amino acid imbalance and diet preference in the hypothalamic–hyperphagic rat

Amino Acid Imbalance and Diet Preference in the

Department of Food and A'utritional Sciences, University of Hawaii, Honolulu, Hawaii 96822

Received May 12,1970

AIPAKAWA, S. M., STANL-IAL, B. R., and BEATON, J. R. 1971. Amino acid imbalance and diet preference in the hypothalamic-hyperphagic rat. Can. J. Physiol. Pharmacol. 49, 752-457.

Diet selection by control and hypothalamic-hyperphagic rats was recorded to examine the hypothesis that the plasma amino acid pattern may act as a satiety signal with respect to the decreased food intake associated with amino acid imbalanced diets. Rats were offered choices between: (a) protein-free and imbalanced diets; (b) imbalanced and corrected diets; ( c ) corrected and basal diets. Although selection by control and operated rats differed with respect to choice a, selection behaviors were comparable for choices b and c. Plasma amino acid patterns were similar in control and operated rats indicating that the same potential satiety signal was present in both groups. Since the "satiety center" was ablated in operated rats, it would seem that if plasma amino acid patterns serve as a satiety signal, this signal must act in some manner other than on the ventromedial area of the hypothalamus.

AWAKAWA, S. M., STANL-IAL, B. R., et BEATON, J. R. 1971. Amino acid imbalance and diet preference in the hypsthalamic-hyperphagic rat. Can. J. Physiol. Pharmacsl. 49, 752-757.

Le type de dibte chsisi par des rats tkmoins et des rats hyperphagiques a 6th not6 en m e &examiner l'hypothbse selsn laquelle la composition plasmatique en acides aminks agirait sur le centre de la satiCtt. Ceci compte tenu de la diminution de la prise alimentaire associCe 2 un rCgime fnon CquilibrC en acides aminks. Les rats ont 2 i leur disposition trois types de dibtes: (a) sans protCines et non Cquilibrbe, (b) non Cqrailibrte mais corrigCe, (c) dikte basale et corrigie. Nous avons not6 une prCfQence de l'un des deux groupes pour la dibte a mais il n'a pas CtC not6 de diffgrences entre les deux groupes quant au choix de la di2te b et c. La composition plasmatique en acides aminCs est similaire chez les deux grsupes indiquant ainsi un m3me potentiel de sig- nalisation vis-his du centre de la satiCtC. Puisque chez les rats hyperphagiques, le centre de la satittk est supprimC, il semble que si la composition plasmatique en acides aminCs constitue un signal de satiCtC, elle agirait ailleurs que sur les zones ventro-mCdiales hypothalamiques.

Food intake is depressed in rats provided with a diet containing an imbalance of amino acids (Harper 1958). Other reported changes include an elevation of blood urea concentra- tion shortly after the ingestion of the imbalanced diet, a fall in the plasma concentra- tion of the limiting amino acid, and an altera- tion in food preference whereby animals select a nongrowth-supporting protein-free diet in preference to a growth-promoting imbalanced diet (Harper el al. 1964). Harper and Rogers

'Journal Series No. 1194 of the Hawaii Agricultural Experiment Statisn.

'Based in part upon a thesis (S.M.A.) submitted to the Graduate Division, University of Hawaii, in partial fulfillment of the requirements for the degree of Master of Science in Nutrition.

3Fresent address: Pacific Research, Queen's Medi- cal Center, Honolulu, Hawaii.

'Present address: College of Human Biology, Uni- versity of Wisconsin-Green Bay, Green Bay, Wis- consin 54301.

(1965) observed an altered free amino acid pattern in plasma due to the amino acid imbalanced diet and suggested that the altered pattern may serve as a satiety signal that r e p - lates food intake. Correction of the imbalanced diet by the addition of the essential amino acid present in the smallest amount in relation to the daily requirement resulted in the choice of the corrected diet over either the imbalanced or the protein-free diets.

Hypothalamic-hyperphagic rats have been reported to retain a relative hyperphagie condi- tion and increased body weight gain when fed an amino acid imbalanced diet (Nasset et ak. 1967; Beaton 1967). However, the presence or absence of diet selection by these animals has not been reported. In this study, an amino acid imbalanced diet was presented to hyperphagic rats along with protein-free or corrected diets. Food choice, duration of hyperphagia, and plasma free amino acid patterns were determined.

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ARAKAWA ET ALr: HYPOTHALAMIC-HYPERPHAGIC RAT

TABLE 1. Composition of the control and experimental diets (g1108 g) - - - - -- - -- --- -- - - - - - -- - - - - - - - - - -- - . - - - .-A -- -

Diets - - -

Constituents Control Protein-free Imbalanced Corrected BasaI - - - - - - - - - - - -. - - - -

Casein 16 - 6 6 -

6 Vitaminized caseina 4 - - - Corn oil 10 5 5 5 5 Saltsb 4 4 4 4 4 Choline 0.4 0.4 0.4 0.4 0 s 4 Inositol 8 .2 0 . 2 0 . 2 0 . 2 0 . 2 DL-Methionine -- - 0.3 0.3 gSC 3 Sucrose 31 - 7 15.8 13 13 15 Vitamin ized sucrose" - 4 4 4 4 Cornstarch 31 - 7 66.6 63.1 63.1 4 3 . 1 Alphacel 2 2 2

- -- 2 2

Imbalanced mixture" 2 - - Corrected mixtured - - - 2 - - -- - -- - - - - - - - .--

~ T h n amount provided in mg: 0.5 thiamine, 4.5 ~aisotinie acid, 1 riboflavin, 1.25 pyridoxine hydrochloride, 2 para- aminobenzoic acid, 2 calcium pantothenate, 0.1 biotin. 0.1 fo11c acid. 0.05 menadione, 10 alphatocopherol and, in L.U., 100 of vitamin A palmitate and 25 of vitamin Da.

bPhillips and Hart (1935). This provided in mg: 4.8 of DL-tryptophai~, 16.8 of L-leucine, 24.0 of DL.-isoleueine, 26.0 of ~ ~ - v a l ~ n e , 7.2 of

r-histidine HCl, 10.8 of DL-ptnenylalanine, and 18:0 ofk-lysine HCI. dBn addialon to the amino acids provided by the nmbalanced mlxture this provided 9.0 mg of^-threonine.

TABLE 2. Amins acid compositisn and amins acid ratios of the experimental diets

Amino acids in the diets Qmg/lW g diet) Amino acid ratios in the diets"

Amino acids Basal Imbalanced Corrected Basal Innbalanced Corrected

Tkreonine Tryptophan Isoleucine Leucine Lysine Methionine Phenylalanine Valine Histidine

'Ratios were computed by taking the amounts of methionine In the diets to be 1.066.

Materials and Methods Male rats of the \Vistas strain were housed in

individual, screen-bottom cages, and during the three test periods had free access to food and water; the animals were maintained in an environmental tem- perature of 24 9 1 '&I, with 12 h light and I2 h darkness daily. Food intake was rneasrared daily and body weights were measured three times each week.

Hypothalamic hyperphagia was produced in 23 male rats of approximately 150 g body weight by placement of bilateral radiofrequency lesions in the ventrornedial area of the hypothalamus as previously described by Birige and Beaton (1969). The hypothalamic-hyperphagic (operated) rats and intact rats were divided into three groups sf 16, 14, and 16 rats, respectively. Groups H and IBI each had eight operated and eight intact rats, and group I1 had seven intact and seven operated rats. All rats were provided with a control 20% casein diet (Table 1) and food intakes were recorded for 4 days. Average daily food intakes of operated rats of all groups were greater than were the food intakes of corresponding intact

rats ( B < B).gB(BB, Table 4) thus demonstrating the presence of hyperphagia in the operated rats.

Test Per isd .~ The test period of 21 days sf food choice was

initiated on the 5th day after operation. Operated and intact rats were offered a choice between two diets placed in separate non-spiller jars in the cage. The positions of the jars in the cage were changed daily to minimize position preference. Rats s f group P were offered a choice between the protein-free and the imbalanced diets, sf group IF between the imbalanced and the corrected diets, and of group TIP between the corrected and the basal diets. The compositions of the experimental and basal diets are given in Table 1. The amino acid compositions and ratios sf the diets are given in Table 2. The additions of eight essential amino acids were in the same ratios as those of Leasng et al. ( 1968) but at an amount of 2.7 times less. This mild manipulation entailed an addition of not more than 1 1 0 mg of free amino acids to 100 g sf the diet and caused only minor changes in the amino acid ratios of the diets.

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CANADIAN JOURNAL OH PHYSIOLOGY AND PHARMACOLOGY. VOL. 49, 1971

TABLE 3. Dietary preference sf operated and intact rats for 21 days (mean + S.E.M.) - -- - - -- - -- -- - -- - - -- - - . - - -. -- -- -- - - - - - - -- - --

Daily food intake (g/rat)

Group No. of rats Diets Days Operated Intact

IIT

8 Imbalanced

Pa 7 Imbalanced

7 Corrected

Pa 8 Corrected

8 Basal

°Probability when comparing dietary preference by rats from each treatment for 1-21 days.

The period of I4 days of recovery began on day 22. All rats were provided with the 20% casein control diet. This period allowed for rehabilitation of the rats that had eaten the physiolsgically dificult diets and also ascertained the presence s r absence sf & yperphagia.

The final period began on day 36 and lasted for 21 days. The rats received one of three experimental diets. Group 1 received the imbalanced diet; group II received the corrected diet; group 11% received the basal diet. On the day that this period eanded, all rats were fasted overnight and in the morning their re- spective diets were oifered far 2 h. They were then anesthetized with ether and blood was removed by cardkc pumctbare. The plasma was deproteinized according to Bovmgaardt and McDonald (1969) using 9% sulfosalicylic acid. The supernatant was removed by centrifuging, lyophiiked, and diluted to f ml with 10% isogropanol in water. The amino acids were separated by one-dimensional, ascending pager chromatography using Whatman 3MWq paper for 16 h in buhnol - acetic acid -- water (4: 1 : 1 v/v). The papers were air-dried and developed according to Levy and Ghrang ( 1953) with ninkydrin - eollidime - acetic acid. The amino acid spots were marked by pencil, cut, and eluted by the method s f Kay et a!. (1956) using 71% ethanol. The bands from the plasma were identified by comparison with individual standard amino acids put through the same ekrep- matographic procedure and judged by colors and Ri values. The ratios of essential to nonessential amino acids were ca8culated.

Results and Discussion

All choices made by the rats of each treatment for particular experimental diets drnring the test period were statistically significant (Table 3 ) . It is evident that both operated and intact rats preferred the corrected over the imbalanced diet in group IT, and selected the basal over the corrected diet in goup '1111%. In group 1, he operated rats preferred the protein-free diet and the intact rats preferred the imbalanced diet. The preference of operated rats for the protein-free diet over the imbalanced diet containing 2 % of the imbalanced amino acid mixture is similar to the choice sf the protein-free diet by intact rats reported by Eeung et al. (2968) when the amount of Lm- balarnced amino acid mixture was raised to 5.496, but dissimilar when their imbalanced mixture was only 2%. This observation sug- gests that operated rats may be more sensitive than are intact rats 40 small amounts of imbalanced amino acid mixture in the diet. At the 2% level our intact rats responded as did those of Leung ct al. (1968) by consuming 68% of the total faud from the diet containing the imbalanced mixture.

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ARAKAWA ET AL. : HYPOTHALAMIG-HYPERPHAGIC RAT

TABLE 4. Average food intake and weight change during three periods (mean + S.E.M.) - -- - -- - -- -- - - -- - -- -.

Food Body intake weight change

Periods Diets Groups (glratlda~) (drat)

Preexperiment Control 4 days Control

Control Control Control Control

Food choice Protein-free 21 days and imbalanced

Imbalanced and corrected

Corrected and basal

Control Control Control Control Control Control

Final Imbalanced 21 days

Corrected

Recovery 14 days

Basal

I Operated I Intact

I1 Operated I1 Tntact

I11 Operated I11 Intact

I Operated I Intact


111 Operated I11 Intact

I Operated I Intact

11 Operated I1 Intact


I Operated I Intact



Since the operated rats of groups I1 and PI1 selected in the same way as did the intact rats, the monitoring of food choice in this situation appears to be independent of the ventromedial area of the hypothalams, i.e. independent of the "satiety center".

The selection of the corrected over the imbalanced diet by intact rats of group 11 confirmed the observation of Sanahuja and Harper (1 963); a similar choice by operated rats was observed although these rats appeared to eat more of the imbalanced diet than did the intact rats. The selection of basal over corrected diet by intact rats of group 111 is not in agreement with the conclusion of Leung et al. ( 1968) that intact rats show no diet preference with similar diet choices. It should be noted, however, that ex- cept for the 5th to the 7th day, food intake histograms reported by Leung et al. (1968) for the basal diet were on a higher level than for the corrected diet. An extension of the experiment for an additional 7 days might have brought out the tendency observed in their histograms more definitely. Examination of the daily food intake during our choice period at two intervals of 1-4 and 5-21 days show no change in the food consumption during the two

intervals. However, it should be noted from data on total food intakes (Table 4) , that operated rats did not exhibit marked hyperphagia during the diet selection test period.

During the recovery period using the 20% casein diet, food intakes were compared with intakes during the preexperimental period of 4 days (Table 4). Operated rats of groups II and III consumed significantly less food during this period ( P < 0.001) while intact rats consumed significantly more food ( P < 0.081). Thus operated rats were no longer in a hyperphagic state. It should be noted, however, that the body weight gain of operated animals of group I during the recovery period markedly exceeded that of control animals. As dem- onstrated by several workers, including May and Beaton ( 19661, an excessive weight gain in the absence of significant hyperphagia is observed in the static phase of hypothalamic hyperphagia. Attainment of a static phase after only about 25 days following operation is sur- prising and remains unexplained since this does not usually occur until 60-90 days after operation (May and Beaton 1966).

The observation of increased body weight gain in operated animals in the absence of

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CANADIAN JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY. VOL. 49, 1971

TABLE 5. Ratio of essential amino acids to nonessential amino acids (mean 9 S.E.M.)

Group Treatment Diet Nonessential/essentiaB

I Operated Imbalanced 0.41 k0.04 Intact Imbalanced 0.32k0.05

I1 Operated Corrected 0.50k 0.09 Intact Corrected 0.38k0.07

I11 Operated Basal 0.43k 8.03 Intact Basal 0.42kO. 18

hyperphagia during the final experimental period (39 days following operation) would indicate again the static phase of hypothalamic hyperphagia in the operated animals. This would appear to rule out the possibility of recovery of the "satiety center9' or failure to ablate the ventromedial area of the hypothalamus at the time of operation.

No significant differences were observed in the plasma free amino acid patterns between operated and intact rats, nor between dietary groups. Consequently, only ratios of the essential to the nonessential amino acids were cal- culated as shown in Table 5 and no significant differences were noted. Since detemina- tion sf amino acids by paper chromatography is less sensitive than by column chromatography, the possibility exists that small but significant changes were not detected.

It has been shown previously (Beaton 1967) that operated rats had difficulty in maintaining their hyperphagia when provided with a 6% protein (fibrin) diet although an amino acid imbalance superimposed on this diet did not cause a reduction in food intake. However, operated rats fed the imbalanced diet gained significantly more body weight than did intact controls fed the same diet. Observations of the present experiment are in agreement, d.e. increased body weight gain in the absence of hyperphagia with an amino acid imbalanced diet in the final experimental period.

Although hypothalamic-hyperphagiG rats are excessive eaters, their acceptance of specific diets is unpredictable. For example, the anorexigenic effect of adding rapeseed oil to the diet of intact rats is not observed in hypothalamic-hyperphagic rats (Beare and Beatton 1967)- In contrast, the anorexigenic effect of a high-protein diet is much greater in operated than in intact rats (Krauss and Mayer 1965); this observation might indicate a regulatory mechanism other than through the ventro-

medial area of the hypothalamus, and further, might explain the apparent preference of operated rats for the protein-frec diet over the imbalanced diet.

Considering the results of these experiments, it would appear that rats with bilateral lesions of the ventromedial area of the hypothalamus behave in a manner similar to intact, control animals with res~ect to diet selection when offered a choice between imbalanced and corrected diets or corrected and basal diets. Some- what different behaviors were noted when the choice was between protein-free and imbalanced diets. It has been suggested that alterations in plasma amino acid pattern may serve as a satiety signal and this may explain diet selection wiLh respect to amino acid imbalanced diets (Harper and Rogers 1965). It is of interest that plasma amino acid patterns of operated and intact rats fed the same diet were not markedly different; thus, the potential "amino acid satiety s i p d " was comparable in both groups. Since the "satiety center'has ablated in operated rats, it would seem that if plasma amino acid patterns serve as a satiety signal, this signal must act in some manner other than on the ventromdial area of the hypothalamus. Recently, similar conclusions were reached by h u n g and Rogers ( 1970) in their study in which the effects of amino acid imbalance and deficiency on food intake of rats with hypothalamic lesions were measured.

BEARE, %. E., and BEATON, I. Ha. 1967. Effect of rapeseed oil on food intake in the rat, Can. J. Physiol. Pharmacol. 45, 1893-1894.

BEATON, J . R. 1967. Effect of increased metaboBism and of hyperphagia on dietary amino acid imbalance in the rat. Can. J. Physiol. Pharmacol. 45, 101 1-1019.

B o o ~ c ~ a a ~ ~ , J., and MCDQNAED, B. E. 1969. Com- parison of fasting plasma amino acid patterns in the pig, rat, and chicken. Can. J . Physioh. Phar- macol. 47, 392-395.

DIRIGE, 0. V., and BEATON, I. R. 1969. Factors

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ARAKAWA ET AL.: HYPBTHALAMIGHYPERPHAGIC RAT 757

affecting vitamin Be requirement in the rat as determined by erythrocyte transaminase activity. J . Nutr. 97, 109-1 16.

HARPER, A. E. 1958. Balance and imbalance of amino acids. Ann. N.Y. Acad. Sci. 69, 1025-1041.

HARPER, A. E., LEUNG, P., YOSHIDA, A., and ROGERS, C). 1964. Some new thoughts on amino acid imbalance. Fed. Proc. 23, 1087-1092.

HARPER, A. E., and ROGERS, Q. 1965. Amino acid imbalance. Proc. Niltr. Soc. 24, 173-190.

KAY, R. E., HARRIS, D. C., and ENTENMAN, C. 1956. Quantification of the ninhydrin color reaction as applied to paper chromatography. Arch. Bio- chem. 63, 14-25.

KRAUSS, K. A{., and MAYER, J. 1965. Influence of protein and amino acids on food intake in the rat. Am. J. Pkysiol. 209, 479-483.

EEUNG, P., ROGERS, Q., and HARPER, A. E. 1968. Effect of anmino acid imbalance on dietary choice in the rat. J. Nutr. 95, 483-492.

EEUNG, P., and ROGERS, Q. 1970. Effect of amino

acid imbalance and deficiency on food intake of rats with hypothalamic lesions. Nutr. Rep. Int. I, 1-10.

LEVY, A. E., and CHUNG, B. 1953. Two-dimensional chromatography of amino acids on buffered papers. Anal. Chem. 25, 396-399.

MAY, K. K., and BEATON, J. R. 1966. Metabolic effects of hyperphagia in the hypothalamic- hyperphagic rat. Can. J. Physiol. Pharmacol. 44, 641-650.

NASSET, E. S., R ~ L E Y , P. T., and SGHENK, E. A. 1967. Hypothalamic lesions related to ingestion sf an imbalanced amino acid diet. Am. J. PhysioB. 213, 645-650.

PHII~LIPS, P. H., and HART, E. B. 1935. The effect of organic dietary constituents upon chronic fluorine toxicosis in the rat. J. BioI. Chem. 109, 657-663.

SANAHUJA, J. C., and HARPER, A. E. 1963. Amino acid balance and innbalance. XIH. Effect of amino acid imbalance on self-selection of diet by the rat. J. Nutr. 81, 363-371.

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amino acid imbalance and diet preference in the hypothalamic–hyperphagic rat

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