Effect of dietary limiting cortex on food intake J. L. BEVERLY,

amino acid in prepyriform

D. W. GIETZEN,

AND

Q. R. ROGERS

Department of Physiological Sciences and Food Intake Laboratory, School of Veterinary Medicine, University of California, Davis, California 95616

Gietzen et al. (3, 4) demonstrated that, although the whole brain content of the DLAA was decreased when an imbalanced diet is fed, the decrease in the concentration of the DLAA occurred only in some brain areas. the reduced intake of an amino acid-imbalanceddiet (imbal- The concentration of the DLAA in the prepyriform coranced diet) appearsto involve a decreasein the content of the tex (PPC) was reduced to nearly 50% of basal levels dietary limiting amino acid (DLAA) in the prepyriform cortex (PPC), Intake of imbalanceddiet was increasedfrom 45-50 to within 2.5-3.5 h of feeding an imbalanced diet (4). Thus, pre70-75% of baseline after bilateral injection of the DLAA di- in the PPC, the decrease in DLAA concentration rectly into the PPC, following an inverted U-shaped dose- ceded the initial decrease in intake of imbalanced diet. to be an responsecurve. Injections had no effect on intake of basaldiets. The PPC has previously been demonstrated Injection of the DLAA into the PPC reversedthe aversion to essential brain area for the reduction in intake of imbalimbalanceddiet in choice studies, as rats selected an imbal- anced diets. Rats with bilateral lesions of the PPC conanced diet over protein-free diet after such injections. Intake sume imbalanced diet at 80-90% of baseline instead of of imbalanceddiet did not increaseafter a nonlimiting amino the -50% of baseline ingested by sham-lesioned rats (10, acid was injected into the PPC or after injections of the DLAA 16, 17). into other brain areas.Resultswere similar when either threThe present experiments were designed to investigate onine or isoleucinewasthe DLAA. These results confirm that the relationship between the DLAA concentration in the the decreasein the concentration of the DLAA in the PPC is PPC and the feeding response to amino acid-imbalanced involved in the reduction in intake of imbalanceddiets. diets. The objective was to measure food intake in rats fed amino acid imbalanced diets when the decrease in food-intake regulation; amino acid imbalance; intracerebral DLAA content in the PPC, measured after ingesting an injection; rats; threonine; isoleucine imbalanced diet, was prevented by microinjection of the DLAA directly into the PPC. BEVERLY,J.L.,D.

W. GIETZEN,AND Q.R. RoGERs.@"~~~

of dietary limiting amino acid in prepyriform cortex on food intake. Am. J. Physiol. 259 (Regulatory Integrative Comp. Physiol. 28): R709-R715, 1990.-The mechanismsunderlying

ADAPTATION TO DIETS varying in protein content and quality requires a mechanism(s) to monitor and adjust

METHODS

intake to maintain adequate, but not excess, quantities of essential amino acids in the blood. Ingestion of an amino acid-imbalanced diet (imbalanced diet), in which a single essential amino acid is limiting, results in a decrease in the circulating concentration of the dietary limiting amino acid (DLAA) (5, 9, 19-21). Animals consistently select against or reduce their intake of an imbalanced diet, unless DLAA concentrations in the blood are maintained (3, 5, 6, 9, 13, 21). Concomitant with the decrease in plasma concentration of the DLAA is a decrease in whole brain content of the DLAA (19, 20). That the decrease in DLAA content of the brain is responsible for the decrease in imbalanced diet intake was demonstrated by Leung et al. (9). Infusion of small quantities of the DLAA into the carotid artery prevented the decrease in intake when an imbalanced diet was fed. When the same quantity of DLAA was infused into the jugular vein, there was no alleviation of the depression in imbalanced diet intake (9,23). Thus, by maintaining supply of the DLAA to the brain the feeding depression was alleviated, presumably by increasing availability of the DLAA to specific brain areas.

Male Sprague-Dawley rats (250-300 g) were housed singly in hanging wire-mesh cages in a temperature- (22 -+ 2°C) and light- (12:12 h light-dark cycle) controlled room; lights were off from 1300-0100 h. Experimental diets (Table 1) and clean water were available ad libitum. Two days after arrival, rats were offered either threonine basal or isoleucine basal that, except where noted, was fed throughout each experiment. After a 14-day adaptation period, rats were anesthetized with sodium pentobarbital (65 mg/kg ip; Steris Laboratories, Phoenix, AZ). Atropine sulfate (ElkinsSinn, Cherry Hill, NJ) was administered at 0.4 mg/kg SC to reduce respiratory distress during anesthesia. Bilateral 24-gauge stainless steel guide cannulas were stereotaxitally positioned 3 mm dorsal to the intended area of the anterior pyriform cortex [ PPC; anteroposterior (AP) = +11.4, lateral (L) = 4.0, dorsal (D) = 6.51, using the coordinates of Paxinos and Watson (18). Cannulas were fixed in place with dental cement and stainless steel screws anchored into the skull. Guide cannulas were kept patent by 29-gauge stainless steel stylets, which were removed and cleaned at regular intervals. After a 7-day

0363-6119/90

$1.50

Copyright 0 1990 the American Physiological Society

R709

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R710 TABLE

AMINO

ACID

INJECTION

INTO

1. Diet formulas Diet,

Ingredient

DAA” BEAA” IEAA’ Thr Ile Corn starch Sucrose Corn oil Vitaminsd Minerals’ Choline Cl

Thr basal

8.1 3.9 0.2 0.4 50.9 25.4 5.0 1.0 5.0 0.1 100.0

Thr imbalance

8.1 3.9 9.4 0.2

0.9 44.3 22.1 5.0 1.0 5.0 0.1 100.0

Ile basal

8.1 3.1 0.3 0.3 51.4 25.7 5.0 1.0 5.0 0.1 100.0

5% Ile imbalance

Protein free

8.1 3.1 9.4 0.7 0.3

44.9 22.4 5.0 1.0 5.0 0.1 100.0

59.3 29.6 5.0 1.0 5.0 0.1 100.0

I1Dispensible Amino Acid (DAA) Mix: 12.4% ArgeHCl; 12.4% Asn; 4.3% Ser; 12.4% Pro; 12.4% Gly; 37.1% Glu; 4.3% Ala; 4.7% NaAcetate. ” Basal Essential Amino Acid (BEAA) Mix: Thr diets: 7.6% His. HCl. H,O; 15.3% Leu; 20.1% Lys HCl; 7.5% Met; 5% Cys; 11.3% Phe; 6.3% Tyr; 2.5% Trp; 12.6% Val; 11.8% NaAcetate. Ile diets: 7% His HCl= H,O; 15.9% Leu; 19.3% Lys HCl; 7.9% Met; 6.3% Cys; 7.9% Phe; 7.9% Tyr; 2.5% Trp; 14.3% Val; 11.1% NaAcetate. ’ Imbalance Essential Amino Acid (IEAA) Mix: 9% His HCl. H,O; 15.8% Leu; 18% Lys. HCl; 6.8% Met; 4.4% Cys; 11.3% Phe; 6.7% Tyr; 2.7% Trp; 11.3% Val; 14.2% ” Total Vitamin Supplement, United States Biochemical. NaAcetate. ” Rogers-Harper Salt Mixture, United States Biochemical. l

recovery period, during which body weights and food intake returned to precannulation levels, 24-h intakes of basal diet were measured, and the mean of 3 consecutive days was used to establish baseline food intake. Intake, measured to the nearest 0.1 g, was determined by differences in food cup weights and corrected for spillage. On the first day of treatment, food cups were removed -30 min before onset of the dark cycle, and rats were lightly anesthetized with Metofane (Pittman Moore, Washington Crossing, NJ). Stylets were removed, and injection cannulas (30-gauge stainless steel) were slowly lowered through the guide cannulas into the PPC. Each injection cannula was attached via Tygon tubing (0.01 in. ID, 0.03 in. OD) to a lo-p1 syringe fitted into a microinjection pump (CMA/lOO, Bioanalytical Systems, West Lafayette, IN). Buffered artificial cerebrospinal fluid (aCSF, in mM: 158 Na+, 2.55 K’, 1.26 Ca2+, 0.93 Mg 2+, 135 Cl-, 20.8 HCO;, 1.26 HP0y2, pH = 7.4), sterilized by filtration, and the L form of the amino acids, dissolved in aCSF, was prepared the morning of each experiment. Bilateral injections of either 0.5 ~1 vehicle (aCSF), the DLAA, or a nonlimiting amino acid were delivered at a constant rate of 0.1 pl/min. Injection cannulas were left in place for 60 s after the injection was completed before being slowly removed. Stylets were then replaced and animals returned to their home cages. Rats routinely appeared to be recovered from metofane exposure by 15 min. In a pilot study, neither 2-h nor 24h food intakes after metofane exposure differed from those of unanesthetized, noninjected rats. At the onset of the dark cycle, a preweighed food cup containing basal diet, Thr-imbalanced diet or Ile-imbalanced diet was placed in each cage. Rats were offered these diets between 2 and 5 days before being refed basal diet. Each rat received a single iniection. administered on the first dav

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of each experiment unless noted, and was naive to the imbalanced diet. Injection sites were verified by injecting 0.5 ~1 india ink, using the injection procedure described above, immediately before decapitation. Whole brains were quickly removed and a series of coronal slices made to expose the injected ink. Injection sites were identified and compared with the stereotaxic atlas of Paxinos and Watson (18). If cannulas were asymmetrical or misplaced, data for that rat were not included in analysis. Experiment 1. Dose response. The effects of various doses of Thr or Ile on intake of the Thr-imbalanced diet or the Ile-imbalanced diet, respectively, were evaluated in a series of trials. Rats fed the Thr-imbalanced diet received bilateral injections of 0, 1, 1.5, 2, 3, or 4 nmol Thr/side. Rats fed the Ile-imbalanced diet were injected with 0, 0.5, 0.75, 1.0, 1.5, or 2 nmol Ile/side. There were seven rats at each dose level, and all rats were fed the imbalanced diets for 2 consecutive days. Experiment 2. Effect on basal diets. After establishing an effective dose on imbalanced diets, the effect of injections into the PPC on intake of basal as well as imbalanced diets was evaluated. In one trial, rats were randomly assigned to receive one of three treatments; vehicle (buffered aCSF), 2.0 nmol Thr, or 4.0 nmol Thr. Rats were then fed either Thr-basal or Thr-imbalanced diet to complete a 3 X 2 experimental design (n = 6 rats/ treatment-diet combination). In another trial, rats received either buffered aCSF or 1 nmol Ile and were fed either Ile-basal or Ile-imbalanced diet in a 2 x 2 design (n = 7 rats/treatment-diet combination). In both trials, rats were fed the diets for 5 days after injection. Experiment 3. Repeated injections. This trial evaluated the effect of injecting 2 nmol Thr for 2 consecutive days on the intake of Thr-imbalanced diet. Thr-imbalanced diet was fed to 21 rats for 3 days. Two nanomoles of Thr were administered to 14 rats and aCSF to the remaining seven rats on day 1. On day 2, one-half of the rats in the Thr group received a second injection of 2 nmol Thr. The second injection to the remaining 14 rats was aCSF. No injections were given on the third day. Experiment 4. Dietary choice. A more sensitive test of an altered feeding response by rats offered imbalanced diets is the dietary choice model (1, 5, 12, 15) in which animals are given a choice between an imbalanced diet and a protein-free diet. This paradigm was carried out to determine whether injections of the DLAA into the PPC reversed the aversion to the imbalanced diet. Thirty-two rats were allowed to choose between a protein-free diet and Thr-basal diet for 14 days. After a baseline choice was established, rats were divided into four groups. One group received aCSF and chose between the basal diet and the protein-free diet. The second group received aCSF and chose between the imbalanced diet and the protein-free diet. The other two groups received injections of Thr and were offered a choice between the protein-free diet and the basal (group 3) or the imbalanced diet (group 4). There were eight rats in each treatment-diet combination. Diet choices were maintained for 2 days before all rats were fed the basal diet ad libitum.

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AMINO

ACID

INJECTION

INTO

Experiment 5. Amino acid specificity. The specificity of the feeding response to the DLAA was determined by injecting a nonlimiting essential amino acid into the PPC. Two trials, employing a 2 X 2 experimental design, were conducted. In the first trial, rats fed either Thrbasal or Thr-imbalanced diets were injected bilaterally with either aCSF or 1 nmol Ile (n = 8/treatment group). In the second trial, aCSF or 2 nmol Thr were administered to rats fed Ile-basal or Ile-imbalanced diets (n = 7/ treatment group). Experiment 6. Specificity of PPC. To determine whether the feeding responses were unique to the PPC, 32 rats received injections of either aCSF or 2 nmol Thr into a more posterior area of the pyriform cortex (AP = 9.4, L = 5.0, D = 7.5); 2 mm posterior to the effective area in the PPC. An additional group of 16 rats received injections into the medial amygdala (AP = 6.5, L = 3.4, D = 9.0). All rats were then fed either Thr-basal or Thrimbalanced diet for 3 days. Statistical analysis. Results from experiment 1 were analyzed by one-way analysis of variance (ANOVA). When the ANOVA showed there was a significant difference, the mean intake at each dose was compared with the intake by controls (0 nmol Thr or Ile) by Dunnett’s two-tailed t test. Results from all other experiments were analyzed by two-way ANOVA with treatment means compared by Tukey’s multiple t test when the ANOVA showed there was a significant difference. In experiment 4, the intakes of each diet within a choice were also evaluated via Student’s t test. All analyses were conducted on version 6.03 of PC-SAS (SAS, Cary, NC). Values reported are means t SE. A4ateriah. Vitamin and mineral mixtures and choline chloride were purchased from United States Biochemical (Cleveland, OH). Purified amino acids were purchased from Ajinomoto USA (Los Angeles, CA). Corn starch (National Starch and Chemical, Bridgewater, NJ), sucrose (Spreckles, San Francisco, CA), and corn oil (Best Foods, Englewood Cliffs, NJ) were obtained locally. All other chemicals were purchased from Sigma Chemical (St. Louis, MO). Rats were purchased from Bantin and Kingman (Lafayette, CA). RESULTS

Experiment 1. Dose response. The response to Thr injection into the PPC on intake of Thr-imbalanced diet, as a percent of baseline food intake of the basal diet (baseline), is presented in Fig. 1. An inverted U-shaped dose-response curve was obtained with intakes of imbalanced diet increasing up to 75% of baseline before decreasing when >2 nmol Thr were administered. Intakes between 50 and 75% of baseline were measured at all doses of Thr tested. Control rats ate 50-55% of baseline, as did sham-injected animals (results not shown). Intake of rats receiving 1.5, 2, or 4 nmol Thr (68, 75, and 64% of baseline, respectively) were significantly greater [ F( 5, rats. The 36) = 10.50; P < O.OOl] than vehicle-injected dose providing the greatest response in intake of Thrimbalanced diet was -2 nmol of Thr. The dose response of Ile injections on Ile-imbalanced diet is presented in Fig. 2. As observed in the Thr dose-response trial, an

PREPYRIFORM

R711

CORTEX

40-r

I

0

. 1

,

. 2

,

. 3

,

4 4

L-Thr (nmoles) injected 1. Intake of Thr-imbalanced diet after bilateral microinjection of various doses of L-Thr into prepyriform cortex in experiment 1. * Intake at this dose was significantly greater (P < 0.05) than intake at 0 nmol Thr. Values are means & SE for 7 rats/dose. FIG.

40-r

0.0



I

0.5



I

1.0

L-Ile (nmoles) 2. Intake of of various doses of * Intake at this dose at 0 nmol Ile. Values FIG.

-

I

1.5

-

1

2.0

injected

Ile-imbalanced diet after bilateral microinjection L-Ile into prepyriform cortex in experiment 1. was significantly greater (P c 0.05) than intake are means * SE for 7 rats/dose.

inverted U-shaped curve was evident. The significant increase in intake of Ile-imbalanced diet between 0.75 and 1.5 nmol [F(5, 36) = 5.20; P < O.OOl]was absent when X.5 nmol Ile was injected. As observed in the Thr dose response, intakes increased to -70% of baseline. In both trials, intakes on the second day of feeding imbalanced diet did not differ among doses; all rats ate -60% of baseline basal intake. Experiment 2. Effect on basal diet. Intakes of basal diet were not affected by aCSF, Thr or Ile injections into the PPC (Figs. 3 and 4). Rats receiving aCSF reduced intakes by 46% when fed Thr-imbalanced diet (Fig. 3) and 51% when fed Ile-imbalanced diet (Fig. 4). Rats receiving injections of 2 nmol Thr into the PPC had significantly greater intakes of Thr-imbalanced diet [70% of basal; F(5, 30) = 28.8, P < O.OOl]; consistent with the results from experiment 1. Also as seen in the first experiment, when 4 nmol of Thr were injected into the PPC, the intake of Thr-imbalanced diet was less than intake after 2 nmol Thr. Rats fed Ile-imbalanced diet and administered 1 nmol Ile ate significantly greater amounts [77% of basal; F(3,24) = 31.06, P < O.OOl] than aCSF controls. In both experiments, there were highly significant diet effects [F(l, 25) = 13.0, P < 0.001 and F(1, 24) = 77.4,

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R712

AMINO

25 -I

ACID INJECTION

0

a

INTO

PREPYRIFORM

CORTEX

0 nmole Thr 2 nmole Thr

40-J 0 Basal

Imbalanced

FIG. 3. Intake of Thr-basal and Thr-imbalanced diets after bilateral microinjection of 0, 2, or 4 nmol L-Thr into prepyriform cortex in experiment 2. Bars represent means + SE for 6 rats/treatment group. Bars having different superscripts differ significantly (P < 0.05).

1 2 days fed thr-imbalanced

4 3 diet

5. Intake of Thr-imbalanced diet after bilateral microinjections of L-Thr (closed squares) of aCSF (closed circles) before 1st and 2nd day of feeding Thr-imbalanced diet in experiment 3. Open squares represent rats receiving Thr 1st day and aCSF 2nd day. * Significant difference (P < 0.05) among treatments. Values are means for 7 rats/ treatment group. FIG.

n Thr Basal 0 Protein Free

q Thr-Imbalanced c 201 ”

Basal

Imbalanced

0 Protein Free Dl

FIG. 4. Intake of Ile-basal and Ile-imbalanced diets after bilateral microinjection of 0 or 1 nmol L-Ile into prepyriform cortex in erperimerit 2. aCSF, artificial cerebrospinal fluid. Bars represent means f SE for 7 rats/treatment group. Bars having different superscripts are significantly different (P < 0.05).

P < 0.001, respectively] and diet-by-treatment interactions [F(l, 24) = 3.32, P = 0.05 and F(1, 24) = 13.5, P < 0.001, respectively]. In both trials, the intake of imbalanced diet by rats receiving the DLAA were reduced on days 2-5; being similar to aCSF-treated rats at -55% of baseline. Experiment 3. Repeated injections. As observed in the first two experiments, injection of 2 nmol Thr into the PPC increased intake of Thr-imbalanced diet (Fig. 5). Injecting 2 nmol Thr on the second day was also effective in increasing intake of Thr-imbalanced diet. Rats receiving Thr ate 48% more imbalanced diet than aCSFtreated rats fed imbalanced diet on the first day [F(l, 18) = 67.1, P < O.OOl] of the trial. Intake of imbalanced diet on the second day by Thr-treated rats was 40% more than rats injected with aCSF [F(2, 18) = 20.9, P < O.OOl]. The effect of Thr was not carried over after the first day as evidenced by the decrease in intake to that of the controls when aCSF was administered to rats that had received Thr only on the first day. Rats in this group ate 66% of baseline on day 2 compared with 84% by rats receiving Thr on both day 1 and day 2. A similar decrease in imbalanced diet on day 2 was observed in experiments

aCSF Thr 6. Intakes of Thr-basal protein-free diets in experiment 6 when aCSF (A) or 2 nmol L-Thr (I?) was administered, and of Thr-imbalanced and protein-free diets when aCSF (C) or 2 nmol L-Thr (I)) was administered bilaterally into prepyriform cortex. Intake of Thr-imbalanced diet was greater and protein-free diet less (P < 0.001) when LThr was applied into prepyriform cortex. FIG.

I and 2. There may have been some carry over from the second to the third day in the group of rats receiving Thr on days 1 and 2 since intake of Thr-imbalanced diet of that group on day 3 was 91% of baseline, a 30% increase in intake, relative to baseline, over the other two groups [F(2, 15) = 44.3, P < O.OOl]. Experiment 4. Dietary choice. During the baseline period, Thr-basal diet accounted for 90%, and proteinfree diet accounted for 10% of total intake. The same proportion of intake was measured in rats choosing between Thr-basal diet and protein-free diet (Fig. 6, A and B), whether aCSF or Thr was injected. However, rats choosing between Thr-imbalanced diet and protein-free diets and injected with Thr (Fig. 6, C and D) selected significantly more Thr-imbalanced diet than did aCSF-

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AMINO

ACID

INJECTION

INTO

2. Intake of Thr- or Ile-based diets in response to injections of Ile, Thr, or aCSF into prepyriform cortex

TABLE

Basal,

Injectate-Diet

Thr-based

g

Ile-based

10.9t1.0t 11.1kO.W

diets (n = 7/treatment) 20.1t0.8* 19.5k1.4”

aCSF Thr (2 nmol)

g

diets (n = g/treatment) 20.5t0.7* 19.6t0.7*

aCSF Ile (1 nmol)

Imbalance,

10.3*0.5t 10.8kO.7”r

Values are means -t- SE. aCSF, artificial cerebrospinal Thr- or Ile-based diet having different superscripts cantly different (P < 0.05). within

fluid. Values are signifi-

3. Intake of Thr-based diets in response to injections of 2-nmol Thr or aCSF into posterior pyriform cortex or medial amygdala TABLE

Injectate-Diet

Basal,

Posterior aCSF Thr

cortex

Imbalance,

amygdala

g

(n = 7ltreatment)

18.8t0.7” 19.4t0.4* Medial

aCSF Thr

pyriform

g

9.9*0.5-F 10.4t0.4 t

(n = 4/treatment)

18.6&0.3* 17.5t0.9*

Values are means k SE. Values superscripts are significantly different

10.6+1.5”r 9.6tO.l t

within an area (P < 0.05).

having

different

treated rats (75% of total vs. 23% of total, tlz = 5.39, P < 0.001). Intake of Thr-imbalanced diet by Thr-treated rats amounted to 86% of the amount of Thr-basal diet selected during the baseline period. On the second day of selections (Thr was injected only on day 1), both groups of rats selected the protein-free (-75% of total intake) over the imbalanced diet. The total intake of Thr-imbalanced diet and protein-free diet was 3-4 g less than the total intake of Thr-basal diet and protein-free diet [ F(3, 24) = 8.6, P < O.OOl]. Experiment 5. Amino acid specificity. Injection of 1 nmol Ile into the PPC did not increase the intake of Thrimbalanced diet nor reduce the intake of Thr-basal diet (Table 2). Injecting Ile at the same quantity as that of Thr, which stimulated intake of Thr-imbalanced diet (ie., 2 nmol), did not increase intake of Thr-imbalanced diet (Beverly, unpublished observations). Intakes of Ilebasal or Ile-imbalanced diets were not affected by injection of Thr (2 nmol) into the PPC. In both trials, intake of imbalanced diets was reduced to GO-55% of basal diet intake [F(3, 28) = 47.65, P < 0.001 and F(3, 24) = 94.55, P < 0.001, respectively]. Experiment 6: Specificity of PPC. Injection of 2 nmol Thr either into a more posterior area of the pyriform cortex or into the medial amygdala (Table 3) did not alter intake of either Thr-basal or Thr-imbalanced diets. As seen in experiments l-5, intakes of Thr-imbalanced diets were reduced 45-50% [ F(5, 40) = 33.5, P < O.OOl]. DISCUSSION

The results of these experiments suggest that the concentration of the DLAA in the PPC is involved in

PREPYRIFORM

CORTEX

R713

the feeding depression when an amino acid-imbalanced diet is fed. The increased intake of the amino acidimbalanced diet when the DLAA was injected into the PPC was consistent across all experiments. Intakes of imbalanced diet, as a percent of baseline, were 20-25% higher than vehicle-injected controls fed imbalanced diet, reversing approximately one-half of the decrease in intake. The shapes of the food intake response curves (Figs. 1 and 2) were surprising in that the maximal response might be expected to plateau when higher quantities of the DLAA were administered. The fact that a normal depression in intake by rats fed the imbalanced diets occurred after injections of the higher doses indicates that the increase in intake of imbalanced diet was not the result of some form of lesion caused by injections into the PPC. When verifying cannulas positions, no apparent lesions were noted within the PPC. Nor was there likely to have been a “metabolic” lesion, in which the PPC experienced a temporary impairment in function due to disruption of cellular metabolism, since injecting ~2 nmol Thr did not increase intake of Thrimbalanced diet nor decrease intake of Thr-basal diet. The lack of an increase in intake of imbalanced diet at the higher doses of injected DLAA is probably not the result of a toxic quantity of the limiting amino acid in the PPC. It is also unlikely that injecting the higher doses of the DLAA created an imbalance among the essential amino acids within the PPC, since the higher quantity of the DLAA did not depress intake of the basal diet. Furthermore, concentrations of Thr in the PPC often exceed the concentration that would be predicted after injection of 4 nmol Thr into the PPC. For example, in rats fed a mild Ile-imbalanced diet, Thr concentration in the PPC increased more than 2.5 times the Thr concentration in the PPC of basal-fed animals (4). Feeding a Thr-corrected diet also increased the concentration of Thr in the PPC to values 1.5 times greater than the calculated concentration of Thr after injection of 2 nmol Thr. Thus Thr concentrations in the PPC often exceed the concentration that would result after diffusion from the injection site with the higher doses of Thr utilized in experiment 1. It is also unlikely that the higher doses created an imbalance among the amino acids within the PPC, since the same concentrations of Thr injected into the PPC in experiments 2 and 5 had no deleterious effects on intake of either Thr-basal or Ile-basal diets. An osmotic effect causing a depression of food intake is unlikely, since the differences in osmolarity within 1 nmol of the optimal dose of Thr were 2 mosmol/l, only 0.6% of the total osmolarity. An increase in the concentration of Thr, per se, in the injectate is also unlikely to have resulted in the feeding response seen in the doseresponse trials. Threonine, at 2 nmol/injection, has been delivered in volumes of 0.25, 0.5, or 1.0 ~1 (8, 4, and 2 mM solutions, respectively), and the increase in Thrimbalanced diet has been similar on all occasions (Beverly, unpublished observations). Furthermore, in experiment 1, injection of 1 nmol Thr, as a 2 mM solution, did not increase intake of Thr-imbalanced diet. Thus the concentration of the DLAA in the iniectate appears to J AI

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R714

AMINO

ACID

INJECTION

INTO

be less critical than the absolute amount of the DLAA administered. Except on the third day of experiment 3, there was no carry over from the day of injection into the second day, since intake of imbalanced diet by rats injected with the DLAA only on the first day was similar to control rats on the second day of each trial. This also argues against possible lesions caused by the injection into the PPC. Thus it appears that the injected amino acids are indeed being metabolized. The absence of an effect on food intake the second day after injection appears to be the result of normal metabolic turnover resulting in a decrease in Thr concentration in the PPC by the second day. In experiment 3, Thr was injected for 2 consecutive days and there was no depression in intake on the third day. Leung et al. (13) suggested that “self correction” of the availability of the limiting amino acid could occur as a result of increased ingestion. When the imbalanced diet is ingested at levels comparable with basal diet, normal growth is maintained (5, 14). Intake of Thrimbalanced diets by rats in experiment 3 was 7585% of baseline, an amount that may have supplied enough Thr to help block the decline in Thr content in the PPC after 2 days of elevated intake but not after a single day of increased imbalanced intake. Allowing rats to choose between diets is a sensitive method to test the relationship between the fall in limiting amino acid content in the PPC and intake of an amino acid-imbalanced diet. Rats placed in a cold environment, and having increased energy expenditure, increase their intake of amino acid-imbalanced diets (1, 7). The mechanism(s) regulating intake of protein and amino acids is subordinate to mechanisms regulating energy intake (21). Nevertheless, cold-exposed rats select a protein-free diet over an imbalanced diet (1, 7), indicating that the mechanism(s) for amino acid-imbalance avoidance involve more than simply decreasing energy intake. As seen in experiment 4 and reported by Leung et al. (15), unchallenged rats readily avoid a protein-free diet when the basal diet is the alternate choice. However, an imbalanced diet is more aversive, since rats chose the protein-free diet over the imbalanced diet when aCSF was administered into the PPC. Rats receiving Thr injections did not avoid the imbalanced diet as did aCSFinjected rats, instead these rats displayed a preference for the protein-containing diet, despite the amino acid imbalance. These results, and those of experiment 2, indicate that the increased intake of imbalanced diet after injection of the limiting amino acid into the PPC was not the result of some perceived need for energy. These results also suggest that when the concentration of DLAA in the PPC is increased, the disproportionate amino acid patterns in the diet may not be recognized. Changes in the concentration of each essential amino acid in the PPC appear to influence the intake of amino acid-imbalanced diets independently. There was no effect on intake of Thr-imbalanced diet when the optimal dose of Ile was injected into the PPC, nor did the optimal dose of Thr increase the intake of Ile-imbalanced diet. The feeding effects were specific for the DLAA, suggesting a mechanism responsive to the changes in each of

PREPYRIFORM

CORTEX

the essential amino acids. Since plasma concentrations of most of the essential amino acids may also influence brain content of catecholamines or indolamines (2), there may be more than one mechanism responding to most conditions involving an imbalance among the essential amino acids. Several amino acids may utili .ze one mechanism whereas other amino acids may use another. A certain amount of crossover may occur, but, as seen in experiment 5, this does not occur between Thr and Ile. The results of these experiments, and those of Gietzen et al. (4), do not rule out the existence of a common mechanism recognizing an imbalance in several of the essential amino acids. An increase in “spontaneous discharge frequency” was reported to occur in neurons in the lateral hypothalamus after electrophoretic application of Thr and most of the other essential amino acids but not after Ile (24). This supports the concept that specific neurons in the brain respond to selected essential amino acids. In the report of Wayner et al. (24), no cells responded to Ile and no cells in the cerebral cortex responded to application of any essential amino acid. The apparent specificity exhibited in experiment 5 and the time course of the-feeding effect argue against a direct neurotransmitter role for the injected amino acids. If Thr was acting as a neurotransmitter in the PPC, an effect on feeding should have been seen when Thr was administered and the Ile-imbalanced diet was fed. It is possible that the responsiveness of a neuron to the presence of a single amino acid differs between different diets or that different populations of neurons become sensitive depending on which essential amino acid is limiting. To our knowledge, there is no evidence to support either of these possibilities. The increased intake of an imbalanced diet is not a general phenomenon in response to amino acid injections into the central nervous system (8). Results from lesion experiments have identified only two brain areas in which lesions increase intake of amino acid-imbalanced diets: the PPC and medial amygdala (10, 11, 16). However, the increase in intake of imbalanced diet after medial amygdala lesions may be related to deficits in learned aversions (16). Thus the medial amygdala may not be specific for the depression in intake of amino acid-imbalanced diets (16), which has been shown to involve learned aversions (2 1, 22). In contrast, rats with lesions of the PPC do increase intake of imbala riced diet while still capable of responding to a conditioned taste aversion paradigm (16). This supports a role for the PPC as a site for recognition of an amino acid-imbalance. There was no effect on the intake of Thr-basal or Thrimbalanced diet when Thr was injected 2 mm posterior to the PPC or into the medial amygdala. It is possible the optimal doses in these two brain areas are different from these in the PPC. Use of an ineffective dose or site specificity may explain why infusions of the limiting amino acid into the lateral ventricles failed to block the feeding depression to amino acid-imbalanced diets (8). The results of experiments l-6 lead us to postulate a causal relationship between the decrease in the content of a DLAA in the PPC and the decreased intake of an amino acid-imbalanced diet. Increased intake of amino

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AMINO

ACID

INJECTION

INTO

acid-imbalanced diet occurred only after injecting the limiting amino acid, in a narrow dose range, into the PPC. The restoration of intake was not complete, some feeding depression still occurred. This may suggest that other brain areas, or mechanism(s) are also involved in monitoring plasma levels of the essential amino acids. Alternatively, a more sustained infusion or affecting a wider area in the PPC may be necessary to block the decreased intake of amino acid .-imbalanced diets completely. Taken together, the results of this study suggest that the depression in intake when an amino acid-imbalanced diet is fed is, in part, the result of a mechanism involving the decrease in the concentration of the limiting amino acid in the PPC. The authors acknowledge the assistance of P. M. B. Leung, Vicki Hammer, Valentine Sworts, Cheri Aune, and Nicki Beierle. This research was supported by National Institutes of Health Grants AM-07355 and DK-13252 and by United States Department of Agriculture Grant CRCR-1-2418. Address for reprint requests: J. L. Beverly, Dept. of Physiological Sciences, School of Veterinary Medicine, University of California, Davis, CA 95616. Received

26 December

1989; accepted

in final

form

11 May

PREPYRIFORM

8.

9. 10.

11.

12.

13.

14.

15.

16.

1990.

REFERENCES 1. ANDERSON, H. L., N. J. BENEVENGA, AND A. E. HARPER. Effect of cold exposure on the response of rats to a dietary amino acid imbalance. J. Nutr. 99: 184-190, 1969. 2. FERNSTROM, J. D. Role of precursor availability in control of monoamine neurotransmitters in brain. Physiol. Reu. 63: 484-546, 1983. 3. GIETZEN, D. W., P. M. B. LEUNG, T. W. CASTONGUAY, W. J. HARTMAN, AND Q. R. ROGERS. Time course of food intake and plasma and brain amino acid concentrations in rats fed amino acid-imbalanced or -deficient diets. In: Interaction of the Chemical Senses zoith Nutrition, edited by M. R. Kare and J. G. Brand. New York: Academic, 1986, p. 415-456. 4. GIETZEN, D. W., P. M. B. LEUNG, AND Q. R. ROGERS. Norepinephrine and amino acids in prepyriform cortex of rats fed imbalanced amino acid diets. Physiol. Behav. 36: 1071-1080, 1986. 5. HARPER, A. E., N. J. BENEVENGA, AND R. M. WOHLHUETER. Effects of ingestion of disproportionate amounts of amino acids. Physiol. Rev. 50: 428-558, 1970. 6. HARPER, A. E., AND J. C. PETERS. Protein intake, brain amino acids and serotonin concentrations and protein self-selection. J. Nutr. 119: 677-689,1989. 7. HARPER, A. E., AND Q. R. ROGERS. Effect of amino acid imbalance

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Effect of dietary limiting amino acid in prepyriform cortex on food intake.

The mechanisms underlying the reduced intake of an amino acid-imbalanced diet (imbalanced diet) appears to involve a decrease in the content of the di...
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