J. Nutr.-1993-Gietzen-610-25.pdf

7/27/2019 J. Nutr.-1993-Gietzen-610-25.pdf

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Critica l ReviewNeural Mechanisms in the Responses toAmino Acid Deficiency1

DOROTHY W. GIETZENDepartm ent of Physiological Sciences, School of Veterinary M edicine andF ood Intake L aboratory, U niversity of C alifornia-D avis, D avis, CA 95616

A BST RA CT Food intake is rapidly and reliably reducedw hen an im als are o ffered d iets th at resu lt in an e ssentialamino acid deficiency, such as those used in the im-balan ced am in o acid diet (1MB )p arad igm. T here se em tobe at least three phases in the responses of rats to 1M B:1) In o rder to resp on d to a d ieta ry ch allen ge, th e a nim alsm ust first recognize that challenge. T he available datasuggest that before the behavioral effects occur, a decline in the concentration of an essential amino acid issensed in a specific brain area, the prepyriform cortex.This recognition phase is associated with localizeddecreases in the concentrations of the lim iting am inoacid, norepinephrine and cyclic A MP and with alteredprotein synthesis. 2) Subsequent to recognition of thedeficiency, a conditioned taste aversion develops,m ediated in part by serotonin at the level of the vagus.3) Finally, in the absence of a choice, the anim als adaptto an 1M B(but not a diet devoid of one or m ore essentialam in o a cid s) in ~1 w k. D am ag e to certain ex trahy po th a-lam ic brain areas or liver denervation accelerates adaptation to 1M B, suggesting both central and peripheralco ntrol in th e ad aptatio n p hase. T he resu ltin g b eh av io ralresponses provide adaptive advantage to an animal inthe selection of a diet w ith an appropriate balance ofamino acids. J. Nutr. 123: 610-625, 1993.INDEX ING KEY WORDS:fe ed in g b eh av io r n eru ou s s ys temano re xia amin o a ci d imbala nc erats serotonin norepinephrineess en ti al am ino acids pro te in s yn thes is

A nim als, particularly om nivores and generalist herbivores, need mechanisms that provide for selectionof a balanced diet to meet all of their nutrient requirements. For most nonruminants, this includes the indispensable (essential) am ino acids. Because they arenot stored (Berg and Rose 1929), in the event of adeficiency, am ino acids are utilized from bodyprotein, resulting in negative nitrogen balance (M unro1976). Clearly, the capacity to distinguish balancefrom imbalance among the am ino acids in the dietand to select for the growth-lim iting essential am ino

acid provides adaptive advantage to anim als. Torii etal. (1987) showed that rats eating a lysine-dficientdiet can learn to select from 15 water bottles containing various solutions (including lysine, otheramino acids and saccharin) the one that containslysine. W hen given a lysine-repl te diet these ratsquickly select a saccharin solution, which rats norm ally prefer. M any authors have reported choice situations in which the animals have demonstratedselection for a necessary amino acid, albeit havingfewer alternatives (e.g., Booth and Simson 1971,Gietzen et al. 1992, Halstead and Gallagher 1962,Rogers and Harper 1970). The basic question underlying the studies to be reviewed here is: Whatmechanism s allow such exquisite sensitivity to dietary amino acids?

H ISTORICAL PERSPECT IVEAmino acid imbalances were defined by Harper(1976) as resulting from "additions to a low protein

diet of one or more amino acids, other than the onethat is grow th lim iting, in am ounts that individuallyare not toxic. They cause depressions in food intakeand grow th that are readily prevented by a supplement of the growth-lim iting amino acid."Nitrogenous constituents have been recognized asimportant in the diet for over 150 y (M agendie 1816),and diets containing inadequate amounts of am inoacids have been associated w ith a m arked depressionof growth and food intake since the early part of the

20th century (Osborne and M endel 1914, Rose 1931,W illcock and Hopkins 1906). Perhaps the first use of

'S upported by U SD A: C SR S 90 37200-5440 and N IH grants no.DK42274; NIH DK35747 to the Food Intake Laboratory via theC lin ic al N utrition R esea rch U nit, U niv ersity o f C alifo rn ia -D av is ,Sandoz R esearch Institute, and a gift of dietary am ino acids fromN utri-Q ue st (C he sterfie ld , MO ).

0022-3166/93 $3.00 1993 A merican Institute of N utrition. Received 9 Septem ber 1992. A ccepted 30 N ovem ber 1992.61 0


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NEURAL RESPONSES TO AMINO ACID DEFICIENCY 611

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60

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n i i i i i i i i i i i i 1 1 i 1111 i i 1 1 i i i i 1 1

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t S ^ eg s . . 2 s t t^ o ? o iiEw - t .*FIGURE 1 Effect of feeding imbalanced amino acid or basal control diet on concentrations of amino acids in plasma.Plasm a w as collected at various tim es, as indicated, after a single m eal of the test diet. Closed circles: basal diet; X : threonine-imbalanced diet; values are ^mol/100 mL plasma, n = 5 per group. Amino acids are indicated by their conventionalabbreviations along the abscissa. From Leung et al. (1968b), reproduced with permission. For comparison with Figure 4,(values given in ^m ol/100 m L)--10 values as nm ol/m L.

an im balanced am ino acid diet (1M B)2'3 was in studiesusing gelatin (which is devoid of tryptophan and deficient in several essential am ino acids) as the source ofdietary protein (reviewed by Jackson et al. 1928).Then, with the isolation of threonine, the final am inoacid discovered to be required for growth (McCoy etal. 1935), the protein fraction of the diet could becomposed entirely of amino acids, and the effects ofeach indispensable amino acid could be studied withprecision.The effects of 1MB on food intake and grow th havebeen observed in several animal species (Harper et al.1970) and have been the subject of several reviews(Elvehjem and Krehl 1955, Gietzen et al. 1986a and1988a, Harper and Rogers 1965, Harper et al. 1970,Jackson et al. 1928, Leung and Rogers 1987, Rogersand Leung 1973 and 1977, Salmon 1958). Aminoacid-im balanced diets can be distinguished from deficient diets, because either reducing the added aminoacids or increasing the lim iting one will restorebalance to an 1MB, whereas only increasing thelim iting amino acid w ill remedy a deficient diet. Itshould be noted that requirement studies usinggraded reductions in the lim iting amino acid can alsoproduce an imbalanced situation (M illward andR ivers 1988 ).The observation that a reduction in food intakeoccurs before the growth retardation led to the reali

zation that the depression in food intake is theprim ary event (Harper and Rogers 1965); anim als fed1MB grow to the level of control animals if foodintake is maintained. This demonstrated that thedepression in food intake is the cause rather than theresult of the growth failure (Harper et al. 1970). Thesehistorical studies show clearly that am ino acids playan important role in the control of food intake.In the search for the mechanism s underlying theresponses to 1M B, the one biochem ical observationthat has consistently been made is a marked imbalance in the plasma amino acid pattern, which becomes apparent within a few hours after a meal of1M B (Leung et al. 1968b; Fig. 1) and which resemblesthat of a severe amino acid deficiency (Harper andRogers 1965). The anorectic response can be seen asearly as 28 min after introduction of a threonine-devoid diet and is well correlated with a decreased

Abbreviations used: AC, anterior cingulate cortex; CNS,c en tral n erv ou s s ys te m; 5H IAA, 5-h yd ro xy in do le a min o a cid; 5HT,serotonin; 1MB, am ino acid-imbalanced diet; LH, lateralhypothalam us; N E, norepinephrine; PPC , anterior prepyriformcortex; P VN , paraventricular nucleus of the hypothalam us; S CO P,s co po la mine ; VMH, v entro me dial h ypo th alam us .3Diets have been described in detail (Beverly et al. 1991b,Ham mer et al. 1990b).


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612 GIETZENconcentration of threonine in the plasma (Gietzen etal. 1986a). Thus, the use of 1M B provides a paradigmfor the study of a rapidly occurring, robust, nutritionally induced decrease in food intake due to recognition of an amino acid deficiency.This review will focus on studies of the neuralmechanisms underlying the responses of rats toam ino acid deficiency. The 1MB feeding paradigmused by the author, as well as by Rogers and Leung(reviewed in 1977) and others, has a distinguishedhistory and was reviewed com prehensively by Harperet al. (1970). The responses will be viewed in thecontext of 1} the initial m etabolic factors occurringw ith ingestion of 1MB, i.e., the recognition phase; 2)the aversive responses that require a period oflearning; and 3) the adaptive phase, which is seen oversev eral d ays.

PHASE ONE: RECOGNITION OFAM INO ACID DEFICIENCY

Recognition of the am ino acid deficiency does notrequire intact olfactory (Leung et al. 1972), hepatic(B ellinger et al. 1992), vagai (Jiang 1992, W ashburn etal. 1992), gastric (Stickney et al. 1976) or adrenal(Hammer et al. 1990b) systems. Several otherpotential physiological variables have been studiedand nearly as many negative results reported(reviewed in Harper and Rogers 1965). By definition(see above), the amino acids individually added to1MB are not toxic, because in the absence of a choiceanimals will adapt and grow on 1MB. Although important in learned responses to 1M B (Chambers 1990,Mori et al. 1991, Rogers and Leung 1977), taste isunlikely to be essential for the initial recognition of1MB that occurs in the first hour after diet introduction (R ogers and Leung 1977); palatability doesnot contribute to the initial recognition of 1MB, because 1M B intake is not reduced during the first meal(Rogers and Leung 1977).Rather, in studies dating back to the late 1960s,several lines of evidence have supported the idea thatthe brain is the site of recognition in this model. Theprimary observation was that the food intakedepression of rats (Leung and Rogers 1969) and cockerels (Tobin and Boorman 1979) fed 1MB wasprevented after a sm all quantity of the m ost lim itingamino acid was infused into the carotid artery, beginning a few hours before 1MB was fed. Infusion ofthe lim iting amino acid into the portal or jugular veinrequired a much greater quantity than that given intothe carotid artery to prevent the food intakedepression (Leung and R ogers 1969). In addition, Tew sand colleagues (Tews et al. 1978, 1979 and 1981) haveshown that the plasma amino acid imbalance createsa disadvantage for the lim iting amino acid in itscompetition for transport into the brain, reducing the

concentration of the growth-lim iting am ino acid inthe brain. Reduced concentrations of the lim itingam ino acid in whole brain were measured within 2-3h after consumption of 1MB (Peng et al. 1972) andwere well correlated with the onset of the feedingdepression (reviewed in Gietzen et al. 1986a). Theseseparate findings demonstrate that the brain has anim portant role in the food intake responses of anim alsingesting am ino acid-im balanced or devoid diets. Thework of Tews et al. (1978, 1979 and 1981) describedthe m echanism for establishing am ino acid im balancein the brain (Tackman et al. 1990) but did not provideevidence for localization within the brain.In a series of neuroanatomical experiments, Leungand Rogers (1987) studied the effects of various brainlesions on intake of 1MB, devoid and high proteindiets (Table 1). Among the results was the observation that limbic-rhinencephalic neural areas thatrelay olfactory information, such as the anteriorprepyriform cortex (PPC ) and am ygdala, are essentialfor the initial depression of food intake in rats fed1MB (Firman and Kuenzel 1988, Leung and Rogers1971, 1973 and 1987, Noda 1975, Noda andChikamori 1976, Rogers and Leung 1973) or devoiddiets (Leung and Rogers 1971). Indeed, the PPC hasbeen suggested to be the body's amino acid"chemosensor" (Beverly et al. 1990a and 1990b,Leung and Rogers 1973).To determ ine w hether the decreased concentrationof the lim iting amino acid, which had already beenobserved in whole brain by Peng et al. (1972), occursuniform ly throughout the brain, 14 microdissectedbrain areas, including the PPC, were assayed for concentrations of the lim iting amino acid after 1MBfeeding. The results (Table 2) show that, althoughdecreases in the lim iting am ino acid in the 1MBgroups were not seen in all brain areas, the concentrations of threonine in the PPC, anterior cingulatecortex (AC), locus ceruleus and nucleus of the solitarytract from the threonine-im balanced diet group weresignificantly reduced. Thus, regional differences inthe concentrations of amino acids were seen withinthe brain at 2.5 h, when food intake had just becomedepressed. It is interesting that after ingestion of 1M Bthe concentration of the lim iting amino acid was notaltered in any of the three hypothalam ic areas form erly thought to control feeding (Gietzen et al. 1989)or in the am ygdala. A reduction of the lim iting am inoacid did occur in other brain areas (listed above and inTable 2), suggesting that those areas, along w ith thePPC, have the potential to serve as sensors of anam ino acid decrease. However, whereas either PPC oramygdala lesions resulted in animals that did notavoid the 1M B, the lim iting am ino acid was decreasedin the PPC but not in the amygdala. Moreover, theamygdala has been linked to conditioned tasteaversion (M eliza et al. 1981), which can develop onlyafter recognition of some negative stimulus


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NEURAL RESPONSES TO AMINO ACID DEFICIENCY 61 3

TABLE 1E ffects of electrolytic lesions in various brain regions, or rem oval of pituitary or olfactory bulbs of rats, on intake ofa min o a cid-u nba la nced , a min o a cid -d evo id o r h ig h p ro tein d iets1

LesionI ni ti al Re sp on se

1MB DEV HIAdaptation

1MB DEV HIAnt eri or amy gd al aA nte rio r c in gu la te c orte xA nte rio r p rep yrifo rm c ortexA re a p os tr em aDo rs ol at er al h ip po campu sL at er al s ep tumMedia l amy gd alaN ucleus of solitary tractPo st er io r amygda laR ap he n uc le iT halam ic taste nucleiV en tra l te gm ental n ucleiVent romed ia l h ypot ha lamusHypophysectomyO l fa ct or y bul be ct omy

4 -*

4

TTTTT

4'S ym bols used: 4 or T , d ecreased or increased intake of indicated diet, relative to that anim al's baseline control intake; ", increased intakewith mild but not severe imbalance; ?*, food intake greater than control over time but not true adaptation. Diet abbreviations: 1M B,

imbalanced amino acid diet; DEV, diet devoid of an essential am ino acid; HI, 75% casein diet. Data taken from Leung and Rogers (1987|,Leung et al. (1986a, 1987a and 1987b), Rogers and Leung (1973 and 1977).

(Chambers 1990). Thus, the PPC remains, from thesestudies, the primary candidate for the brain area thatmay serve as the essential am ino acid chemosensor.

The limiting amino acid in thep re py rifo rm corte xThe concentration of the lim iting amino acid inthe PPC, measured after feeding basal diet or 1MB(Gietzen et al. 1986b), was used in determ ining theconcentration of amino acid to use for injections intothe PPC. In this series of studies, the effects ofreplacing the lim iting am ino acid precisely into thePPC were established (Beverly et al. 1990a, 1990b,1991a, 199Ib and 1991c). A fter injections of threonineinto the PPC, rats did not decrease their intake of athreonine-imbalanced diet whereas, as expected,threonine had no effect on intake of an isoleucine-im balanced diet. Sim ilarly, isoleucine injected intothe PPC reversed the feeding depression with anisoleucine- but not a threonine-imbalanced diet. Theoptimum dose of threonine, in improving intake of athreonine-imbalanced diet, was ~2 nmol (given inbilateral injections of 0.5 ^L), and intake of the basaldiet w as not affected using sim ilar injections (Beverlyet al. 1990a and 1990b; solid bars in Fig. 2). S im ilar

TAB LE 2Concentration of threonine in rat brain areas taken 2.5 ha ft er in tr od uc ti on o f t hr eo ni ne -b as al , - imba la nc edo r -c or rec te d diets1

DietB ra in are a Basal Im balanced C orrected

\unolfg w et tissueAmygdalaA nte rio r cin gu late co rtexA r ea pos tr emaHippocampusLa te ral hypo tha lamusLo cu s c oe ru lu sN ucleus of the solitarytractPar ab ra ch ia l n uc le usPa ra vent ri cu la r n uc le usP re py rif orm c or te xR ap he n uc le iSeptumTegmentumVentromedialhypothalamus

0 .1 4 0 .00 0 .1 5 0 .010 .1 2 0 .30 0 .0 6 0 .02 '0 .2 0 0 .0 3 0 .2 1 0 .060 .2 7 0 .05 0 .2 8 0 .040 .2 5 0 .0 3 0 .2 9 0 .070 .2 5 0 .0 4 0 .1 5 0 .00 *0.210.370.180.140.250.35

0.040.140.020.020.070.11

0 .1 2 0 .0 5-0.280.180.080.220.30

0.150.040.02'0.040.030.2 9 0 .0 3 0 .2 2 0 .0 4

0 .2 4 0 .0 10 .3 0 0 .0 80 .3 6 0 .0 60 .3 1 0 .0 50 .3 0 0 .0 20 .2 4 0 .0 20 .3 2 0 .0 30 .4 2 0 .0 90 .3 1 0 .0 40 .2 5 0 .0 50 .3 3 0 .0 40 .4 8 0 .0 70 .4 4 0 .1 3

0.24 0.02 0.26 0.04 0.36 0.04'V alu es a re m ea ns SE,n = 4 -6 /g ro up. 'S ig nific an tly re du ce d, P< 0.05 by F isher's protected least significant difference test after

A NO VA . D ata taken from G ietzen et al. (1986a, 1986b, 1988b and1989).


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614 GIETZEN

CDS

10 0

75

S O

25

VEHICLEPUnOMYCIN

a C S F THREONINEFIGURE 2 Effects of threonine, with and withoutpuromycin, injected into the prepyriform cortex in0.3-fjL volum es, on intake of a threonine-im balanced diet.Bars represent means for food intake over 24 h as a percentage of each animal's baseline intake of the basal diet.Error bars: SE. Solid bars: artificial cerebrospinal fluid(aCS F), injected w ith a control injection of aC SF (vehicle) orL-threonine (6.67 m mol/L), as indicated on the abscissa; n =5 p er g rou p. H atche d b ars: p uromy cin (10 0 / m ol/L )in jected

with aCSF or threonine. Injections were made 0.5 h prior tointroduction of the imbalanced diet. Adapted from data inB everly et al. (1991a).

injections made 2 mm posterior to the PPC or intothe amygdala were ineffective, showing regionalspecificity as well (Beverly et al. 1990a).Injection of threonine 6 h before onset of the darkcycle also resulted in increased intake of thethreonine-imbalanced diet during the first 6 h of thedark cycle. Total intake of the 1MB was higher (85%of baseline) than that seen with injections madecloser to the time of diet introduction, althoughtim ing of a vehicle injection did not alter intake ofthe 1MB at any time point. Thus, the effect of injecting the lim iting am ino acid into the PPC lasted atleast 6 h, and we have noted a delay of at least 3 hbefore increased intake of the 1MB was observed, ifthe threonine was injected into the PPC just beforefeeding. Delivering two half-optimal doses, severalhours apart, brought intake to the same level as asingle injection of the optimal dose given just beforefeeding. Conversely, when threonine (2 nmol) wasinjected twice (6 h and 0.5 h before feeding), theorexigenic effect of the 2-nm ol dose was reduced, as itwas in dose-response studies using 4 nmol ofthreonine in a single dose. The results indicate thatsome integral of the changes in the concentrations ofamino acids is recognized in the PPC, influencingintake of 1MB (Beverly et al. 1993).It was hypothesized that diffusion or metabolismcould account for the time lag between injection ofthe lim iting amino acid into the PPC and the increased intake of 1MB. To exam ine these questions,first, diffusion of the lim iting amino acid was determ ined by m easuring the amount of L-[14C]threonine

in the PPC and surrounding areas at various timesafter injection. Diffusion of label was rapid (


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NEURAL RESPONSES TO AMINO ACID DEFICIENCY 615

ILE BAS ALILE IM BA LA NC EDILE C OR RE CTE D

5OOUl0 ) 40Hl_ 3 00UJo , e oox .e *c 100

NE DA 5H TFIGURE 3 Concentrations of the neurotransmittersnorepinephrine (N E), dopam ine (D A) and serotonin (5H T) inthe prepyriform cortex of rats fed isoleucine-lim iting dietsas indicated. Bars: mean; error bars: SE for 6-7 rats pergroup. Values are ng/g wet tissue wt. *Significantly lower

NE in the group fed the imbalanced diet (P < 0.05). Ratswere given access to the test diets for 3.5 h after onset of thedark period prior to collection of the tissues. Reproducedwith permission from Gietzen et al. (1986b).

which the animals are given a choice between 1MBand a protein-free diet, was returned to normal (i.e.,the animals preferred the protein-free diet, as do un-injected animals) after either puromycin or ac-tinomycin D. Thus, both the increased intake of an1M B a nd the reversal of aversion to the 1M B, when thelim iting am ino acid was injected into the PPC, require de novo protein synthesis (Beverly et al. 1991a).These findings, in association with the am ino acidprofile described above and in Gietzen et al. (1986b),support the suggestion that a reduction in the concentration of the lim iting amino acid, specifically in thePPC, but only with attendant changes in protein synthesis, is involved in the initial recognition of 1MB.The data are consistent w ith a rapidly turning-overprotein, which is )not synthesized after ingestion of1MB, and 2) increased in IMB-fed animals after injection of the lim iting amino acid. Inability to make acrucial protein in the PPC could lead to a neuralsignal that protein synthesis is at risk. Thus, effectson neurotransm ission are clearly indicated.

norepinephrine in the prepyriform cortexThe m onoam inergic neurotransm itter system s playimportant roles in the control of feeding (reviewed in

1 voC

F R E E AMINO ACID S IN P R E PYR IF O RMCOR TE X

15.013.011.09.07.05.03.01.0

0.200.150.100.05O

- T HR B A SAL D IE To o TH R IMB ALA NC ED DIE T

FIGURE 4 Free amino acids in the prepyriform cortexfrom animals killed 2.5 h after introduction of threonine-basal, -corrected or -im balanced diets. V alues are m eans innmol/mg fresh tissue. As in Figure 1, amino acids areabbreviated along the abscissa, but note differing order.Samples were microdissected samples of prepyriformcortex, homogenized in acid medium, centrifuged and analyzed in an automated amino acid analyzer (Beckman, PaloAlto, C A); n = 5-6 animals per group. *P < 0.05 by ANOVA.Reproduced with permission from Gietzen et al. (1986a).

Hoebel 1984). To estimate the role of monoamineneurotransm itters in the neural responses to 1M B, theconcentrations of several monoamines and theirmetabolites were measured by HPLC with electrochemical detection in microdissected brain areasthat were taken 2.5 h after introduction of basal, 1MBor corrected diet. Amino acid concentrations in ali-quots of the same samples were also measured byautom ated am ino acid analyses. The concentrationsof both norepinephrine (NE, Fig. 3) and the lim itingamino acid (Table 2) were decreased in the PPC of ratsfed either of two 1MB (Gietzen et al. 1986b). Adecreased concentration of NE could be due to eitherincreased release or decreased synthesis. A lthough theincreased tyrosine in the PPC of IM B-fed anim als (Fig.4) could reflect a lower rate of tyrosine utilization forN E synthesis, the available evidence favors increased


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616 GIETZENNE release (see below). The lim iting amino acid wasalso decreased in the AC, a nearby cortical area (Table2), but NE levels were unchanged in the AC. As notedin Table 1, the AC is involved in the adaptive phaseof the response but is not critical for the initialfeeding depression. These findings suggest that either1) both the lim iting amino acid and NE must bealtered in concert to serve as the initial signal to thebrain,- or 2) the depression in the lim iting amino acid(or the disproportion of amino acids) causes the activation of the NE system in specific responsive brainareas, such as the PPC. Support for the involvementof NE in the ventral cortex, in protein selection, hasalso been provided by Gibson et al. (1987). In anyevent, it seems that reduction of the lim iting aminoacid in a responsive brain area is a necessary first stepin the recognition of am ino acid deficiency (Beverly etal. 1990a and 1990b, G ietzen et al. 1986b). Examination of the am ino acid profile in the PPC (Fig. 4)shows that any amino acid disproportion must be dueto reduction, rather than increase, in the concentrations of amino acids in that area at 2.5 h after 1MBfeeding. This is in keeping with the work of Harperand Tews (1988) showing that the brain is protectedfrom large influxes of amino acids (compare Fig. 1, forplasma, and Fig. 4, for PPC, after threonine 1MB).In the ventromedial hypothalamus (VM H), an areaearlier thought to contain the "satiety center" (Stellar1954), the concentration of NE was increased in IM B-fed animals, suggesting that release was inhibited inthat area. Inhibition of NE release from cells in theVMH would be consistent with a depression infeeding, because G rossm an (1960) show ed an increasein feeding after extracellular NE injections into theVM H. This is also consistent with an integrative role,such as assim ilation of input from other brain areas inthe co ntrol of feedin g, rather than a signal-rec ognitionrole for the VMH. Also, the level of the lim itingamino acid was not depressed in any of the threehypothalam ic areas exam ined, and N E concentrationswere unchanged in the lateral hypothalamus (LH) andparaventricular nucleus (PVN), which are two otherhypothalam ic areas thought to be associated withfeeding (Gietzen et al. 1989). The lack of depression ofthe lim iting amino acid in the hypothalamus is consistent with the lack of effect of both VMH and LHlesions on 1MB intake (Leung and Rogers 1970,Scharrer et al. 1970) and again supports the proposalthat extrahypothalam ic structures support the initialrecognition of amino acid deficiency. Still, the increased concentration of NE in the VMH suggeststhat hypothalam ic areas may be involved in otheraspects of the response. Evidence for effects in the LHwill be discussed later.In studies such as the amino acid injectionsdescribed above, clonidine was injected bilaterallyinto the PPC (see Table 3 for dosage) prior to feedingtrials with an 1M B. C lonidine is an alpha2 agonist that

Time2Dose33h6h12 h2 4%

ofontrol0.001150569.069.791.8107.2 12.5a6.2a24.3b11.6b68.070.04.4a5.8a5.1a4.5b73.877.277.887.92.84.62.94.1

TABLE 3E ffect o f C lo nidine in jection in to th e p re pyrifo rm cortex (P PC )o n cumu la tive in ta ke o f iso leu cin e im ba la nced diet by rats1

'V alues are means SE for imbalanced diet intake as a percentage of control (baseline basal diet) intake for each anim al; n = 6for the 1.0- and 1.5-pg dose groups, n = 7 for the 0.0- and0.5-/ig dose groups. V alues for a tim e interval bearing differentsuperscript letters are significantly different, P < 0.05 by leastsquares m eans after significant overall A NOVA . D ata are adaptedfrom G ietzen and B everly (1992).Tim e after introduction of the im balanced diet, at the onset ofdarkness.3Doses in g/L = total dose per rat. Injections were 1/2 of eachdose, given in 0.5-^L volum es, bilaterally, sim ultaneously into thep re py ri fo rm cor te x.

decreases NE activity by acting at a presynaptic site,providing feedback inhibition onto presynaptic au-toreceptors of NE neurons (Scatton 1990). If the injection was made within the precise stereotaxic coordinates identified by early lesion studies (Leung andRogers 1971), intake of the 1MB was increased(Gietzen and Beverly 1992). The results (Table 3)provide additional evidence for activation of the NEsystem in the PPC in the anorectic response, becauseinhibition of NE release resulted in increased intakeof 1M B.Clonidine can inhibit the production of cAMP viathe alpha2 receptor (Starke et al. 1989), and cAM P isessential for the activity of catabolite activatorprotein, which is necessary to initiate transcription,at least in prokaryotes (Lew in 1990). Therefore, cAMPconcentrations were measured in the PPC of rats fedeither threonine-im balanced or basal control diets andkilled at differing times after dark onset, using aprotein binding assay (A mersham , A rlington H eights,IL). To control for any generalized changes in cAMPsuch as would be expected from stress or otherstimuli, the data for the PPC were compared withthose for the AC from each animal. The AC was usedas a control area, because it had not shown anychanges in NE concentration after ingestion of 1MB(Gietzen et al. 1986b). The concentration of cAMPwas decreased in PPC, reaching a nadir at 2.5 h afterintroduction of 1MB (Fig. 5). It is tempting tohypothesize that this decrease in cAMP in the PPC,after consumption of 1M B, provides a link betweenthe neurochemical changes and protein synthesis inthe PPC after ingestion of 1MB.


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NEURAL RESPONSES TO AMINO ACID DEFICIENCY 6172 00

3BO.eCL

10 0

1 .5 2HOURS 2.5 3.5

FIG URE 5 Concentrations of cyclic AM P (cAM P) in theprepyriform cortex (PPC) as a percentage of the cAM P concentration in the control tissue: anterior cingulate cortex(AC). Solid line: basal diet; broken line: threonine-im-balanced diet. M arkers: means; error bars: SE; n = 4-5 ratsper group. Brain areas were microdissected from brains ofanimals killed at times indicated on the abscissa (h) afterintroduction of basal or imbalanced diets. T issues weretaken into acidified ethanol, hom ogenized and centrifugedand the supernatant assayed for the concentration of cAM Pusing an assay kit from Amersham (Arlington Heights, IL).'Significantly reduced concentration of cAM P at 2.5 h afterintroduction of the imbalanced diet, P < 0.05.

Our findings are consistent with the view that areduction in the concentration of the lim iting am inoacid in the PPC is necessary but not sufficient forproviding the initial signal to the animal that itshould find an alternative dietary source for its am inoacids. Subsequent neurochem ical events, includingactivation of the NE system , inhibition of adenylcyclase activity and alterations in protein synthesis,also seem to be involved. W ithin the PPC, roles forNE, cAMP and probably other neuroactive compounds seem to be indicated. Still, a full understanding of the neurochemical events in the PPC,which will explain its role as the amino acidchemosensor in the recognition of amino acid deficiency, aw aits further research.

THE SECOND PHASE:LEARNED RESPONSESAfter recognition of the am ino acid-deficient state,the animals develop an aversion to the 1MB, or tocues associated with the diet, and a preference forcues associated with repletion (Booth and Simson1971, Halstead and Gallagher 1962, Leung et al.1968a, Rogers and Harper 1970, Torii et al. 1987,Treneer and Bernstein 1981). It is generally assumedthat taste cues are used in these conditioning situations (Garcia et al. 1955, Mori et al. 1991, Rogers andLeung 1977). However, that taste is not the cue forrecognition of the initial metabolic event signaling

amino acid deficiency is indicated by the followingobservations. Food intake is rapidly depressed inanimals that have been given an intravenous infusionof im balanced am ino acids, which bypasses oral inputand therefore the taste pathways (Peng and Harper1969). A lso, rats choose a corrected diet adulteratedwith quinine over an 1MB without any added tastecues (Leung et al. 1986b). Choice studies have shownthat the animals cannot distinguish the corrected dietfrom the 1MB by taste or smell, although they canbecome confused for a few days if the diets containadded flavorings as cues and the flavor cues aresw itched (Rogers and Leung 1977). Thus, rats seem touse taste as a marker for identification of acceptableor unacceptable diets, but only after they have madethe appropriate metabolic distinctions. Baker andBooth (1989) demonstrated that olfactory conditioning is also involved, and Deutsch et al. (1989)reported that rats have an unlearned specific appetitefor protein that is mediated by olfactory systems(Heinrichs et al. 1990). Whatever the sensory modality used, the ability to make appropriate associations would support selection of a more adequate diet.B ooth and colleagues (Baker et al. 1987, Gibson andBooth 1986) have argued that learning is required todemonstrate protein preference. A preference forflavors associated w ith threonine repletion clearly required a period of learning (Gietzen et al. 1992). Wehave also observed that the preference for thethreonine-associated flavor was not seen in animalseating a threonine-adequate diet, but could be shownlater, in the absence of additional training, if theanim als were again depleted of threonine. That is, thelearned preference for threonine w as state dependent,occurring only in deficient animals (unpublished observation). A lso, birds selected diets adequate inlysine or valine over diets containing only 25% oftheir requirements. These results did not becom e significant until after 2-4 d (Murphy and K ing 1989),again suggesting that learning had taken place.Additional evidence for separation of the initialrecognition phase from a subsequent learned component is shown in the exhaustive series of brainlesion studies by Leung and colleagues, in which thebrain areas that were shown to be associated with theinitial responses were not the sam e as those involvedin adaptation to the 1MB (Leung and Rogers 1987).Accelerated adaptation to an 1M B after lesioning wasthought to result from impaired learning. The brainareas in which lesions improved adaptation but hadno effect on initial recognition of 1MB include theAC, the thalam ic taste nuclei, dorsolateral hippocampus, septum and ventral tegmentum (Table 1).As reviewed in Leung and Rogers (1987), the AC,hippocampus and septum are brain areas that havebeen associated with learning, lesions of the ventraltegm entum would damage NE pathways (Ungerstedt1971), and the implicated thalam ic nuclei are, by


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