Brain TRH and cyclo (his-pro) and brain protein in the newborn rat are altered by maternal liquid protein feeding

Life Sciences, Vol. 32, pp. 1607-1612 Printed in the U.S.A.

Pergamon Press

BRAIN TRH AND CYCLO (HIS-PRO) AND BRAIN PROTEIN IN THE NEWBORNRAT ARE ALTERED BY MATERNAL LIQUID PROTEIN FEEDING M. Mori*, J.F. Wilber*, and T. Nakamoto** Departments of Medicine* and Physiology** Louisiana State University Medical Center New Orleans, Louisiana 70112 (Received in final form December 28, 1982)

Summary Liquid protein d i e t (LPD) has been shown previously to produce maternal and f e t a l weight loss and fetal congenital anomalies, including cataracts and craniofacial malformations. Therefore, to examine the e f f e c t s of LPD in pregnancy upon the central nervous system of pups, pregnant dams were fed e i e t h e r a 20% casein d i e t ad l i b i t u m , a 20% LPD, or pair-fed with a 20% casein d i e t . LPD was associated with s i g n i f i c a n t maternal weight loss, and pups had s i g n i f i c a n t l y lower b i r t h weights (5.14 ± 0.64) than pups from the pair-fed controls (5.70 ± 0.46, p < 0.05). Total brain protein content was reduced s i g n i f i c a n t l y in pups of both sexes from pregnant fed LPD. Moreover, the concentrations of two brain peptides neurotransmitters, t h y r o t r o p i n releasing hormone (TRH), and i t s b i o l o g i c a l l y active metabolite, h i s t i d y l - p r o l i n e diketopiperazine Cyelo (His-Pro), were elevated in the pups from LPD-fed mothers. In contrast, there was no sign i f i c a n t difference in brain protein or brain peptides in pups from pair-fed mothers vs. pups from mothers fed ad libitum. These data suggest that q u a l i t a t i v e a l t e r a t i o n s of the protein component in maternal dietary composition have deleterious e f f e c t s upon the ontogeny of the rat f e t a l CNS, as r e f l e c t e d by reduced total protein and elevated concentrations of TRH and Cyelo (His-Pro). Since l i q u i d protein d i e t (LPD) has been introduced as a means of weight control,numerous deaths associated with this d i e t have been reported ( I - 3 ) . I t has been estimated that approximately I00,000 persons have used l i q u i d protein f o r weight control as t h e i r source of nourishment for at least one month ( I ) . Until recently, however, s c i e n t i f i c studies using animals models to c l a r i f y whether or not this dietary regimen may be harmful have been limited. In the growing male r a t , l i q u i d protein feeding has been found to provide suboptimal n u t r i t i o n (4). Importantly, no studies have been performed to date to explore potential affects of LPD upon f e t a l brain protein or neuropeptide concentrations. The present study was prompted because women taking these products f o r weight c o n t r o l , not advised f o r pregnancy (5), may become pregnant accidentally without detection from 1 to 3 month's gestation. Data herein, showing that LPD does have s i g n i f i c a n t affects upon both f e t a l brain protein, TRH and Cyclo (His-Pro) metabolism, forms the basis of the present report.

0024-3205/83/141~07-06503.00/0 Copyright (c) 1983 Pergamon Press Ltd.

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Material and Methods A total of 15 timed-pregnant rats were fed regular laboratory chow until day 13. On day 13, dams were randomly divided into 3 groups. Six rats were given a 20% liquid protein diet (LPD) (Table I ) , 4 were given a 20% casein diet ad libitum, and the remaining 5 were pair-fed with the liquid protein group with 20% casein diet. The l i q u i d protein nutrient source was purchased from a health food store. This is commercially available across the United States as "Liquid Protein" (Vangard Products Liquid Protein Wild Cherry Flavor). Dietary composition was based on previous research involving casein diets administered to laboratory rats (6), which contained appropriate carbohydrate, f a t , vitamins and minerals to permit normal growth and development of rats (6). "Liquid Protein" was substituted i s o c a l o r i c a l l y for casein as the protein component in the LPD. The remaining dietary components were e n t i r e l y identical in both diets. Total caloric content was adjusted with the carbohydrate component (Table I ) . Thus, any detectable differences in fetal development could be attributed solely to the composition of the protein component, without the added complication of possible effects resulting from negative caloric balance or induced deficiency states. Food intake and weight in the pregnant females were recorded daily. Within 8 hours of b i r t h , offsprings were weighed and separated into male and female groups. Newborns were k i l l e d by decapitation. Brains from offsprings of each dietary groups were randomized with respect to maternal origin to minimize the influence of maternal differences. Six to 12 brains of both male and female offsprings were used for determinations of total brain protein and brain TRH and Cyolo (His-Pro) in each group. TABLE I Composition of Diets* 20% Casein (9)

20% Liquid Protein (g)

Casein

200

200 (400 ml)

Dextrose

192

192

Sucrose

178

178

Dextrin

192

192

Mazola Corn Oil

150 ml

150 ml

Mineral Mix** Choline Chloride

(5o% w/v)

40 4 ml

40 4 ml

Cellulose

35

35

Vitamin Mix***

I0

I0

* T h i r t y m i l l i l i t e r s of l i q u i d protein correspond to 15 g of soluble protein hydrolysate. The amino acid composition is approximately the following in milligrams: L-Alanine, 1300; L-Arginine, 1200; L-Aspartic acid, 900; L-Cystine, I0; L-Glutamic acid, 1500; Glycine, 3500; L-Histidine, 70; L-Hydroxylysine, 150; L-Hydroxyproline, 2200; L-Leucine, 450; L-Isoleucine, 200; L-Lysine, 650, L-Methionine, I I 0 ; L-Phenylalanine, 350; L-Proline, 2300; L-Serine, I000; L-Threonine, 300; L-Tryptophan, 65; L-Tyrosine, I00; L-Valine, 350. **Rogers-Harper mineral mix (Teklad Test Diets, Madison, WI) ***AIN vitamin mixture 76 (ICN Pharmaceuticals, Cleveland, Ohio)

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Fetal whole brain was i ~ e d i a t e l y placed into 1 ml of O.4M perchloric acid kept on ice, and homogenized. Then, 20 ~l sample was taken from each homogenate and diluted with O.IN NaOH for measurement of protein as described previously (7). The remaining homogenates were centrifuged and neutralized with saturated KHCO3 and used for TRH and ~elo (His-~o) by our specific radioimmunoassays (8). All samples were run in the same assay to minimize interassay v a r i a b i l i t y . Co-efficients of intraassay variations for RIAs and protein measurements were less than 6 and 2%, respectively. Data were analyzed by Student's t - t e s t , and the level less than 5% is considered s i g n i f i cant s t a t i s t i c a l l y . Experimental Results Liquid protein feeding of dams were associated with significant diminution in caloric intake and weight gain in comparison to coptrol pregnant rats fed ad libitum (Fig. l ) . As anticipated, the group pair-fed 20% casein, in lieu of LPD, ingested less calories and also lost weight relative to the ad libitum group. I t was observed, however, that animals fed LPD, which was isocaloric to the 20% pair-fed group, nevertheless gained less weight than the pair-fed controls between days 16 and 21. ~'1 ~Ad

/

]0 0

1

I

I

I

I

I

I

iibitum group

~~",~fPair-f~ group l~Liquid protein group I I

~j.f

t

~Ad

hbltum group "

group

-IO

I 14

I ~

I 15

I 17

I 18

I Ig

I ~

I 21

I 22

GESTATiO~L AGE IDAYS)

Figure 1 D a i l y Food Intake and Increments ( A ) of Body Weights in Pregnant Rats Beginning at day 13 of pregnancy, dams were fed e i t h e r a 20% casein ad l i b i t um, a 20% l i q u i d p r o t e i n d i e t , or p a i r - f e d with a 20% casein d i e t through the remainder of the g e s t a t i o n . Body weights are expressed as i n c r e m n t s ( A ) over the weights obtained on day 13. vs. ad l i b i t u m group, * = p < 0.01, * * = p < 0.05 vs. pair-fed group, + = p < 0.01, ++ = p < 0.05

Body weight of offsprings of mothers fed the LPD regime exhibited a ~an of 5.14 ± 0.64 (±SD), N = 33, which was significantly less than the weight of both offsprings from pair-fed controls (5.70 ± 0.46 g, N = 19) (p < 0.05) and

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offsprings derived from ad l i b i t u m controls (6.90 ± 0.52, N = 20) (p < O.OOl). Concentration and content of t o t a l brain protein and brain TRH and Cyelo (His-~o) of pups from the 3 experimental groups are summarized in Table I I . LPD feeding resulted in s i g n i f i c a n t reductions (p < 0.01) in brain protein concentrations in comparison to the ad l i b i t u m controls. This reduction in brain protein was not simply a function of reduced t o t a l c a l o r i c intake because protein concentrations in the mothers p a i r - f e d with LPD rats were not d i f f e r e n t s t a t i s t i c a l l y from the ad l i b i t u m controls. There were no sex d i f ferences noted in the reduced protein contents of the LPD group. TABLE I I Protein, TRH and Cyolo (His-Pro) Concentrations in the Newborn Rat Brain

Ad Libitum

Pair-Fed

Liquid Protein

Protein (mg/whole brain ± SD) Male Female

13.96 ± 1.01(8) 13.51 ± 0.79(10)

13.47 ± 0.50(8) 12.91 ± 0.50(8)

12.79 ± 0.45(12)*+ 11.76 ± 0.55(12)*+

TRH (pg/mg protein ± SD) Male Female

20.4 22.9

± 5.3(8) ± 2.5(10)

25.1 25.9

± 3.3(8) ± 3.9(8)

28.4 30.3

58.6 56.6

± 18.5(6) ± 17.5(6)

60.7 61.1

± 18.6(6) ± 12.7(6)

117.4 ± 34.5(6)*+ 125.7 ± 31.8(6)*+

± 4.5(12)* ± 5.8(12)*

Cyclo (His-Pro) (pg/mg protein ± SD) Male Female

Newborn whole brains from each group of dams fed e i t h e r a 20% casein d i e t ad l i b i t u m , a 20% l i q u i d protein d i e t , or p a i r - f e d with a 20% casein d i e t were extracted in perchloric acid and measured f o r TRH and Cyclo (His-Pro) by RIAs. The number of animals/group are in parentheses to the r i g h t of each datum. Liquid protein vs. ad l i b i t u m group, * = p < 0.01; l i q u i d protein vs. p a i r fed group, + = p < 0.01. In association with reduced brain protein, LPD was also accompanied by s i g n i f i c a n t elevations in brain TRH and Cyclo (His-~o) concentrations. In both sexes, LPD caused s i g n i f i c a n t (p < 0.01) elevations in TRH and Cyelo (His-Pro) in comparison to peptide concentrations in the ad l i b i t u m controls. As with brain p r o t e i n , TRH and Cyolo (His-~o) values, in contrast, were not elevated in the p a i r - f e d group in comparison to ad l i b i t u m controls. Discussion The present study demonstrates f o r the f i r s t time that feeding pregnant rats a l i q u i d protein d i e t , i s o c a l o r i c to a control 20% casein d i e t , is attended by s i g n i f i c a n t abnormalities in the metabolism of not only brain t o t a l protein but also in the metabolism of the two neuropeptides, TRH and OwoZo (His-Pro), in the central nervous system of r e s u l t a n t offsprings. This decrease in brain protein was anticipated in part from previous studies demons t r a t i n g reduced brain protein and brain DNA in offsprings from mothers fed

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low protein diets during pregnancy (9-11). However, in the present studies the quantity of protein, dextrose, lipid, minerals, cellulose and vitamins in LPD and pair-fed controls was identical. Therefore, the differences in brain composition observed are presumed to be related to qualitative differences in the amino acid composition of the liquid protein component of the dietary regimens. Thus, the quality of dietary protein may be a critical new variable in the ontogeny of the fetal CNS, and studies are ongoing to ascertain what specific differences in amino acid composition may have been implicated in the reductions in brain protein concentrations. We speculate that these reductions in brain protein reflect diminished rates of protein synthesis and not accelerated release or proteolysis, but this interpretation must remain conjectural at present. In the face of reduced brain protein, i t was of great interest that both TRH and ayolo (His-Pro) concentrations, relative to protein, were elevated significantly by the administration of LPD to mothers. Although the neurobiological significance of this finding is presently not known, TRH and Cyelo (His-Pro) exemplify putative CNS peptide neurotransmitters and/or neuromodulators, whose wide-ranging behavioral effects not dependent upon integrity of the hypothalamic-pituitary-thyroid axis (12). Since infant rats from malnourished dams have been demonstrated to exhibit the behavioral changes (13-14) and also exhibt reduced norepinephrine and dopamine accumulation in their brains (15), alterations in the availability of TRH induced by liquid protein diet, likely linked to catecholamine turnover rates (16), have potent i a l l y profound implications for the functions of the fetal nervous system post-partum. Furthermore, Cyelo (His-pro), which is derived from TRH by proteolytic enzymatic processing, has been found also to possess a number of neuropharmacological properties (22). Thus, the present observations suggest that high concentrations of Cyclo (His-pro) are likely to result in neurophysiological modulations in the fetal brain. Our findings of elevated brain peptides are not a reflection of simple negative caloric balance, which has been demonstrated by one of us previously to be associated with lowered hypothalamic TRH (17). Moreover, we would not anticipate any changes in pituitary-thyroid regulation per se, since the fetal pituitary-thyroid axis appears to be regulated indepe~ently of TRH influences prenatally and at parturition (18-19). However, later influences of elevated TRH concentrations upon thyroid function cannot be excluded at present. To exemplify, in earlier studies Bakke and co-workers have shown that elevations in hypothalamic TRH produced by thyroxine administration to newborns were associated with the occurrence of hypothyroidism in the adult (20). Whether or not the observed elevations in TRH reflect increased synthesis or reduced catabolism and/or release is not known at present. In a previous report, the deleterious effects of LPD in pregnancy from day 13-18 were underscored, which included cataract formation, spinal curvature, and craniofacial anomalies (21). In the present report, LPD was continued beyond day 18 through term (day 22) and was attended by s t i l l further reductions in pup weight in comparison to those whose mothers were fed LPD until day 18. This may indicate that longer periods of LPD administration could be more injurious to the developing CNS than the shorter dietary schedules utilized earlier. Studies are presently underway to examine specifically what quantity and duration of LPD dietary regimens are needed to produce the alterations in brain protein and peptide metabolism observed herein.

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Acknowledgments Supported in part by a grant from the Cancer Association of Greater New Orleans. References I. 2. 3. 4. 5. 6. 7. 8. 9. lO. II. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

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