 3 days (ps < .OOl) with recovery occurring by days 6-7. Vehicle injections had no major effects on any parameter studied. Therefore, these findings replicate and extend to female rats the research by Howard et al (1980) in male rats that i.c. EOS in this dose range produces graded, reversible anorexia. More important to the theme of this paper, all three models of chronic overeating showed The possible exception essentially the same dose-dependent anorexia as did control subjects. was Zucker fatties, who ate slightly more than lean littermates under the effect of i.c. EOS. However, as only one dose was studied no firm conclusions can be drawn about in these animals, the significance of this finding. Indeed, since all models displayed dramatic overeating prior to and after drug injections, the approximate control levels of anorexia attained These suggest a temporarily complete reversal of those processes driving excessive feeding. results add further indirect support for the conclusion of our acute overeating studies in suggesting that elevating whole brain GABA may effectively impede excessive eating under certain conditions.

Specificity

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anorexia

An important consideration in determining the utility of a potential anorexigen is its specificity of action. Optimally, any such drug’s effects should be limited to reducing sensations of appetite and/or hunger, or conceivably the motivation to respond to those specific states. In practice it has been difficult to obtain such effects without Well known concomitantly impairing general sensory, motor and/or motivational processes. examples of this problem include the once widely-prescribed sympathomimetic, amphetamine, or its more recent derivative, fenfluramine. In the former case, a primary side-effect is psychomotor excitation; in the latter case, untoward sedation has been common. it is difficult to make definitive statements about At this early stage of investigation, the potential side-effects of GABA-elevating drugs like EOS. On the one hand, it is clear that administering very high doses would produce sedation or profound depression of many motor behaviors (see Palfreyman et al 1981). However, in the lower doses studied here, such an We have recently reported an in-depth explanation seems unlikely to account for our results. behavioral study in normal rats to determine if the anorexia induced by i.c EOS can be clearly associated with non-specific motor impairments (Nobrega and Coscina 1983). The results of that work imply that this is not the case. such experiments will have to be Of course, repeated using the various obese rodent models studied here before more firm conclusions can be drawn. However, informal observations of these animals suggest that the profound EOSinduced anorexia is not likely due to general motor impairments (see also Howard et al 1980 for additional documentation).

Conclusions This brief review of recent research serves to identify a relatively novel approach to the potential treatment of overeating disorders. While considerable additional work is required to assess this possibility, the current lack of effective clinical treatments for this major problem should provide smple impetus for future investigations. However, the conceptual viability of such treatment approaches most probably requires first a much better basic understanding of the intricate factors which appear to contribute to CNS GABA metabolism.

Acknowledgements Portions of this work were supported by the Clarke Institute of Psychiatry and grant MA-6579 from the MHC of Canada. I thank Dr. J. Nobrega for his many contributions to the research summarized here.

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References COSCINA, D.V. and DIXON, L. (1983) Bodyweight regulation in anorexia nervosa: Insights from an animal model. In: Anorexia Nervosa: Recent Developments in Research. P. Darby, P. Garfinkel, D. Garner and D. Coscina, Eds. Alan Liss: New York (in press). COSCINA, D.V. and LLOYD, K.G. (1980) Medial hypothalamic obesity: Association with impaired hypothalamic GABA synthesis. Brain Res. Bull. 1, S2: 793-796. COSCINA, D.V. and MUIR, C. (1981) Intracisternalethanolamine-O-sulfate(EOS) reverses overeating in three different animal models. Eastern Psychol. Assoc. Abstr. 52: 108. COSCINA, D.V., SEGGIE, J., GODSE, D.D. and STANCER, H.C. (1973) Inductionof rage in rats after central injection of 6-hydroxydopamine. Pharmac. Biochem. Behav. 1: l-6. ENNA, S.J. (1980) GABA in neuropsychiatricdisorders. Can. J. Neurol. ScT. 7: 257-259. HOWARD, J.L., COOPER, B.R., WHITE, H.L., SOROKO, F.E. and MAXWELL, R.A. (1985) A role for GABA in the control of ingestive behavior: Effects of ethanolamine-O-sulfateand muscimol. Brain Res. Bull. 1, S2: 595-599. LEIBOWITZ, S.F. (1980) Neurochemical systems of the hypothalamus. Control of feeding and drinking behavior and water-electrolyteexcretion. In: Handbook of the Hypothalamus. Vol. 3 - Part A. Behavioral Studies of the Hypothalamus. P.J. Morgane and J. Panksepp, Eds. Marcel Dekker: New York, 299-437. MCGEER, P.L., ECCLES, J.C. and MCGEER, E.F. (1978) Molecular neurobiology of the mammalian brain. Chapter 7. Inhibitory amino acid neurons: GABA and glycine. Plenum: New York, 199-231. NOBREGA, J.N. and COSCINA, D.V. (1982) Inhibition of acute feeding responses to systemic 2deoxyglucose or insulin in rats pretreated with the GABA-transaminaseblocker ethanolamineO-sulfate (EOS). Pharmac. Biochem. Behav. 17: 1145-1148. NOBREGA, J.N. and COSCINA, D.V. (1983) Cent=1 in'ections of the GABA transaminase inhibitor ethanolamine-O-sulfate(EOS): Effects on brain [ 14C]-2 deoxyglucose uptake and behavior in rats. Brain Res. 262: 243-252. PALFREYMAN, M.G., SCKHTER, P.J., BUCKETT, W.R., TELL, G.P. and KOCH-WESER, J. (1981) The pharmacology of GABA-transaminase inhibitors. Biochem. Pharmacol. -30: 817-824. Inquiries and reprint requests should be addressed to: Dr. Donald V. Coscina Section of Biopsychology Clarke Institute of Psychiatry 250 College Street Toronto, Ontario, Canada M5T lR8