Proc. Nad. Acad. Sci. USAVol. 82, pp. 6320-6324, September 1985Neurobiology

Glucostatic regulation of (+)-[3H]amphetamine binding in thehypothalamus: Correlation with Na',K+-ATPase activity

(amphetamine binding site/anorectic drugs/D-glucose)

ITZCHAK ANGEL*, RICHARD L. HAUGER*, MY Do Luu*, BRIDGET GIBLIN*, PHIL SKOLNICKt,AND STEVEN M. PAUL*t*Clinical Neuroscience Branch, National Institute of Mental Health, and tLaboratory of Bioorganic Chemistry, National Institute of Arthritis, Diabetes, andDigestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20205

Communicated by Seymour S. Kety, May 13, 1985

ABSTRACT Preincubation of rat hypothalamic slices inglucose-free Krebs-Ringer buffer (370C) resulted in a time-dependent decrease in specific (+)-[3H]amphetamine bindingin the crude synaptosomal fraction prepared from these slices.The addition of D-glucose resulted in a dose- and time-dependent stimulation of (+)-[3H]amphetamine binding,whereas incubations with L-glucose, 2-deoxy-D-glucose, or3-O-methyl-D-glucose failed to increase the number of (+)-[3HWamphetamine binding sites. Ouabain potently inhibited theglucose-induced stimulation of (+)-[3H]amphetamine binding,suggesting the involvement of Na',K+-ATPase. Preincubationof hypothalamic slices with glucose also resulted in an increasein Na+,K+-ATPase activity and the number of specific "high-affinity" binding sites for [3H]ouabain, and a good correlationwas observed (r = 0.89; P < 0.02) between the glucose-stimulated increase in (+)-[3H]amphetamine and [3H]ouabainbinding. Similar-increases in (+)-[3H]amphetamine binding,[3HJouabain binding, and Na+,K+-ATPase activity were ob-served in the hypothalamus after parenteral administration ofglucose to rats. The administration of anorectic doses ofamphetamine (0.1-5.0 mg/kg of body weight) also increasedNa+,K+-ATPase activity in the hypothalamus. These datasuggest that the (+)_[3HJamphetamine binding site in hypo-thalamus, previously linked to the anorectic actions of variousphenylethylamines, is regulated both in vitro and in vivo byphysiologial concentrations of glucose. Glucose and amphet-amine appear to interact at common sites in the hypothalamusto stimulate Na+,K+-ATPase activity, and the latter may beinvolved in the "glucostatic" regulation of appetite.

Amphetamine and structurally related phenylethylaminesproduce a variety of pharmacological effects including vaso-constriction, motor stimulation, and anorexia (1). Recentstudies in our laboratory have demonstrated the presence ofspecific low-affinity, high-capacity binding sites for (+)-[3H]amphetamine in membrane preparations from rat brain(2, 3). Specific (+)-[3H]amphetamine binding is saturable,stereospecific, temperature sensitive, highly enriched in thecrude synaptosomal fraction, and unevenly distributed invarious brain regions (3). Further, the relative potencies of aseries of phenylethylamine derivatives in displacing (+)-[3H]amphetamine binding from hypothalamic membranes ishighly correlated with their anorectic but not motor-stimulantpotencies (2). These findings suggest that (+)-[3H]ampheta-mine binding sites in the hypothalamus may mediate theanorectic actions of amphetamine and related phenylethyl-amines. Recently, we have observed that the binding of(+)-[3H]amphetamine to hypothalamic membranes is re-duced after periods of food deprivation (unpublished data).

Further, when food-deprived rats are allowed access to eitherrat chow or a 10% glucose solution, there is a rapid reversalof the food deprivation-induced decrease in hypothalamic(+)-[3H]amphetamine binding, and this effect is highly cor-related with changes in blood glucose concentration. Glu-cose, the principle energy source of brain, has been postu-lated to directly regulate "appetite" or food intake through acentral mechanism (the so-called "glucostatic hypothesis offeeding") (4, 5). In the present study we have investigated theeffects of glucose on hypothalamic (+)-[3H]amphetaminebinding in vitro and in vivo and now report that glucoserapidly and stereoselectively increases the number of (+)-[3H]amphetamine binding sites in the hypothalamus. Thisstimulation is highly correlated with increases in Na+,K+-ATPase activity and the number of specific "high-affinity"[3H]ouabain binding sites, suggesting that glucose and am-phetamine may interact through a coupled system in thehypothalamus to regulate Na+,K+-ATPase activity, and thatthis site may also be involved in the "glucostatic" regulationof appetite.

MATERIALS AND METHODS

Tissue Preparation. Adult male Osborne-Mendel rats(120-200 g) housed under diurnal lighting conditions (12:12)with free access to food and water were killed by decapita-tion, and their brains were quickly removed and dissected onice. Pooled hypothalami were chopped into 350 x 350 Atmslices using a McIlwain tissue chopper (Brinkmann). Theslices were washed twice in 10 ml of Krebs-Ringer bicar-bonate buffer (pH 7.4) and incubated at 37°C in the samevolume in a gently shaking water bath under a constantstream of 95% 02/5% CO2. D-Glucose and (or) other com-pounds were added, and after various times the tubes weretransferred to an ice bath, the buffer was aspirated, and theslices were washed twice in fresh Krebs-Ringer bicarbonatebuffer. Hypothalamic slices were then gently homogenized in10 volumes (wt/vol) of ice-cold 0.32 M sucrose by using aTeflon/glass homogenizer, and a crude synaptosomal pelletwas prepared as described (3).

Radioreceptor and Enzyme Assays. The binding of (+)-[3H]amphetamine sulfate was carried out as described byusing both filtration and centrifugation assays (3). Briefly,100-200 ug of membrane protein (crude P2 fraction) and 50 ,ulof (+)-[3H]amphetamine (200-300 nM unless otherwise spec-ified; specific activity, 20-30 Ci/mmol; New England Nu-clear; 1 Ci = 37 GBq) were added in a total incubation volumeof 300 ,ul. After a 30-min incubation at 0-4°C, the tubes wererapidly filtered over Whatman GF/B glass fiber filters under

tTo whom reprint requests should be addressed at: Section onMolecular Pharmacology, Clinical Neuroscience Branch, NIMH,Bldg. 10, Rm. 4N 214, Bethesda, MD 20205.

6320

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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vacuum as described (3). By the filtration technique, specificbinding [defined as the difference between the bindingmeasured in the presence and absence of unlabeled amphet-amine sulfate (100 MM)] was -80% of the total binding at aligand concentration of 200 nM. Centrifugation assays wereperformed in an identical manner, except that specificallybound (+)-[3H]amphetamine was separated from free ligandby centrifugation (40C) at 27,000 x g for 20 min. The resultingsupernatant was aspirated, and the pellet was washed super-ficially three times with 0.5 ml of ice-cold 50 mM Tris HClbuffer (pH 7.4). Ready-Solv (4 ml) (Beckman) was addeddirectly to the assay tubes (Beckman Bio Vials) and mixed,and the radioactivity was measured in a Beckman LS 9000liquid scintillation spectrometer. A comparison of the filtra-tion and centrifugation methods for measuring specific (+)-[3H]amphetamine binding has been reported (3). Similiarresults were obtained with either the filtration or centrifuga-tion assays.The specific binding of [3H]ouabain (specific activity, 20.9

Ci/mmol, New England Nuclear) was carried out in bothcrude brain homogenates and the crude synaptosomal (P2)fraction as described (6). The binding of [3H]imipramine wasconducted essentially as described (7) with a [3H]imipramineconcentration of -3.5 nM (specific activity, 70.7 Ci/mmol,New England Nuclear). The binding of [3H]dihydroalpreno-lol to B-adrenergic receptor was carried out by using themethod of Bylund and Snyder (8) and a [3H]dihydroalpreno-lol concentration of -1.5 nM (specific activity, 48.1Ci/mmol, New England Nuclear). The binding of [3H]diaze-pam to benzodiazepine receptors was carried out as de-scribed (9) with a [3H]diazepam concentration of -1.0 nM(specific activity, 85.4 Ci/mmol, New England Nuclear).Na+,K+-ATPase activity was measured in lysed crude

synaptic membranes by using a modification of the methoddescribed by Logan and O'Donovan (10). Inorganic phos-phate released by the enzyme was assayed by the method ofPeterson (11).

A

'-

0

- ~~~10 20 -30

RESULTS

Incubation of hypothalamic slices in oxygenated Krebs-Ringer bicarbonate buffer in the absence of-glucose resultedin a time-dependent decrease in the number of (+)-[3H]amphetamine binding sites in crude synaptosomal mem-branes prepared from these slices (Fig. 1A). The addition ofglucose resulted in a dose- and time-dependent stimulation of(+)-[3H]amphetamine binding. This stimulation was ob-served at glucose concentrations from 1 to 10 mM (Fig. 2) andthus occurs at physiologically relevant glucose concentra-tions. The decrease in (+)-[3H]amphetamine binding sitesafter incubation of hypothalamic slices in glucose-free bufferwas rapidly reversed by the addition of glucose (Fig. LA),which demonstrates an action ofglucose to not only maintainthe number of (+)-[3H]amphetamine binding sites but also todirectly increase binding to levels above that of tissueincubated in glucose-free buffer. In contrast, L-glucose,2-deoxy-D-glucose, or 3-O-methyl-D-glucose did not increaseor maintain (+)-[3H]amphetamine binding under identicalincubation conditions (Fig. 1A and Table 1). Kinetic analysisof the glucose-induced stimulation of (+)-[3H]amphetaminebinding revealed an increase in the density (BmaQ) of bindingsites with no change in the apparent affinity constant (Kd)(Fig. 3). In a representative experiment, the apparent Bmaxincreased from 72 pmol of (+)-[3H]amphetamine bound per

mg of protein in the absence of glucose, to 238 pmol/mg ofprotein in the presence of glucose (10 mM). In the sameexperiment, the apparent Kd values were 9.4 ,uM and 7.7 ,uM,respectively. Qualitatively similar results were obtained witheither a filtration or centrifugation assay (data not shown).The stimulation of hypothalamic (+)-[3H]amphetamine

binding by glucose was inhibited by coincubation with2-deoxy-D-glucose or ouabain (Table 1). Fig. 4 shows theeffects of various concentrations ofouabain on both the basaland glucose-stimulated levels of (+)-[3H]amphetamine bind-ing in hypothalamic slices. Ouabain, at concentrations up to1 mM failed to significantly alter the number of binding sites

3 4 5 6Incubation time, min [3H]Ouabain bound, pmol/mg of protein

FIG. 1. (A) Effect of preincubation of hypothalamic slices with glucose stereoisomers on (+)-[3H]amphetamine binding. Hypothalamic sliceswere incubated for the indicated time in the presence (o) or absence (o) ofeither D-glucose (10 mM) or L-glucose (10 mM) (o). The arrow indicatesaddition of D-glucose (10 mM) to tubes already preincubated without glucose, and the incubation was stopped 10 min later. Crude synaptosomalmembranes were subsequently prepared, and the binding of (+)-13H]amphetamine was carried out as described in the text. Data represent themean ± SEM of four separate experiments. Statistical analysis by Student's t test; **, P < 0.05; ***, P < 0.01 (indicate differences betweenhypothalamic slices incubated in the presence of glucose and the corresponding time points in the absence of glucose). Incubation with L-glucose(10 mM) failed to significantly alter (+)-[3H]amphetamine binding. (B) Correlation between the glucose-stimulated increase in (+)-[3H]amphetamine and [3H]ouabain binding. Preincubation of hypothalamic slices with or without D-glucose (10 mM) was carried out as describedin A. [3H]Ouabain binding to the crude homogenate and (+)-[3H]amphetamine binding to the crude synaptosomal fraction was carried out asdescribed in the text. A good correlation (r = 0.89, P < 0.01 Pearsons product moment) was observed between (+)-[3H]amphetamine and[3H]ouabain binding in the hypothalamic slices incubated at various time points in the presence and absence of D-glucOse.

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100

r._0

-& 60*._xZ

1 2 5 10 20D-Glucose, mM

50 100

FIG. 2. The effects of various concentrations of D-glucose (1-100mM) on (+)-[3H]amphetamine binding. Hypothalamic slices werepreincubated for 10 min in Krebs-Ringer bicarbonate buffer at 37°Cwith the indicated concentrations of D-glucose. After the incubation,the crude synaptosomal fraction was prepared as described in textand Fig. 1. Data represent the change in (+)-[3H]amphetaminebinding as a percentage of the maximal stimulation and are from a

typical experiment repeated three times with similar results **, P <0.01; ***, P < 0.005 (compared to incubations in the absence ofglucose).

in slices incubated in the absence of glucose. However,ouabain potently (EC50, 10 tkM) inhibited the glucose-inducedstimulation of (+)-[3H]amphetamine binding (Fig. 4). Similarresults were obtained with 2-deoxy-D-glucose (Table 1).Furthermore, incubation of crude (P2) membranes withouabain (up to 1 mM added directly to the binding assay) hadno effect on (+)-[3H]amphetamine binding (data not shown).The antagonism of the glucose-stimulated increase in

(+)-[3H]amphetamine binding by ouabain suggests the in-volvement of Na+,K+-ATPase. In subsequent experiments,the effects of preincubation with and without glucose was

examined on the Na ,K+-ATPase activity of hypothalamic

Eco .E

co .

x

+ o_-E'a c;r.

0m

2 5 10 20

(+)-[3H]Amphetamine, ,uM

Table 1. Effect(s) of various D-glucose analogues on the glucose-induced stimulation of specific (+)-[PH]amphetamine binding inhypothalamic slices

D-Glucose Bound [3H]amphetamine,D-Glucose analogs (10 mM) pmol/mg of protein

Control - 1.77 ± 0.30+ 3.35 ± 0.55*

2-Deoxy-D-glucose - 1.55 ± 0.38t+ 1.40 ± 0.41tt

3-O-Methyl-D-glucose - 1.48 ± 0.31t+ 2.75 + 0.52t§

L-Glucose - 1.90 ± 0.23t+ 3.67 ± 0.44*§

Hypothalamic slices were incubated for 10 min at 370C inKrebs-Ringer bicarbonate buffer (pH 7.4) in the presence or absenceof D-glucose (10 mM) and with 100 mM of the indicated glucoseanalogues. Subsequently, crude synaptosomal membranes wereprepared, and specific (+)-[3H]amphetamine binding was measuredas described in the text. Statistical analysis was done using Student'sf-test. Values represent the mean ± SEM of three separate incuba-tions from a typical experiment repeated four times with similarresults.*P < 0.005 compared to control.tNot significant compared to control.tP < 0.05 compared to control with glucose.§Not significant compared to control with glucose.

slices. The stimulation of (+)-[3H]amphetamine binding inhypothalamic slices by D-glucose was also associated with anincrease in Na+,K+-ATPase activity (Figs. 1 and 3). Asignificant increase in Na+,K+-ATPase activity after incu-bation of hypothalamic slices with glucose was observed witheither an assay for direct measurement of Na+,K+-ATPaseactivity (data not shown) or the number of high-affinity[3H]ouabain binding sites (Figs. 1 and 3 and Table 2); the latteris a specific and selective ligand for measuring the number ofNa+,K+-ATPase units (cf. refs. 6, 11, and 13). Furthermore,there was a good correlation (r = 0.89, P < 0.01) between theglucose-induced stimulation of (+)-[3H]amphetamine and[3H]ouabain binding over time (Fig. 1B). Kinetic analysis of theeffect(s) ofglucose on [3H]ouabain binding revealed an increasein the number of sites with no significant change in the apparentaffinity (Kd) (Fig. 3B).

w

4-

0

0

2 4 6 8 10Bound, pmol/mg of protein

FIG. 3. Kinetic analysis of the D-glucose-stimulated increase ifl specific (+)-[3H]amphetamine and [3H]ouabain binding. Hypothalamic sliceswere incubated in Krebs-Ringer bicarbonate buffer for 10 min in the presence (e) or absence (0) of D-glucose (10 mM). Crude synaptosomalmembranes were subsequently prepared (see text), and the binding of (+)-[1H]amphetamine (A) and [3H]ouabain (B) were measured as describedby using ligand concentrations from 0.5 to 100 ,uM and from 5 to 200 nM, respectively. (A Inset and B) Kinetic data was plotted by the methodsof Klotz and Scatchard (cf. ref. 12).

20fr

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4

C

C)

.1 r 3

E2

E~~~123-

FIG. 4. Effect of ouabain on the D-glucose-stimulated increase in(+)-[3H]amphetamine binding. Hypothalamic slices were preincu-bated for 10 min in the absence (bars 1 and 4) or presence (bars 2 and3) of D-glucose (10 mM) and with ouabain at 10 1LM (bar 3) or 1 mM(bar 4). In this experiment D-glucose (10 mM) resulted in a 4-foldincrease in the (+)-[PH]amphetamine binding (**, P < 0.01).Coincubation with ouabain at 10 ,uM significantly inhibited thisstimulation [tt, P < 0.02 (compared to D-glucose alone)]. Datarepresent the mean ± SEM of quadruplicate incubations from arepresentative experiment repeated three times with similar results.

In related experiments, we studied the effects of D-glucoseadministration on the number of hypothalamic (+)-[3H]amphetamine binding sites in vivo. After parenteraladministration of glucose (750 mg/kg i.p., administered as a10% solution at 45-min intervals over 3 hr) significantincreases were observed in the binding of (+)-[3H]ampheta-mine and [3H]ouabain to hypothalamic membranes whencompared to membranes prepared from saline-injected rats(Fig. 5). No significant changes in (+)-[3H]amphetaminebinding were observed in other brain regions such as thestriatum, cerebral cortex, or cerebellum (Fig. 5). The effectsof parenteral glucose administration on (+)-[3H]ampheta-mine and [3H]ouabain binding also represent changes in thenumber of binding sites, with no significant changes in theapparent Kds for either ligand (data not shown). L-Glucoseadministered to rats in an identical fashion failed to increase(+)-[3H]amphetamine binding in the hypothalamus (data notshown).The effect(s) of preincubating hypothalamic slices in the

presence or absence of glucose on various other neurotrans-

Table 2. Effect(s) of glucose on various drug/neurotransmitterreceptors in hypothalamic slices

Specifically boundligand,

fmol/mg of protein

Without WithRadioligand glucose glucose Significance

[3H]Dihydroalprenolol 140 ± 10 167 ± 28 NS[3H]Ouabain 1079 ± 13 1664 ± 173 P < 0.02[3H]Diazepam 465 ± 26 538 ± 37 NSPHIAmphetamine 1680 ± 330 4617 ± 586 P < 0.005[3H]Imipramine 472 ± 61 400 ± 34 NS

After the incubation of hypothalamic slices with or withoutD-glucose (10 mM) in Krebs-Ringer bicarbonate buffer (pH 7.4) for10 min at 370C, crude synaptosomal membranes were prepared asdescribed in the text. The various receptors were assayed by usingligand concentrations at approximately their apparent Kd values.Statistical analyses were done by using Student's t-test. Valuesrepresent the mean ± SEM of triplicate determinations from a typicalexperiment repeated twice with similar results.

....

T

E10

T

. .:.

HypothalamuLs Striatum Cortex Cerebellum

FIG. 5. (A) Effect of parenteral D-glucose administration onspecific (+)-[3H]amphetamine binding in various brain regions.(Inset) [3H]Ouabain binding in the hypothalamus. Rats were injectedi.p. with a 10% glucose solution (in 0.9% NaCl) administered in fourinjections (750 mg/kg of body weight in each injection) at 45-minintervals. Forty-five minutes after the last injection the rats werekilled and (+)-[PH]amphetamine and [3H]ouabain binding weremeasured in membranes prepared from the various brain regions asdescribed in the text. Data are from a representative experiment,repeated three times with similar results. Values represent the mean± SEM of triplicate determinations from individual animals (n = 6).**, P < 0.02 [compared to saline controls (Student's t test)].

mitter/drug receptors was also studied. Preincubation ofhypothalamic slices with D-glucose (10 mM) had no effect onthe specific binding of [3H]dihydroalprenelol to 83-adrenergicreceptors, [3H]diazepam to benzodiazepine receptors, and[3H]imipramine to imipramine binding sites (Table 2).

Since glucose stimulates both (+)-[3H]amphetamine bind-ing and Na+,K+-ATPase activity in the hypothalamus andsince ouabain potently inhibited the glucose-stimulated in-crease in (+)-[3H]amphetamine binding in vitro, we examinedwhether the administration of pharmacologically relevantdoses of amphetamine would alter Na+,K+-ATPase activity.Fig. 6 shows the effects of amphetamine administration(0.1-5.0 mg/kg) to rats on Na',K+-ATPase activity and[3H]ouabain binding in the hypothalmus. A significant stim-ulation of Na+,K+-ATPase activity and [3H]ouabain bindingwas observed with doses of amphetamine as low as 0.1mg/kg. No change in Na+,K+-ATPase activity was observedin the striatum from these same animals (data not shown).

DISCUSSIONSaturable and stereospecific binding sites for (+)-[3H]am-phetamine that are enriched in crude synaptosomal mem-branes from the hypothalamus have been described (2, 3).These sites appear to be related to the anorectic but notmotor-stimulant properties of various phenylethylamine de-rivatives and are modulated by food deprivation andrefeeding.

In the present study, we observed a dose-dependentstimulation of (+)-[3H]amphetamine binding by glucose inhypothalamic slices (Fig. 1A). Parenteral administration ofglucose also results in an increase in hypothalamic (+)-[3H]amphetamine binding (Fig. 5). The onset of this stimu-lation is rapid and represents an increase in the number (Bmax)

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14

121

**Ul.0 10

24^-12~0-.

0 ~~~100

6-

8

3 6 9

Na' ,K'-ATPase activity,

Amiol of Pi/mg of protein per hr

~~~~~~5

Amphetamine, mg/kg

FIG. 6. Effect of parenteral administration of amphetamine torats on specific [3H]ouabain binding and Na+,K+-ATPase activity(Inset) in the hypothalamus. (+)-Amphetamine sulfate (0.1-5.0mg/kg, i.p.) was administered to rats in 0.9% NaCl, and 30 min laterthe animals were killed. [3H]Ouabain binding was measured in thefreshly prepared crude synaptosomal membrane fraction (P2) andNa+,K+-ATPase was measured in these same membranes afterfreezing (-20°C). Values represent the mean ± SEM of triplicatedeterminations from a typical experiment repeated three times withsimilar results. [*, P < 0.05; **, P < 0.01 (compared to saline-injectedcontrols)]. (Inset) r = 0.83; P < 0.01. Correlation between[3H]ouabain binding and Na+,K+-ATPase was carried out by usingPearson's product moment.

of (+)-[3H]amphetamine binding sites. The mechanism re-

sponsible for this relatively rapid induction of binding isunclear, although the high capacity of (+)-[3H]amphetaminebinding in brain membranes suggests that these binding sitesmay be associated with a membrane-bound enzyme (3).Thus, the stimulation of (+)-[3H]amphetamine binding byglucose could be related to the induction or allosteric acti-vation of an enzyme that is labeled by this radioligand.Moreover, there were no changes in the number of benzo-diazepine receptors, P-adrenergic receptors, or [3H]imipra-mine binding sites in hypothalamic membranes incubated at37°C in the presence or absence of glucose (Table 2),suggesting that the effects of glucose in stimulating (+)-[3H]amphetamine and [3H]ouabain binding were not due to anonspecific alteration of membrane protein.Ouabain completely antagonized the effects of glucose in

stimulating (+)-[3H]amphetamine binding but had no effecton the basal binding in the absence of glucose (Fig. 4). Thesedata suggest that the stimulation of (+)-[3H]amphetaminebinding in hypothalamic slices may be dependent on

Na+,K+-ATPase activity. Subsequent experiments revealedthat glucose also stimulates Na+,K+-ATPase activity and[3H]ouabain binding in hypothalamic slices and that therewas a good correlation between the increase in (+ )-

[3H]amphetamine and [3H]ouabain binding in vitro (Figs. 1Band 3B). It is possible, therefore, that the effect(s) of glucosein stimulating (+)-[3H]amphetamine binding are secondary toan action on Na+,K+-ATPase activity. These results furthersuggest that amphetamine (as well as other drugs that bind tothe (+)-[3H]amphetamine binding site) may also regulateNa',K+-ATPase activity. The administration of relativelylow doses of amphetamine (Fig. 6) to rats, in fact, doesincrease Na',K+-ATPase activity and [3H]ouabain bindingin the hypothalamus.

Since our previous data suggests that the (+)-[3H]amphet-amine binding site in hypothalamus mediates the anorecticproperties of amphetamine and related phenylethylamines(2), the regulation of hypothalamic (+)-[3H]amphetaminebinding by glucose may be related to the previously reportedeffects of glucose in regulating feeding and satiety (14, 15).The elevation of circulating or brain glucose levels afterfeeding (or by intraventricular administration of glucose) infood-deprived rats, is followed by a rapid termination offoodintake (15). Glucoprivation, induced by 2 deoxy-D-glucose, isassociated with an increase in food consumption, and thiseffect is blocked by pretreating rats with intraventricularalloxan, implying the presence of central glucoreceptors,which mediate glucoprivic feeding (16). The presence offunctional glucoreceptors in the hypothalamus also has beendemonstrated by electrophysiological techniques (5). More-over, the effect of glucose in hyperpolarizing hypothalamicneurons is reversed by ouabain, suggesting that the hyper-polarizing action of glucose is secondary to an activation ofNa+,K+-ATPase (5). Taken together, our data suggest thatglucose and amphetamine interact through a tightly coupledsystem in the hypothalamus to regulate Na+,K+-ATPaseactivity. Whether or not the activation of hypothalamicNa+,K+-ATPase activity by glucose and (or) amphetamine isresponsible for their appetite-suppressant actions will requirefurther study.

1. Biel, J. H. & Bopp, B. A. (1979) in Handbook ofPsychophar-macology, eds, Iversen, L. L., Iversen, S. D. & Snyder, S. H.(Plenum, New York), Vol. 11, pp. 1-39.

2. Paul, S. M., Hulihan-Giblin, B. & Skolnick, P. (1982) Science218, 487-490.

3. Hauger, R. L., Hulihan-Giblin, B., Skolnick, P. & Paul, S. M.(1984) Life Sci. 34, 771-782.

4. Mayer, J. (1953) Ann. N. Y. Acad. Sci. 63, 15-43.5. Oomura, Y. (1983) in Advances in Metabolic Disorders, ed.

Szabo, A. J. (Academic, New York), Vol. 10, pp. 31-65.6. Hauger, R., Luu, H. M. D., Meyer, D. K., Goodwin, F. K. &

Paul, S. M. (1985) J. Neurochem. 44, 1709-1715.7. Rehavi, M., Paul, S. M., Skolnick, P. & Goodwin, F. K.

(1980) Life Sci. 26, 2273-2279.8. Bylund, D. B. & Snyder, S. H. (1976) Mol. Pharmacol. 12,

568-580.9. Paul, S. M. & Skolnick, P. (1978) Science 202, 892-894.

10. Logan, J. G. & O'Donovan, D. J. (1980) Biochem. Pharmacol.29, 2105-2112.

11. Peterson, G. L. (1978) Anal. Biochem. 84, 164-172.12. Klotz, I. M. (1982) Science 217, 1247-1249.13. Hansen, 0. (1984) Pharmacol. Rev. 36, 143-163.14. Geiselman, P. J. & Novin, D. (1982) Appetite 3, 203-223.15. Davis, J. D., Wirtschafter, D., Asin, K. E. & Brief, D. (1981)

Science 212, 81-83.16. Woods, S. C. & McKay, L. D. (1978) Science 202, 1209-1211.

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