Effect of dietary GABA and protein on growth, food intake and GABA metabolism in the rat

Brtrirl R~scwch Bukrin,

Vol. 5,

Suppl.2,

pp. 245-251.

Printed

in the U.S.A

Effect of Dietary GABA and Protein on Growth, Food Intake and GABA Metabolism in the Rat’ JEAN K. TEWS, ELIZABETH

Dqxzrtment

c$Biochemistry,

A. RIEGEL

AND ALFRED

College of Agricultural and Life Sciences, ~~~di.~~~z,WI 53 7%

E. HARPER

University of Wisconsirr-Madison,

TEWS, J. K., E. A. RIEGEL AND A. E. HARPER. &fi~ct t~d~e~u~~ GABA ~t~tdpr~~~~~tt on ~r~ti~.~h,.~)~~~i inttrkc ctntf GABA in the vnf. BRAIN RES. BULL. 5: Suppl. 2,245-251, 1980.-GABA, when present as4..5% of a6% casein diet, decreased food intake and almost completely suppressed growth in young rats. Growth and food intake increased as dietary protein content increased; growth of rats receiving a 6m casein diet+GABA was 86% of that in the absence of GABA. Plasma, liver and kidney concentrations of GABA became lower as dietary protein content increased; brain GABA levels were unaltered by dietary GABA. GABA transaminase (GABA-T; E.C. 2.6.1.19) activity per mg tissue protein increased 2-3 fold in liver as dietary protein content increased but activity in brain and kidney did not change. Total hepatic enzyme activity in rats fed 8% casein was about 7 fold greater than in rats fed a protein-free diet. Rats fed 20% or 6@%casein converted in 6 hrs about twice as much ‘F-GABA to YYO, as did rats fed 6% casein. Urinary excretion of jJC-GABA was not altered by changes in dietary protein content. Relationships between the suppression of food intake and growth by dietary GABA and its neurotransmitter functions are not clear.

mrtahofism

Brain Liver

Dietary GABA Rat

Dietary protein

Food intake

GABA transaminase

Growth

Kidney

_” .._. ______ PREVIOUS studies f33] have shown that supplementation of a threonine-limiting, low protein diet with other 3- or 4-carbon small neutral amino acids that inhibit threonine transport by brain slices in vitro (e.g., serine, alanine or ~-amino-n-buty~~ acid 1321) will depress food intake and growth, and brain threonine content 133,341. These effects can be prevented by adding threonine to the diet. Although GABA has no effect on threonine transport by brain 1321, a severe growth depression also occurEd when GABA was added to the diet; however, there were no changes in tissue threonine content and additional dietary threonine did not alleviate the condition, implying that the mode of action of dietary GABA on growth differed from that of the other small neutral amino acids which were tested. Such effects of GABA might be related to its probable role as a neurotransmitter. Feeding a high protein diet often lessens the toxicity associated with administration of large amounts of certain individual amino acids [13. 15, 24, 291; this effect is usually accompanied by an increase, especially in liver, in the activity of enzymes involved in amino acid catabolism 16, 8, 16, 301. Experiments of this nature have now shown that the deleterious effects of dietary GABA can be lessened by increasing the protein content of the diet. The decrease in GABA toxicity is associated with a decrease in plasma and tissue GABA content and a corresponding increase in the

activity of hepatic 4-aminobuty~te-2-oxoglutarate transferase (GABA-T, E.C. 2.6.1.19).

amino-

METHOD

Young, male rats (Holtzman) were housed individually in metal cages with wire mesh bottoms in a room lighted from 0700 to 1900 hr and maintained at about 23°C. They were fed ud lib diets containing various levels of casein plus supplements of methionine and threonine as indicated in the appropriate figures. The diets also contained, in percent by weight, vitamin mix [27], 0.5; mineral mix [27],.5; corn oil, 5; choline chloride, 0.2; and equat amounts of glucose monohyd~te and cornstarch to make 100%. Where indicated, GABA was usually added at 4.5% of the diet (at the expense of the carbohydrates). At various times during the feeding periods tissue samples were taken and frozen for later analysis of GABA content by ion exchange chromatography (Technicon Autoanalyzer or Beckman Model 119 CL) or of GABA-T activity. Enzyme activity of tissue homogenates was measured by the method of Wu et al. [42] with minor modifications. This method involves incubating ‘*C-cY-ketoglutarate and GABA with tissue homogenate, followed by determination of radioactivity in the product, “C-glutamate, after its separation from unreacted ‘*C-cY-ketoglutarate by ion exchange

‘Supported in part by the College of Agricultural and Life Sciences, University of Wisconsin, Madison, and by Grant AM 10747 from the National Institutes of Health, Bethesda, MD.

Copyright

c 1980 ANKHO

Inte~ational

Ins.-0361-9230/80/080245-07$01.20/O

‘I‘EWS, RIEGEL AND HARPER

r .

12%

LL 20%

150

4

40%

q

60%

---+4.5%

GABA

DAYS

FIG. 1. Cumulative change in weights of rats fed diets containing different levels of protein with or without added GABA. Methionine (0.2%) and threonine (0.4%) were added to all diets except that containing 60% casein (when fed at low levels, casein is limiting in these two amino acids). On Day 7, shown by the left vertical dashed line, haIf of the rats of each of the 5 dietary groups were switched to the appropriate diet also containing 4.S% GABA; on Day 15, shown by the right vertical dashed line, GABA was removed from the diets. Results are expressed as the mean for 12-16 rats for Days i-7,6-10 rats for Day 8-15, and 4-6 rats for Days 16-22. On Day 15 some of the rats were taken for determination of plasma, liver and kidney GABA concentrations (Fig. 3) and others were used for the ‘?C-GABA oxidation study shown in Fig. 6.

chromatography.

Tissues were homogenized in a medium containing 2-~in~thylisothiouronium bromide (0.1 mM), EDTA. (0.2 mM) and pyridoxal phosphate (0.02 mM). The

reaction was started by adding 0.1 ml of tissue homogenate (containing approximately 0.7 mg protein) to the incubation medium so that, in a total volume of 1 ml, the final concentrations of reagents were: Tris, 100 mM, pH 8; pyridoxal phosphate, 0.1 mM; 2-aminoethylisothiouronium bromide, 0.5 mM; EDTA, 1 mM; GABA, 50 mM (or water for blanks); ~-ketoglutarate, 2 mM including 0.2 &i of U-Y-cuketoglutarate (New England Nuclear, specific activity of 287 mCi per mMole). After a 15 min incubation at 37°C in a shaking water bath the reaction was terminated by adding aminooxyacetic acid (0.1 ml of a 10 mM solution); 0.2 ml of the mixture was placed on glutamate-washed Biorad l-X8 or 1-X 10 resin (chloride form) contained in the wefls of a multiple sampling manifold (emigre). The resin bed was washed twice with 2 ml each of SO mM glutamate (pH 2.8), and 3 ml of the resulting combined filtrate containing 14C-glutamate were counted using Aquasol as the scintillation medium. Enzyme activity was expressed as the amount of glutamate recovered in 15 min either per mg tissue, per mg protein or per organ. In a few cases enzyme activity was also determined using %I-GABA as the substrate followed by recovery of “C-

succinate from Biorad 50-X8 resin (ah reagents were at the concentrations listed above); in these experiments Tris buffer gave unsatisfactory results, in agreement with White and Sato [40]. When glycylglycine buffer was substituted as in the similar method of Hall and Kravitz [ 1 I], results were identical to those obtained with the assay of Wu et al. [42]. Alternatively, addition of NAD (1 mM) to the Triscontaining system also restored activity for liver and brain; however, only partial restoration was obtained for kidney. Protein con~entmtions in the tissue homogenates were determined by the method of Lowry cr al. 1221 with bovine serum albumin as the standard. Production of “‘CO, and urinary excretion of radioactivity were determined after administration of 14C-GABA to rats previously fed f%, 20% or 60!%casein diets for 2 weeks. Rats were placed in glass metabolism cages immediately after the int~pe~tone~ injection of 2 mMofes GABA ~ont~ning f &i l-‘*C-GABA (New Engiand Nuclear; specific activity of 49.4 mCi/mMole) per 100 g rat. Respired *4C0, was collected at hourly intervals in fresh portions of a CO:! trapping solution composed of methylcellosolve:ethanolamine (2: 1, v/v); urine samples were taken every 3 hr and were diluted to 25 ml. Radioactivity was determined in portions of these samples and recovery of ad~nistered ~dioactivity was calculated .

DIETARY

GABA ON FOOD INTAKE

247

AND GROWTH

I

t

I

0 6% cosetn l 12% A 20% A 40% q 60% ----+45% GABA

6 12

% Casein

Fed

FIG. 3. Plasma, liver and kidney concentrations of GABA in rats fed for 8 days diets containing different levels of protein plus 4.5% GABA. Samples were taken at 08WO900 hr (Fig. 1, Day 15) and protein-free filtrates were prepared using sulfosalicylic acid. Values represent 1 pooled sample containing plasma from 2-3 rats, or the mean t SE of 2 or 3 liver or kidney samples analyzed individually.

i

I-

1

16

1

I

I8

FIG. 2. Cumulative food intakes of rats fed diets containing different levels of protein with or without added GABA. Further details are given in Fig. 1.

RESULTS

In the first experiment we examined in further detail our original observation [35] that dietary GABA depressed growth of young rats (Fig. 1). During the first week of the experiment several levels of dietary protein were fed without added GABA; growth rate was much slower in the group fed 6% casein than in those fed the higher levels of protein. During the second week, when GABA was present at 4.5% of the diet, little further growth occurred in the group fed 6% casein (the average total weight gain was 3 g, or 10% of that seen in the group fed 6% casein alone). However, gradually increasing growth rates were observed in the presence of GABA as the protein content of the diet was increased. The growth-depressing effects of added GABA in rats fed 60% casein were slight; the average total weight they gained during the second week was 51 g or 86% of that seen in the corresponding control group. Upon removal of GABA from the diets on Day 15 all rats immediately resumed growing at rates similar to those seen for the corresponding groups fed the GABA-free diet. These results show that dietary GABA had no apparent long-lasting deleterious effects, even in the animals in which growth was severely retarded.

Our results suggest that the decreased growth rate seen when GABA was added to the diet reflected decreased food consumption (Fig. 2). This effect, which was especially apparent in rats fed the 6% casein-GABA diet, was immediately corrected by removing GABA. As in the growth study, the effect of GABA on the group fed the diet containing the greatest amount of protein was not severe. In another study (not shown), we examined the growth responses of rats fed different levels of GABA. After 10 days the average total weight gain of rats fed a diet containing 1.5% GABA was only depressed to 88% of the control value; growth of rats receiving diets containing 3% or 4.5% GABA was depressed to 1% or 4%, respectively, of the control value. Food intakes decreased correspondingly, with consumption by rats fed 3% or 4.5% GABA being about 50% of that for the control group. Normally, GABA content of plasma and peripheral tissues is so low that this amino acid is not detectable by conventional ion-exchange chromatography. However, feeding GABA raised tissue levels to easily measureable concentrations which became gradually lower as dietary protein levels increased (Fig. 3). For example, when rats were fed the 60% casein-GABA diet, GABA concentrations in plasma, liver and kidney were 14%, 41% and 4%, respectively, of the concentrations found when rats consumed the 6% caseinGABA diet. Dietary GABA (6% casein diet) did not appear to alter GABA content of the limited number of frozen brains (without cerebellum, medulla and pons) which were analyzed, in agreement with earlier observations [2]. Since tissue GABA levels fell as the amount of protein in

TEWS. RIEGEI.

2

4

6

8

2

IO 12 14

4

6

8

AND HARPER

IO 12 14

DAYS 6% coswn

- 60%

2

cosetrl

6 DAYS

FIG. 4. GABA-T activity in liver, kidney and brain of rats fed 2 levels of protein. Rats weighing 60-65 g were fed a diet containing 6% casein plus methionine (0.2%) and threonine (0.4%) for 3 days before half the rats were switched on day zero to a diet containing 60% casein. Ten days thereafter (shown by the vertical dashed fine), all remaining rats were refed the 6% casein diet for the following 4 days. Tissue samples were taken at intervals over the Z-week period and GABA-T activitv determined as described in METHOD. Each point represents the mean + SE

for 3 rats.

-

the diet was increased, it seemed likely that GABA metabolism might also be stimulated under these conditions. GABA-T, the enzyme catalyzing the first step in the conversion of GABA to succinic acid, is known to occur in peripheral tissues such as liver, kidney and platelets [ 12,20,39,40, 421. We therefore examined the changes with time after first feeding the 6% or 60?& casein diets in the activity of the enzyme in liver, kidney and brain (Fig. 4). Hepatic activity began to increase within the first day after the high protein diet was fed and after 4 days was approximately double that seen in the rats receiving the low protein diet. The increase in activity appeared to be greater when results were expressed on the basis of the tissue itself rather than on tissue protein; this is the result of the increased protein content of livers of rats fed increasing amounts of protein. When GABA-T activity was calculated for the entire liver, a continuing increase in liver size and total enzyme activity occurred during the 10 day intake of the high protein diet. Enzyme activity tended to decline when the animals previously receiving the 6m casein diet were subsequently fed the low protein diet for 4 days. However, this effect was less apparent when results were calculated on the basis of liver protein concentration. In another experiment we found that, after rats had been

fed for 3 days diets containing increasing amounts of protein, enzyme activity (per mg liver protein) increased gradually over the range from 15% to 80% casein (Fig. 5); activity in rats consuming a diet containing 80% casein was about twice that in rats eating zero or 6% casein. Total liver activity in rats fed 80% casein was calculated to be at least 7 fold greater than that in livers of rats receiving no protein. The activity of GABA-T per mg kidney or brain tissue or per rng N was not significantly affected by the protein content of the diet (Fig. 4). In some instances, administration of the substrate amino acid can increase the activity of a catabolic enzyme [Sl. When rats were fed the 6% casein diet with added GABA, hepatic GABA-T activity was 72 -c 5 nmoles glutamate per mg protein per 15 min as compared with a value of 51 + 4 for the control group (n=7, p
DIETARY

GABA ON FOOD INTAKE

249

AND GROWTH

URINE ‘VO,

6-

20

% Cosein

60

Fed

FIG. 6. Recovery of radioactivity in CO, and urine after administration of W-GABA to rats fed dserent amounts of dietary protein for

0

iLL 0

*I&

6

Cosein

Fed

the preceding 14 days. Threonine (0.4%) and methionine (0.2%) were added to all diets but that containing 60% casein. Values are,given as the mean 2 SE for 5 rats fed 6% casein; 2 rats, 20% and 4 rats, 60%. Other details are given in METHOD.

80

(7doys)

FIG. 5. Hepatic GABA-T activity in rats fed different amounts of dietary protein for 7 days. Threonine (0.4%) and methionine (0.2%) were added to all diets except those containing 0, 60 or SC% casein. Each point represents the mean t SE for 4 rats. Other details are given in Fig. 4.

‘VJO, from rats fed 6% casein while about 40% was recovered from rats fed 20% or 6@?&casein. Urinary excretion of label was not altered by prior high protein consumption (although not rigorously identified as GABA, the radioactive material in the urine behaved as did authentic GABA on Biorad AG-I or AC-50 ion exchange resins). Six hours after rats previously fed the 6%, 20% or 60% casein diet were injected with the load of “C-GABA, an average of 7C%, 87% or 8%, respectively, of the total administered radioactivity had been recovered in the expired CO, and in the urine. Little or no radioactivity remained in the rats after 24 hr.

DISCUSSION Ingestion by rats of diets containing large amounts of various individual amino acids has long been known to have adverse effects on food intake and growth [14,28]. In general the mechanisms responsible for these effects are not well understood. It is not clear whether our results showing that dietary GABA depressed food intake and growth are related to the neurotransmitter function of this amino acid or have some other explanation. An earlier study showed that when low dietary GABA (0.5% in drinking water and in chow diets) was fed to mice for 75 days after birth, the weights of these animals increased significantly to about 110% of that for the control group [37]. We found a similar, but statistically insignificant, trend in rats fed 0.75% GABA for 10 days. Any possibly beneficial effects of dietary GABA no longer occur once its concentration is increased to about 1.5% of the diet. Several reports have indicated that the deleterious effects of diets containing large amounts of individual amino acids are lessened by increasing the protein content of the diet [13, 15, 24, 291. This effect of increased protein intake is associ-

TEWS. RIEGEL ated, in part, with an accompanying increase to varying degrees in the activities of many enzymes involved in amino acid metabolism f6, 8, 16, 301. The fact that increasing protein intake lessened the depression by GABA of food consumption and growth and lowered plasma GABA concentration is consistent with the observation of the concomitant increase in hepatic GABA-T activity. Enzyme activity, when related to liver protein content, was increased only about 2 fold in rats fed high levels of protein. However, the total capacity of such animals to metabolize peripherally administered GABA was more strikingly increased since both Iiver and kidney became enlarged relative to body size in the rats fed the high protein diets. We are not aware of other reports showing GABA-T activity to be stimulated by dietary means; both hepatic and renal activity were increased in rats treated with ethanol [31]. Regardiess of the level of dietary protein, GABA administered either in the diet or by injection was rapidly cleared from the animal. Other workers have also noted that peripherally administered GABA is cleared from the blood [ 19, 2 1, 38,411 and have found much of the amino acid to appear in the urine [38]. When the administered dose of ‘“C-GABA was smaller than in our experiments, larger amounts (about 70%) of the dose were recovered in expired r4C0, in 4 hr [4 11. In rats fed rtd lib we found plasma GABA to be highest in the morning, presumably because the greatest food intake occurs in the dark. Therefore, variability will be likely in the GABA content of tissues such as liver, kidney and plasma unless samples are taken under standardized conditions. Van Gelder and Elliott [38] found that, after administration of a single dose of GABA, levels of the amino acid in the tissues never exceeded that in the plasma. However, in our experiments in which tissue samples were taken after the rats had been consuming the GABA-containing diets for 1 week, GABA concent~tions in total tissue water of liver and kidney (water content=72% and 78%, respectively) were much higher than in plasma. Furthermore, as dietary protein content was increased, the ratio of GABA in tissue water to that in plasma also increased; these ratios were 5 and 8 for liver and kidney, respectively, from rats fed the 6% caseinGABA diet but increased to 15 and 33 in the group receiving the 60% casein-GABA diet. Increased dietary protein content has also been shown to increase hepatic uptake of the amino acid transport model, ff-aminoisobutyric acid 1361. However, disposition of GABA by the intact rat is different from that of a-aminoisobutyric acid, an amino acid which is not metabolized in mammals but which is instead slowly lost from the body; its urinary excretion is increased by in-

AND HARPER

creased dietary protein content [25] in contrast to the failure of increasing dietary protein content to alter urinary excretion of GABA. Recent reports suggest that GABA may have some role in regulation of food intake. GABA content is relatively high in the hypothalamus [I, 5, 181, an area of the brain which is known to modulate food intake. Circadian rhythms, which appear specific to this region of the brain, occur in GABA concentration with the highest levels seen during the early light period [5]. It was suggested that the increase in GABA may contribute to the decreased feeding which normally occurs during the light period. Although peripheral administration of GABA is thought not to change its concentration in whole brain 131, hypothalamic GABA content has been reported to increase 121,431; this effect was accompanied by a decrease in the content of norepinephrine and an increase in that of serotonin 1431, neurotransmitters which have also been implicated in the regulation of food intake [4, 10, 231. Consistent with this depression by GABA of hypothalamic norepinephrine concentration is the observation that superfusion with GABA of the hypothalamus previously loaded with OH-norepineph~ne causes release of this catecholamine into the perfusing medium [26]. A similar decrease in norepinephrine and an increase in serotonin occurred in the hypothalamus when brain GABA tevels were elevated after administration of aminooxyacetic acid, an inhibitor of GABA-T and other aminotransferases [43]. Cooper c’t (II. [7] found that ethanolamine-O-sulfate, another inhibitor of GABA-T, increased brain GABA content as expected, as well as decreasing food intake and body weight. These workers also reported that aminooxyacetic acid or muscimol, a GABA agonist, similarly reduced food intake and body weight. However, food intake was stimulated when GABA [ 171, muscimol or norepinephrine ]9] was injected directly into the ventromedial a GABA antagonist, hypothalamus, while bicuculline, tended to reduce food intake [17]. Lateral hypothalamic injections of bicuculline increased food consumption 1171. Decreased GABA concentration has been found in the lateral hypothalamus in hypoglycemic rats, while an increase occurred in hyperglycemia; GABA content of the ventromedial hypothalamus increased in hypoglycemia [ISI. These results are not inconsistent with involvement of GABA in regutation of food intake. These results from several laboratories imply that GABA may have an important function in the control of food intake: however, further studies are essential for clarification of this point.

REFERENCES 1. Balcom, G. J., R. H. Lenox and J. L. Meyerhoff. Regional y-aminobutyric acid levels in rat brain determined after microwave fixation. J. Neurochem. 24: 60%613, 1975. 2. Baxter, C. F. The nature of y-aminobutyric acid. In: Handbook of Neurochemistry III, edited by A. Lajtha. New York: Plenum, 1970, pp. 289-353. 3. Biswas, B. and A. Carlsson. The effect of int~pe~tone~ly administered GABA on brain monoamine metabolism. NaunynSchmiedebergs Arch. Pharmac. 299: 47-51, 1977. 4. Blundell, J. E. and C. J. Latham. Pharmacological manipulation

of feeding behavior: Possible infkrences of serotonin and dopamine on food intake. In: Central Mechanisms ofAnorectic Drugs, edited by S. Garattini and R. Samanin. New York: Raven Press, 1978, pp. 83-109.

5. Cattabeni, F., A. Maggi, M. Monduzzi, L. DeAngeIis and G. Racagni. GABA: Circadian fluctuations in rat hypothalamus. .I. Neurochem. 31: 565-567, 1978. 6. Chu, S-H. W. and D. M. Hegsted.

Adaptive response of lysine and threonine degrading enzymes in adult rats. J. Nutr. 106: 1089-1096, 1976, 7. Cooper, B., J. Howard, H. White, F. Soroko, K. Ingold, J. McDermed and R. Maxwell. Anorexic effect of ethanotamineO-sulfate (EOS): A possible relationship of GABA to control of body weight. Pharmacologist 19: 224, 1977. 8. Freedland, R. A. and B. Szepesi. Control of enzyme activity: Nutritional factors. In: Enzyme Synthesis urzd Degrudat~on in Mummaliun Systems, edited by M. Rechcigl, Jr. Baltimore: University Park Press, 1971, pp. 103-140.

DIETARY

GABA

ON FOOD

INTAKE

9. Grandison, L. and G. Guidotti. Stimulation of food intake by muscimol and beta endorphin. Ncuropharmcrcolo~~ 16: 533536, 1977. 10. Grossman, S. P. Eating or drinking elicited by direct adrenergic or cholinergic stimulation of hypothalamus. .Sc,ic,nc,cJ 132: 301302, 1960. Il. Hall, Z. W. and E. A. Kravitz. The metabolism of y-aminobutyric acid (GABA) in the lobster nervous system. I. GABA-glutamate transaminase.J. Nenrochem. 14: 45-54, 1967. 12. Hargreaves, B. M. C. and D. F. Evered. Metabolism ofo-amino acids by rat tissues it1 vitro. Xcnohioticcl 3: 219223, 1973. 13. Harper. A. E., R. V. Becker and W. P. Stucki. Some effects of excessive intakes of indispensable amino acids. Proc. Sot,. c.xp. Bid. Mrtl. 121: 695-699, 1966. 14. Harper, A. E.. N. J. Benevenga and R. M. Wohlhueter. Effects of ingestion of disproportionate amounts of amino acids. Phy.sio/. Ra,. 50: 428-557, 1970. 15. Ip, C. C. Y. and A. E. Harper. Effects of dietary protein content and glucagon administration on tyrosine metabolism and tyrosine toxicity in the rat. J. Nlctr. 103: 1.594-1607, 1973. 16. Kang-Lee. Y-A. and A. E. Harper. Threonine metabolism iu I,~LYJ:Effect of threonine intake and prior induction of threonine dehydratase in rats. .I. Nutr. 108: 163-175, 1978. 17. Kelly, J.. G. F. Alheid, A. Newburg and S. P. Grossman. GABA stimulation and blockade in the hvoothalamus and midbrain: Effects on feeding and locomotor activity. Phrrrmclc,. Biochem. Brhtrt,. 7: 537-541, 1977. 18. Kimura, H. and K. Kuriyama. Distribution of gammaaminobutyric acid (GABA) in the rat hypothalamus: Functional correlates of GABA with activities of appetite controlling mechanisms. .I. Nrrrroc,hem. 24: 903-907, 1975. 19. Kuriyama, K. and P. Y. Sze. Blood-brain barrier to HI-yaminobutyric acid in normal and amino oxyacetic acid-treated animals. Nertrophcrmlcr~oIr,,~~ 10: 103-108, 1971. 20. Lancaster, G., F. Mohyuddin, C. R. Striver and D. T. Whelan. A y-aminobutyrate pathway in mammalian kidney cortex. Biochim.

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21. Levi, G., P. Amaldi and G. Morisi. Gamma-aminobutyric acid (GABA) uptake by the developing mouse brain in t’i\,o. Brcrin RPS. 41: 435-451. 1972. 22. Lowry, 0. H.. N. J. Rosebrough, A. L. Farr and R. J. Randall. Protein measurement with the folin phenol reagent. J. Bid. C’hern. 193: 265-275, 195I. 23. Marshall, J. F. The role of central catecholamine-containing neurons in food intake. In: Rrc,c,nt Ad~~tnc,~.s in Obesity Rr.sc,crrc,h II. edited by G. Bray. London: Newman Publishing, 1977, pp. 6-16. 24. Maramatsu. K., H. Odagiri, S. Morishita and H. Takeuchi. Effeet of excess levels of individual amino acids on growth of rats fed casein diets. J. Nutr. 101: 1117-l 126, 1971. 25 Nimni. M. E., P. O’Day and L. A. Bavetta. Tissue distribution and excretion of alpha-aminoisobutyric acid in the rat. 1. Effect of dietary protein level and composition. J. Nutr. SO: 91-98, 1963.

26. Philippu, A., H. Przuntek and W. Roensberg. Superfusion of the hypothalamus with gamma-aminobutyric acid. Effect on release of norepinephrine and blood pressure. NuunvnSchmirdrhrrgs Arch. Phurmnc. 276: 103-118, 1973. 27. Rogers, Q. R. and A. E. Harper. Amino acid diets and maximal growth in the rat. J. Nutr. 87: 267-273, 1965. 28. Rogers, Q. R. and P. M. B. Leung. The control of food intake: When and how are amino acids involved? In: The Chemical SCV~SC~S cd Nutrition. New York: Academic Press, 1977, pp. 213-249. 29. Sauberlich, H. E. Studies on the toxicity and antagonism of amino acids for weanling rats. J. Nmr. 75: 61-72, 1961. 30. Schimke, R. T. and D. Doyle. Control of enzyme levels in animal tissues. Antz. Rev. Biochem. 39: 92$976, 1970. 31. Sytinsky, 1. A., B. M. Guzikov, M. V. Gomanko, V. P. Eremin and N. N. Konovalova. The gamma-aminobutyric acid (GABA) system in brain during acute and chronic ethanol intoxication. J. Ncurochrm. 25: 4348, 1975. 32. Tews, J. K., S. S. Good and A. E. Harper.

Transport of threonine and tryptophan by rat brain slices: Relation to other amino acids at concentrations found in plasma. J. Neurochem.

31: 581-589, 1978. 33. Tews, J. K., Y-W. L. Kim and A. E. Harper.

Induction of threonine imbalance by dispensable amino acids. Relation to competition for amino acid transport into brain. .I. Nutr. 109: 304-315, 1979. 34. Tews, J. K., Y-W. L. Kim and A. E. Harper. Induction of threonine imbalance by dispensable amino acids: Relationships between tissue amino acids and diet. J. Nutr. 110: 394-408, 1980. 35. Tews, J. K., E. A. Riegel and A. E. Harper. Dietary protein and GABA-transaminase (GABA-T) activity. Trtrns. Am. SW. Nrurochem. 10: 138, 1979.

36. Tews, J. K.. N. A. Woodcock and A. E. Harper. Effect of protein intake on amino acid transport and adenosine 3’,5’monophosphate content in rat liver. J. Nutr. 102: 409A17, 1972. 37. Van Gelder, N. M. Brain weight and growth of mice fed gamma aminobutyric acid, glycine or L-glutamic acid diet. Bruin RPS. 33: 571-577, 1971. 38. Van Gelder, N. M. and K.

y-aminobutyric

acid administered

A. C. Elliott. Disposition of to mammals. .I. NrurochPm.

3: 13%143, 1958. 39. White, H. L. 4-Aminobutyrate: 2-oxoglutarate aminotransferase blood platelets. Sckncu 205: 696698, 1979.

in

40. White, H. L. and T. L. Sato. GABA-transaminase of human brain and peripheral tissues-Kinetic and molecular properties. J. Neurochc~m. 31: 41-47, 1978. 41. Wilson, W. E., R. J. Hill and R. E. Koeppe. The metabolism of y-aminobutyric acid-4-l% by intact rats. J. Biol. Chum. 234: 347-349, 1959. 42. WU, J-Y., L. G. Moss and 0. Chude. Distribution and tissue specificity of 4-aminobutyrate-2-oxoglutarate aminotransferase. Ncrrrockent. Rrs. 3: 207-219, 1978. 43. Yessaian, N. H., A. R. Armenian and H. Ch. Buniatian. Effect of y-aminobutyric acid on brain serotonin and catecholamines. .I. Nctrrochc,m. 16: 1425-1433, 1969.