Effect of dietary protein on hepatic mitochondrial function and cardiac muscle protein turnover in uremic rats

NUTRITION RESEARCH, Vol. 5, pp. 65-79, 1985 0271-5317/85 $3.00 + .00 Printed in the USA. Copyright (c) 1985 Pergamon Press Ltd. All rights reserved.

EFFECT OF DIETARY PROTEIN ON HEPATIC MITOCHONDRIAL FUNCTION AND CARDIAC MUSCLE PROTEIN TURNOVER IN UREMIC RATS Robert S. Tyzbir, Ph.D., Arthur S. Kunin, M.D. Deborah E. Hewett, M.S., and Jean R. Hewitt, B.S.

Departments o f Human N u t r i t i o n and M e d i c i n e , C o l l e g e s of A g r i c u l t u r e and Medicine U n i v e r s i t y o f Vermont, B u r l i n g t o n , VT 05405 ABSTRACT

To d e f i n e t h e m e t a b o l i c c o n s e q u e n c e s o f d i e t s low in p r o t e i n in c h r o n i c r e n a l f a i l u r e , young uremic and sham o p e r a t e d r a t s were fed i s o e n e r g e t i c d i e t s f o r t h r e e weeks v a r y i n g in p r o t e i n c o n t e n t from 22~ t o 8% c a s e i n a t t h e expense o f c a r b o h y d r a t e . The r e s u l t s i n d i c a t e t h a t r e d u c i n g c a s e i n c o n t e n t from 221t ( c o n t r o l ) t o e i t h e r 16%, 12% or 8~ in uremic r a t s d i d n o t r e s u l t in g r o s s d i f f e r e n c e s i n r e n a l f u n c t i o n or d e c r e a s e s i n l i v e r w e i g h t , m i t o c h o n d r i a l c o n t e n t or r e s p i r a t i o n . Atrial muscle p r o t e i n s y n t h e s i s was i n c r e a s e d 26% w h i l e d e g r a d a t i o n was s l i g h t l y , but not s i g n i f i c a n t l y , d e c r e a s e d in uremic and s h a m - o p e r a t e d r a t s f e d t h e 8% c a s e i n d i e t compared t o c o n t r o l . In both uremic and s h a m - o p e r a t e d r a t s f ed t h e 8% c a s e i n d i e t , serum triiodothyronine (T3) concentrations and hepatic mitochondrial glycerolphosphate (GP) shuttle activity were significantly increased while malate-aspartate shuttle activity was d e c r e a s e d compared t o a l l o t h e r d i e t g r o u p s. The i n c r e a s e d serum T 3 level and GP shuttle activity produced by feeding the 8% casein diet suggests a diet induced alteration in hepatic mitochondrial intermediary metabolism which may be of benefit in protecting against the potentially serious c o n s e q u e n c e s of lo w e r e d t h y r o i d hormone l e v e l s in c h r o n i c r e n a l insufficiency. KEY WORDS:

Chronic uremia, Dietary f u n c t i o n , Muscle t u r n o v e r

protein,

Mitochondrial

INTRODUCTION

The b i o c h e m i c a l mechanisms u n d e r l y i n g c l i n i c a l improvement of uremic symptoms in c h r o n i c r e n a l f a i l u r e f o l l o w i n g d i e t a r y p r o t e i n r e s t r i c t i o n have not been c o m p l e t e l y e l u c i d a t e d . P r e v i o u s s t u d i e s in our l a b o r a t o r y (1) have i n d i c a t e d t h a t f e e d i n g a low p r o t e i n , high c a r b o h y d r a t e , normal f a t d i e t (8% c a s e i n / 7 3 % c a r b o h y d r a t e / l O % f a t ) t o uremic a d u l t r a t s r e s u l t s in a d i e t induced, triiodothyronine (T 3) mediated stimulation of the hepatic mitochondrial $1ycerolphosphate dihyroxyacetone phosphate (GP) shuttle system. We h y p o t h e s i z e d t h a t t h e enhanced a c t i v i t y of t h i s pathway (whose

R e p r i n t r e q u e s t s t o Dr. A.S. Kunin, Department of M e d i c i n e , Vermont C o l l e g e of M e d i c i n e , B u r l i n g t o n , VT, 05405, U.S.A. 65

University

of

66

R.S. TYZBIR et al.

f u n c t i o n i s t o t r a n s p o r t r e d u c i n g e q u i v a l e n t s from NADH produced in t h e c y t o p l a s m i n t o t h e m i t o c h o n d r i a might improve e f f i c i e n c y o f m i t o c h o n d r i a l e n e r g y p r o d u c t i o n which, when u l t i m a t e l y e x p r e s s e d a t t h e c l i n i c a l level, c o u l d be m a n i f e s t e d as symptomatic improvement. To f u r t h e r our u n d e r s t a n d i n g o f t h e c o n s e q u e n c e s o f l o w e r i n g d i e t a r y p r o t e i n c o n c e n t r a t i o n s in c h r o n i c r e n a l f a i l u r e , we s t u d i e d t h e e f f e c t s o f feeding a series of isoenergetic diets varying stepwise in protein concentration from 22% t o 8% a t the expense o f c a r b o h y d r a t e , while maintaining fat constant at 10~ in young chronic uremic rats and sham-operated c o n t r o l s . Specifically, we wanted t o a s s e s s t h e r e l a t i v e i n f l u e n c e o f t h e s e d i e t a r y m a n i p u l a t i o n s on o v e r a l l w e i g h t g a i n , r e n a l f u n c t i o n , serum T3 c o n c e n t r a t i o n s , h e p a t i c m i t o c h e n d r i a l r e s p i r a t i o n and GP shuttle activity, and a t r i a l muscle p r o t e i n t u r n o v e r . We a l s o measured t h e e f f e c t o f v a r i o u s d i e t a r y m a n i p u l a t i o n s on t h e a c t i v i t y of the h e p a t i c m i t o c h o n d r i a l m a l a t e - a s p a r a t e (MA) s h u t t l e t o d e t e r m i n e w h e t h e r i t responded in a manner s i m i l a r t o t h a t o f t h e GP s h u t t l e .

NK"rHODS

Weanling male r a t s I w e i g h i n g a p p r o x i m a t e l y 50g were made c h r o n i c a l l y uremic by a one s t a g e b i p o l a r l e f t nephrectomy p l u s a c o n t r a l a t e r a l total nephrectomy, l e a v i n g a f u n c t i o n i n g remnant of a p p r o x i m a t e l y 30~ o f t h e o r i g i n a l t o t a l r e n a l mass. To p r e s e r v e t h e a d r e n a l g l a n d s , t h e k i d n e y s were decapsulated, and t h e resultant loose connective tissue, anatomically c o n t i n u o u s w i t h t h e r e n a l h i l a r f a s c i a and c o n t a i n i n g t h e a d r e n a l s , was r e i n s e r t e d p r i o r to r e n a l a b l a t i o n . The k i d n ey s o f l i t t e r m a t e c o n t r o l s were similarly exteriorized, d e c a p s u l a t e d and r e s t o r e d i n t r a a b d o m i n a l l y . Both groups were t h e n f e d a commercial l a b o r a t o r y r a t i o n ( P u r i n a Rat Chow, Ralson P u r i n a Co., St. Louis, MO) a_dd l i b i t u m . Seven days l a t e r , partially n e p h r e c t o m i z e d and s h a m - o p e r a t e d c o n t r o l r a t s were each d i v i d e d i n t o two grou p s . I n i t i a l mean body w e i g h t s were s i m i l a r w i t h i n t h e n e p h r e c t o m i z e d and s h a m - o p e r a t e d groups but mean body w e i g h t s o f t h e n e p h r e c t o m i z e d groups were slightly lower than the age matched, sham-operated groups. In each e x p e r i m e n t a l c y c l e , h a l f o f t h e n e p h r e c t o m i z e d and h a l f o f t h e s h a m - o p e r a t e d r a t s were f e d one o f t h e t h r e e e x p e r i m e n t a l d i e t s (2) f o r t h r e e weeks (each diet containing % casein/ % carbohydrate/ % fat: 16/65/10, 12/69/10, 8/73/10) and t h e o t h e r h a l f of t h e two groups were f e d t h e c o n t r o l d i e t c o n t a i n i n g 22/59/10%.

Sprague-Dawley r a t s were o b t a i n e d from C h a r l e s River Laboratories (Canadian B r e e d i n g L a b o r a t o r i e s , S t . C o n s t a n t , Quebec). The r a t s were housed in w i r e mesh cages equipped w i t h a u t o m a t i c w a t e r i n g in a room c o n t r o l l e d f o r c o n s t a n t t e m p e r a t u r e (230 ) and h u m i d i t y w i t h a 1 2 - h o u r light-dark cycle.

DIETARY PROTEIN IN UREMIA

67

Each d i e t s u p p l i e d 17.4 k J / g . 2 In each c y c l e , one group o f p a r t i a l l y n e p h r e c t o m i z e d r a t s were f e d t h e e x p e r i m e n t a l d i e t ad l i b i t u m . The r e m a i n i n g groups of s h a m - o p e r a t e d and p a r t i a l l y n e p h r e c t o m i z e d r a t s were p a i r - f e d t h e i r respective diets in the amounts spontaneously consumed by the nephrectomized group fed the experimental diet during the previous 24 hours. Thus, in each feeding cycle, there was always a group of partially nephrectomized and sham-operated rats fed the specific experimental diet under study and a group of partially nephrectomized and sham-operated rats fed the control diet; the latter 3 groups pair-fed to the partially nephrectomized rats fed the e x p e r i m e n t a l d i e t ad l i b i t u m . The treatment history of these animals is represented by the code: C22, L16, L12, Lg; N or S. The number 22, 16, 12 or 8 r e p r e s e n t s t h e p e r c e n t of d i e t a r y p r o t e i n , N or S r e p r e s e n t p a r t i a l l y nephrectomized or s h a m - o p e r a t e d r a t s r e s p e c t i v e l y , L signifies one of t h e low p r o t e i n e x p e r i m e n t a l d i e t s and C r e p r e s e n t s t h e c o n t r o l d i e t . At t h e end o f each t h r e e week f e e d i n g p e r i o d , t h e r a t s were weighed, k i l l e d by d e c a p i t a t i o n , e x s a n g u i n a t e d and bloods c o l l e c t e d . The l i v e r and h e a r t were inuuediately removed. Serum was f r o z e n f o r hormone a n a l y s e s . Following rapid excision, the liver was washed in ice-cold mannitol/sucrose (0.25M/O.OTM) blotted and weighed. A 3.0-3.1g sample was homogenized in cold mannitol/sucrose (I0~ w/v) in a glass-Teflon, Potter-Elvehjem homogenizer. Homogenization was divided into two 15-sec. periods with an interval for recooling. Hepatic mitochondria were isolated at 00 by differential centrifugatlon (3). The mitrochondrial pellet was resuspended in a volume of mannitol/sucrose equal to half the original tissue sample weight. Mitochondrial protein was determined by the microbiuret procedure ( 4 ) . Diluted aliquots of freshly prepared hepatic mitochondria were used in the determination of mitochondrial oxygen consumption and in reconstituting the glycerol phosphate-dihydroxyacetone phosphate (GP) and malate-aspartate (MA) shuttle systems (1,2). L i v e r m i t o c h o n d r i a l oxygen consumption was a s s a y e d a t 300 in a 3 ml incubation volume u s i n g a C l a r k - t y p e oxygen e l e c t r o d e (Yellow S p r i n g s I n s t r u m e n t Company, Y e l l o w S p r i n g s , OH). The i n c u b a t i o n media c o n t a i n e d 0 .1 ml (0.25 mE) m i t o c h o n d r i a l suspension, 150 mM KCl, 5 mM MgC12, 5mM K2HPO4-KH2PO4 p l u s 10 mM t r i e t h a n o l a m i n e c h l o r i d e b u f f e r , pH 7 . 4 , lmM EGTA, 0.1% b o v i n e serum albumin; in a d d i t i o n , 1 g/m1 r o t e n e n e was added when

The c o n t r o l d i e t (C22) s u p p l y i n g 17.4 k J / g , c o n t a i n e d 22% p r o t e i n in t h e form o f v i t a m i n - f r e e c a s e i n , 59% c a r b o h y d r a t e d i v i d e d i n t o e q u a l p a r t s o f d e x t r o s e , s u c r o s e , d e x t r i n and c o r n s t a r c h , and 101t f a t in t h e form o f corn o i l and l a r d . The e x p e r i m e n t a l d i e t s , L16, L12, L8 c o n t a i n e d 16, 12 or 8% v i t a m i n - f r e e c a s e i n r e s p e c t i v e l y , and were made i s o e n e r g e t i c t o t h e c o n t r o l d i e t by a d j u s t i n g t h e c o n c e n t r a t i o n o f each o f t h e c a r b o h y d r a t e sources. A l l d i e t s c o n t a i n e d 5% m i n e r a l mix, ( R o g e r s - H a r p e r S a l t Mix), f u r n i s h i n g i n o r g a n i c p h o s p h a t e o t 0.4% of t h e d i e t ; a t r a c e e l e m e n t mix 0.5%; v i t a m i n f o r t i f i c a t i o n m i x t u r e (A.O.A.C.) 1%; L - m e t h o n l n e , a t 0.2%; c e l l u l o s e 2.5%. S u c r o s e , corn o i l and l a r d were o b t a i n e d l o c a l l y , a l l o t h e r components were p u r c h a s e d from Teklad M i l l s , Madison, WI. The c o n t r o l and e x p e r i m e n t a l d i e t s were p r e p a r e d in our l a b o r a t o r y weekly t o e n s u r e u n i f o r m i t y and a c c u r a c y o f c o m p o s i t i o n .

68

R.S. TYZBIR et al.

succinate was used as substrate. State 3 and state 43 were determined using as substrate(s) either 10 ram pyruvate plus 1.0 mM malate, or 3.3 mM succinate, and 0.5 moles ADP respectively. The GP and NA s h u t t l e system were constituted by i n c u b a t i n g intact h e p a t i c m i t o c h o n d r i a ( 0 . 2 5 ms i n 0 . 1 ml) s u s p e n d e d i n t h e same i n c u b a t i o n medium d e s c r i b e d a b o v e b u t w i t h t h e a d d i t i o n o f 0 . 1 5 mN NADH ( 2 ) . The GP s h u t t l e was r e c o n s t i t u t e d by a d d i n g 10 mM D L - 2 - g l y c e r o p h o s p h a t e , I mM ADP and 5 u n i t s o f g l y c e r o p h o s p a t e d e h y d r o g e n a s e t o a f i n a l volume o f 3 m l . The NA shuttle was reconstituted by adding 1 mM malate, 5mM glutamate, 2 mM asparate, 1 mM ADP, 20 units of malate dehydrogenase and 25 units of glutamate-oxaloacetate transaminase to a final volume of 3 ml. Shuttle system activity was expressed as nanomoles of NADH converted to NAD/min/mg mitochondrial protein. Blanks which omitted either mithochondria or shuttle components were routinely assayed and their results subtracted from those of the complete reconstituted system.

Inunediately following sacrifice, the hearts were rapidly removed and placed in Krebs-Ringer bicarbonate buffer (KRB) saturated with 95% 02 - 5% CO 2, pH 7.4 Both atriae were dissected from the heart following the method of Curfman et al. ( 5 ) . The two pooled atria (each weighing approximately IO ms) from each rat were rapidly rinsed, gently blotted, weighed on a torsion balance and placed in sequence into 25 ml flasks containing 3 ml of gassed KRB at 370 with 20 mM glucose, 0.5 mM l-phenylalanine, and 0.85 mM l-leucine. The flasks were restoppered and the tissues pre-incubated for 30 minutes at 370 in a shaking water bath. Following this, they were rapidly transferred to new flasks containing 3 ml of gassed KRB, 20 mM glucose (5 mM) and [U-14C] phencylalenine (0.5 Ci/ml) l-leuclne (0.85 raM) and incubated at 370 in a shaking water bath for 2 hours (I00 oscillation/minute). The tissue was then removed from the incubation fluid, blotted, placed in 0.2 M perchloric acid, homogenized and centrifuged. The remaining pellet was washed twice in 0.2 M perchloric acid, once in 95% alcohol-ether (I:I) and dissolved in Soluene, (Packard, Downers Grove, IL) counted in a liquid scintillation spectrometer and corrected for quenching to assay for 14c-phenylalanine incorporation into acid insoluable muscle protein. After removal of tissue, the incubation medium was decanted into test-tubes, covered and boiled for two minutes, centrifuged and the supernate analyzed for tyrosine (6). This tyrosine, released into the incubation media from the incubated tissue, was used as a measure of net muscle protein breakdown as this amino acid is neither synthesized nor degraded by muscle. Since the intracellular pool of tyrosine remains constant during such incubations, the appearance of tyrosine in the media reflects net protein breakdown (7,8). The rate of protein synthesis was determined by measuring the rate of incorporation of 14C-phenylalanine into muscle protein after correcting for intracellular specific activity (9,10) and is expressed as nanomoles of tyrosine incorporated/mg wet tissue/2h, utilizing the tyrosine: phenylalanine ratio in muscle myosin of 0.77 to convert results obtained for phenylalanine incorporation to that of tyrosine (11). The absolute rate of protein degradation was calculated algebraically as: net protein breakdown plus the ate of protein synthesis, and is expressed as nanomoles of tyrosine released/mg wet tissue/2h. Thus, protein synthesis and degradation are assayed in the same muscle at the same time without the potentially complicating use of metabolic inhibitors in the incubation medium.

S t a t e 3 i s d e f i n e d by Chance and W i l l i a m s as r e s p i r a t i o n in the presence o f an a c c e p t o r (ADP) s y s t e m and s t a t e 4 as t h e r e s p i r a t i o n which is o b s e r v e d a f t e r t h e a d d e d ADP h a s b e e n c o n v e r t e d t o ATP.

DIETARY PROTEIN IN UREMIA

69

Serum urea nitrogen was assayed following the technique of Crocker (12) and serum creatinine assayed according to a procedure based on the Jaffe reaction. Serum T3 concentration was quantitated by a double radioimmunossay procedure. 4 Data were stored and analyzed statistically either by an analysis o f variance or unpaired t-test u s i n g a TRS-80 R a d i o S h a c k m i c r o c o m p u t e r ( R a d i o S h a c k , F o r t W o r t h , TX). The u n p a i r e d t - t e s t was u s e d t o d e t e r m i n e w h e t h e r significant differences existed between results obtained from either the sham-operated or partially nephreetomized rats fed the same d i e t . The analysis o f v a r i a n c e was u s e d t o d e t e r m i n e w h e t h e r s i g n i f i c a n t differences e x i s t e d among t h e v a r i o u s g r o u p s o f n e p h r e c t o m i z e d s h a m - o p e r a t e d r a t s f e d t h e different diets. After i t was d e t e r m i n e d t h a t t h e r e w e r e no s i g n i f i c a n t differences between the results obtained in each cycle in the sham-operated rats f e d t h e C22 d i e t , the results were combined and considered as a group (N=19). Similar treatment of the data obtained from the partially nephrectomized rats f e d t h e C22 d i e t during each cycle also showed no significant differences, and t h e r e s u l t s w e r e c o m b i n e d and c o n s i d e r e d a g r o u p

(N=17).

RESULTS

The mean t o t a l f o o d i n t a k e p e r r a t o v e r t h e 21 d a y f e e d i n g p e r i o d ( T a b l e 1) was s i m i l a r i n a l l g r o u p s e x c e p t L8-N whose t o t a l f o o d c o n s u m p t i o n was greater than that of all other groups. The a v e r a g e w e i g h t gained o v e r t h e 21 d a y f e e d i n g period differed noticeably between the dietary groups. A n i m a l s f e d t h e 8% p r o t e i n d i e t (L8) w h e t h e r s h a m - o p e r a t e d (S) o r p a r t i a l l y n e p h r e c t o m i z e d (N) g a i n e d l e s s w e i g h t than all of the other groups. This is reflected in the food efficiency ratios (FER) w h i c h i s l o w e s t i n t h e L8 d i e t a r y g r o u p , s u g g e s t i n g t h a t a n i m a l s fed the lowest level of protein were least efficient at using non-protein food energy for weight gain. Deceased metabolic efficiency has been reported in rats fed similar protein restricted diets and involved sympathetic activation of thermogenic mechanisms originating i n brown a d i p o s e t i s s u e (13). A s i m i l a r m e c h a n i s m may e x p l a i n t h e r e d u c e d FER i n b o t h t h e L8 N and S groups. The p r o t e i n e f f i c i e n c y r a t i o (PER) a p p e a r e d somewhat h i g h e r i n t h e L g r o u p s c o m p a r e d w i t h t h e C22 g r o u p s , s u g g e s t i n g that t h e low p r o t e i n fed animals were at least as efficient at utilizing available p r o t e i n a s t h e 22~t protein fed animals. The e f f e c t s of feeding nitrogen (SUN), c r e a t i n i n e , shown i n T a b l e 2.

the various diets f o r 21 d a y s on s e r u m and t r i i o d o t h y r o n i n e (T 3) c o n c e n t r a t i o n s

urea are

The p r o c e d u r e was d e v e l o p e d by A n t i b o d i e s , I n c . o f D a v i s , CA a n d m o d i f i e d by Dr. L . B . Carew, B i o r e s e a r c h C e n t e r , U n i v e r s i t y o f V e r m o n t , B u r l i n g t o n , VT. Antibodies required were rabbit antiserum t o T 3 a n d g o a t gamma g l o b u l i n s u p p l i e d by A n t i b o d i e s , I n c . , D a v i s , CA.

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R.S. TYZBIR et al.

TABLE 1 Mean Total Food Intake, Weight Gain, Food Efficiency Ratio (FEE) 1 and Protein Efficiency (PER) 2 in Chronically Uremic (N) and Sham-Operated (S) Rats Fed Either a Low Protein (L16,L8 F8) or Control (C22) Diet for Three Weeks.3, 4

C22

L16

L12 N

L8

Diet Groups

N

S

N

S

S

N

Food Intake (8)

265.4 (37)

286.7 (47)

286.7 (16)

279.8 (18)

291.1 283.6 (12) (14)

358.2 (19)

293.3 (19)

Weight Gain

97.4

121.7

131.2

128.3

78.6 110.5

57.9

63.7

(g) FER

0.37

0.42

0.46

0.46

0.27

0.39

0.16

0.22

PER

1.67

1.93

2.86

2.87

2.25

3.25

2.02

2.72

IFER = weight gain in srams/total food intake in grams. 2pER = weight 8ain in grams/total protein intake in grams. 3Diets contained % proteinl~carbohydrate/%fat: C22=22/59/I0; L12=12/69/I0; L8=8173/I0. 4Numbers in parentheses indicate number of animals studied.

L16=16165110;

TABLE 2 Serum Urea Nitrogen (SUN), Creatinine and Triiodothyronine (T 3 ) Concentrations in Chronically U r e m i c (N) a n d Sham O p e r a t e d (S) R a t s Fed Either a Low P r o t e i n ( L 1 6 , L12, L 8 , ) or Control (22) Diet for Three Weeks a , b ,

Diet Groups

C22

L16

H SUN (msldl)

s

50.2 +3.1

11.4 +_0.8

(17) (19) p
T3

0.64 • 0.08 (16) ns 209.0 • (12)

b

(14)

208.0 +-6.0 (12) ns

a

0.46

•

s 32.6 +-2.9

L8

LI2

s_

N_

S

8.6 +-0.9

24.8

7.4

11.1

5.2

+-4.0

+_0.9

+1.2

+-0.6

(7) (7) p< 0.001

(6) (7) p< .001

0.93 0.66 +-0.08 • (7) (7) p<0.05

+-0.09 +-0.06 (6) (7) p<0.05

155.9 +8.6 (4)

153.4 +,11.5 (4) ns

0.77

143.8 +,12.6 (4)

N

(11) (I0) p< .001

0.41

207.6 +,31.2 (10)

0.72

•

0.43

+,0.0 (12) (13) p<0.01

250.4 +-9.0 (12)

ns

V a l u e s a r e mean • SEM. Numbers i n p a r e n t h e s i s animals. Diets contained % protein/% carbohydratel% L16=16/65/10; L12=12/69/10; L8=8/73/10.

S_

247.5 +-8.6 (4) ns

indicate fat:

number

of

C22=22/59/10;

DIETARY PROTEIN IN UREMIA

71

The SUN l e v e l s w e r e g e n e r a l l y proportional to the dietary protein fed and the degree of renal insufficiency. A l l o f t h e N r a t s h a d SUN l e v e l s significantly different f r o m o n e a n o t h e r a n d f r o m t h e shams a t e a c h d i e t a r y protein level. Among the S groups, SUN level was significantly increased in rats fed the 22% protein diet compared to S rats fed any of the other diets. In all experimental groups, the serum creatinine concentrations were significantly elevated in the N animals compared to S ones and were moderately but not significantly increased 39% in the N versus S rats fed C22 diet.

Consistent with our previous observations in older rats ( I ) , the concentration of serum T 3 was significantly increased (p <0.05) in both the N and S rats fed the L8 diet when compared to all other groups (Table 2). There were no significant differences in serum T 3 levels as a result of partial nephrectomy regardless of diet.

After feeding the various diets for 3 weeks, the final mean body weights of the S rats were not significantly different from one another irrespective of diet composition (Table 3).

Similarly, there w e r e no s i g n i f i c a n t weight differences among t h e various N rats regardless of diet. Within each dietary group however, the mean body w e i g h t o f t h e N r a t s was c o n s i s t e n t l y l o w e r t h a n t h e mean o f t h e S rats reflecting the difference in initial mean b o d y w e i g h t s b e t w e e n t h e N and S groups.

T h e r e w e r e no s i g n i f i c a n t differences i n l i v e r w e i g h t s b e t w e e n t h e N and S animals irrespective of diet (Table 3). The l o w e s t mean l i v e r w e i g h t s w e r e f o u n d i n t h e low p r o t e i n fed N rats (L12N, L8N). The mg o f m i t o c h o n d r i a l protein isolated p e r gram o f l i v e r t i s s u e was n o t s i g n i f i c a n t l y different in any of the groups indicating that the quantity of mitocbondria isolated per gram was n o t a f f e c t e d by d i e t o r r e n a l s t a t u s .

The a v e r a g e k i d n e y w e i g h t s r e p r e s e n t t h e c o m b i n e d w e i g h t o f t h e two kidneys in the S rats and t h e w e i g h t o f t h e s i n g l e h y p e r t r o p h i e d remnant kidney from the N rats. At every dietary protein level, there was a significant difference between the remnant single kidney weight of the N g r o u p a n d t h e c o r r e s p o n d i n g w e i g h t o f t h e two k i d n e y s o f t h e S g r o u p (p< 0.001) (Table 3). In general, the kidney weights in the S rats increased with an increase in dietary protein, those i n t h e C22 S g r o u p w e r e significantly g r e a t e r t h a n L12 o r L8 S g r o u p s . Similarly, the remnant kidney weights of the N-group tended to increase w i t h an i n c r e a s e in dietary protein. Significant differences w e r e o b s e r v e d b e t w e e n L8N a n d C22N o r L16N (p < 0 . 0 5 ) .

Data summarizing the results obtained for protein synthesis and degradation in the atrial appendages (atria) a r e shown i n T a b l e 4. Quantity of mitochondria isolated p e r gram was n o t a f f e c t e d by d i e t o f r e n a l status.

72

R.S. TYZBIR et al.

TABLE 3 Body weight, liver weight, mitochondrial protein per g liver tissue and kidney weight 1 in chronically uremic (N) and sham-operated (S) rats fed either a low protein (LI6, LI2, L8) or control (C22)diet for Three

Weeks 2,3, Diet Groups

C22

Body Weight (g)

191.0 •

L i v e r Weight

(g)

LI6

224.9 • (15) (18) ns

8.25 • 0.29 (15)

219.7 • (8)

9.42 •

(15)

(8)

Kidney Weight •

1 2 3

22.20 • (13) ns

(8)

1.20 2.02 0.09 • (13) (14) p < 0.01

20.72 18.31 +1.9 • 1.4 (8) (8) ns 1.15

7.33

1.88

•

(10)

(8) (8) p < 0.001

(10) ns

18.71 • (10)

21.57 +_1.4 (10) ns

0.88

•

8.30

•

17.55 18.27 • 1.6 • 1.7 (6) (7) ns

•

•

8.79 +_0.42 (6) (7) ns

169.8 222.5 • • (I0) (I0) p < 0.05

7.50 •

ns 20.64 • (14)

L8

163.2 204.0 • • (6) (7) p < 0.05

9.25 •

ns Mitochondrial Protein

229.4 • (8) ns

8.97 •

LI2

1.60 +_0.09 (6) (7) p< 0.001

0.76

1.63

•

• (10) (I0) p< 0.000

Chronically Uremic (N)= single kidney remnant weight; Sham-operated (S) = weight of two k i d n e y s . Values are mean • SEM. Numbers in parenthesis indicate numbers of animals. Diets containing % protein/ % carbohydrate/ % fat: C22=22/59/10; L 1 6 / 6 5 / 1 0 ; L12=12/69/10; L8ffi8/73/lO. TABLE 4

Muscle Protein Degradation and Synthesis in Pairs of Atria from Chronically Uremic (N) and Sham-operated (S) Rats Fed Either a Low Protein (LI6, LI2, L8) or Control (22) Diet for Three Weeks 1,2,3 Diet Groups

C22

LI6

ATRIA Degradation 0.447 0.468 0.508 0.541 (Tyros• • • • • released (22) (27) (15) (18) nmollm$/2n) ns ns Synthesis (Tyrosine incorp. nmol/mg/2n

1 2 3

0.125 0.140 0.168 0.171 • • • • (22) (27) (15) (18) ns ns

LI2

0.389 •

L8

0.431 •

0.419 •

(5) (6) p <0.05 0.125 • (5)

0.157 •

(6)

ns

(iO) (II) p < 0.05

0.119 •

0.405 •

0.130 •

(I0) (II) p <.05

Values a r e mean • SEN. Numbers in p a r e n t h e s i s i n d i c a t e numbers o f a n i m a l s . Diets contained % protein/ % carbohydrate/ % fat: C22=22/59/10; L16=16/65/10: L12=12/69/10; L8=8/73/10; D i f f e r e n c e s between means in th e same d i e t a r y group a r e i n d i c a t e d .

DIETARY PROTEIN IN UREMIA

73

Within each d i e t group, t h e r e were no s i g n i f i c a n t d i f f e r e n c e s in p r o t e i n degradation between the N and S rats at any dietary protein level. Among the different dietary groups, statistical analysis i n d i c a t e d no s i g n i f i c a n t d i f f e r e n c e s in p r o t e i n d e g r a d a t i o n even though d e g r a d a t i o n a v e r a g e d 22% lower in L8 f e d r a t s compared t o r a t s f e d t h e L16 d i e t and was s l i g h t l y r ed u ced 10% compared t o t h e c o n t r o l f e d , C22 g r o u p s . P r o t e i n s y n t h e s i s in a t r i a a t t h e 16% d i e t a r y p r o t e i n l e v e l was s i g n i f i c a n t l y g r e a t e r than t h a t a t t h e 22 and 12% p r o t e i n l e v e l s in both N and S group% (p < 0 . 0 0 1 ) . In n e p h r e c t o m i z e d r a t s f e d t h e 8% p r o t e i n d i e t (L8N), a t r i a l p r o t e i n s y n t h e s i s was s i g n i f i c a n t l y g r e a t e r than in th e L8 S r a t s (p< 0 . 0 5 ) , n o t s i g n i f i c a n t l y d i f f e r e n t than L16N and s i g n i f i c a n t l y g r e a t e r than C22N or L12N (p 0.01) i n d i c a t i n g t h a t a t this lower l e v e l o f d i e t a r y p r o t e i n muscle p r o t e i n s y n t h e s i s was n o t compromised in t h e h e a r t . Oxygen consumption, expressed as nanomoles 02 consumedlmin/m8 mitochondrial protein in state 3 and state 43 utilizing the pyridine-linked substrate pyruvate plus malate are shown in the upper portion of Table 5. Hepatic mitochondrial respiration in state 3 was similar in all N groups. Among the S groups, liver mitochondria isolated from rats fed the Lg diet consumed significantly less 02 than those fed the C22 (p< 0.005) and LI2 (p< 0.05) diets. With the exception of rats fed the L8 diet, the mean state 3 respiratory rate tended to be lower in the N compared to the S rats within the various dietary groups; however, the only significant difference noted was between the N and S group fed the 22% protein diet, C22 (p< 0.05). In state 4, no constant trends were observed between the N and S pairs within each dietary group. Group L8N comsumed significantly more 02 than N animals fed the LI6 and LI2 diets (p<0.005). Mitochondrial respiration in states 3 and 4 utilizing the flavin-linked substrate succinate is shown in the lower half of Table 5. In general the N rats within each dietary group had slightly lower rates of 02 consumption in states 3 and 4 than the S groups; however, these differences were not statistically significant. Oxygen consumption in state 4 was similar for all N rats. Oxygen consumption of the C22S rats was significantly increased when compared to LI6S or L8S. Oxygen consumption in Lgs was significantly less than LI2S (p< 0.01).

In Figure 1, the activity of the reconstituted mitochondrial malate-aspartate shuttle system (top panel) glycerol phosphate shuttle system (middle panel), and serum T 3 concentrations (bottom panel) are plotted a g a i n s t the v a r i o u s d i e t a r y r e g i m e n s . The most dramatic changes occurred at the 8% dietary protein level, where serum T 3 concentrations were significantly increased in both S and N rats. Similarly the activity of the GP shuttle in both these N and S animals are also at its highest, whereas the activity of the MA shuttle in both groups was at its lowest rate. In the L8 group, GP shuttle activity in mitochondria from the N rats was significantly greater than that of the S animals (p<0.05). DISCUSSION

On the basis of studies in a chronic uremic rat model, which "exhibited changes of thyroid function typical of man with chronic renal failure," Lim et al. ( 1 4 ) reported decreased total thyroxine (T4) serum concentrations, markedly reduced total triiodothyronine.

R.S. TYZBIR et al.

74

TABLE 5 Nitochondrial Respiration in State 3 and 41 Expressed as nmoles of Oxygen Consumed/min/nig Mitochondrial Protein Using Pyruvate plus Malate 2 of S u c c i n a t e 3 as S u b s t r a t e in Chronically U r e m i c (N) a n d Sham O p e r a t e d (S) R a t s Fed E i t h e r a Low P r o t e i n ( L I 6 , L12, L8, F8) o r C o n t r o l (C22) D i e t f o r T h r e e Weeks 4 , 5

Diet Groups

C22

L12

LI6

L8

Substrates p~ruvate + Malate State 3

State 4

20.21 24.77 • • (14) (11) p< 0.05 4.93 • (14)

19.53 • (5)

21.38 •

(6)

18.15 • (6)

n8

20.73 • (7)

ns

ns

4.73 3.00 +__0.30 • (I0) (5)

22.39 • (7)

4.58 •

(6)

3.75 +-0.20 (6)

16.96 • (9) ns

3.61 +0.26 (7)

6.01 • (7)

ns

ns

4.08 +_0.45 (8) ns

Succinate

State 3

46.65 • (13)

54.57 • (13)

44.08 • (5)

ns State 4

12.02 +-0.76 (13)

55.48 • (5) ns

12.59 9.66 +-0.57 • (13) (5) ns

9.03 • (5) ns

42.18 +_4.9 (6)

53.04 +._3.4 (7) ns

Ii. I0 13.71 +--0.91 • (6) (7) ns

44.10 +._4.4 (9)

45.81 • (9) ns

9.73 •

9.91 •

(9)

(9) ns

State 3 = respiration i n t h e p r e s e n c e o f an a c c e p t o r s y s t e m (ADP); State 4 = respiration a f t e r a d d e d ADP h a s b e e n c o n v e r t e d t o ATP. I0 nM pyruvate plus 1.0 nM malate. 3.3 mM succlnate plus 1 ug/ml rotenone. V a l u e s a r e mean • SEN. Numbers i n p a r e n t h e s i s indicate numbers of anlmals. Diets contained % protein/ % carbohydrate/ % f a t : C22 2 2 / 5 9 / 1 0 ; L16= 1 6 / 6 5 / 1 0 ; L 1 2 = 1 2 / 6 9 / 1 0 ; L 8 = 8 / 7 3 / 1 0 . (T 3) c o n c e n t r a t i o n s , normal or slightly r e d u c e d s e r u m TSH l e v e l s and significantly decreased hepatic T3 c o n t e n t in the uremic rat. In these studies, both the uremic and sham-operated, control rats were fed unrestricted amounts of "regular chow" d i e t containing a "normal" protein concentration f o r 4 - 6 weeks a f t e r 5 / 6 n e p h r e c t o m y o r sham operation. Hepatic mitochondrial ~-glycerophosphate dehydrogenase (~-GPDH) a c t i v i t y was r e d u c e d i n t h e u r e m i c g r o u p b e g i n n i n g a t 4 weeks post nephrectomy indicating a biochemical response to the altered hormonal status. If the development of a reduction in hepatic T3 content could be modified or prevented by diet manipulation without seriously affecting other metabolic parameters, one of the potentially serious consequences of chronic renal insufficiency might be avoided.

DIETARY PROTEIN IN UREMIA

40

6 O=

,

75

p

M - A Shuttle

30

E r,,-., <[

z

20

IC I

o

o

%%%

I

I

I

G P - DHAP Shuttle

/I /#

J

I0

Z

=E I

250

I

i

J

"r3

7

2OC

.... I

22% Diet-

I

16% Cosein Content

j,' I

J

12% ....

8% Nephr~'niztKI

Shem

FIGURE 1

The activity of the reconstituted malate-asparate shuttle, glycerol phosphate shuttle and serum T 3 related to vacious dietary regimens.

76

R.S. TYZBIR et al.

T h i s p r o b l e m was i n i t i a l l y addressed, in part, in a previous study from our laboratory (I) utilizing partially n e p h r e c t o m i z e d and s h a m - o p e r a t e d r a t s fed either C22 o r L8 d i e t a t s i m i l a r caloric levels. A f t e r s i x weeks o f feeding, t h e s e r u m T3 c o n c e n t r a t i o n s o f b o t h sham a n d n e p h r e c t o m i z e d r a t s f e d t h e L8 d i e t was s i g n i f i c a n t l y greater than that in the sham-operated rats pair-fed t h e C22 d i e t . The a c t i v i t y of both mitochondrial a-GPDH and cytosolic m a l i c enzyme, p r e s u m p t i v e indices of thyroid hormone function in liver, was e l e v a t e d in the rats f e d t h e 8% p r o t e i n diet. Moreover the increased activity of the reconstituted GP s h u t t l e was significantly correlated with increased serum T3 concentrations in these older chronically uremic rats. This observation, t h a t f e e d i n g a low p r o t e i n - h i g h carbohydrate diet caused a hormone-mediated change in hepatic mitochondrial function, i s c o n f i r m e d a n d e x t e n d e d by t h e r e s u l t s of the present study. The increase i n s e r u m T3 a n d h e p a t i c GP s h u t t l e activity seen in uremic rats f e d t h e L8 d i e t occurred as e a r l y as t h r e e weeks o f f e e d i n g and are a c c c o m p a n i e d by a c o n c o m i t a n t d e c r e a s e i n t h e a c t i v i t y o f NA s h u t t l e . These changes appear to be independent of total energy intake and a r e not significantly a u g m e n t e d o r r e d u c e d by c h r o n i c uremia. The i m p o r t a n c e o f formulating a diet of precise composition to induce the hormone-mediated alteration in hepatic mitochondrial shuttle activity is well illustrated in F i g u r e 1. Manipulations o f L8 d i e t w h i c h i n c r e a s e d i e t a r y protein content iso-energetically at the expense of carbohydrate r e d u c e b o t h s e r u m T3 a n d hepatic GP shuttle activity toward control level regardless of renal status. The increased activity of the unidirectional GP shuttle, in light of the decreased activity of the reversible NA shuttle, in L8 fed rats suggests that the GP shuttle may function in the control of both aerobic glycolysis and the balance between lipogenesis and lypolysis similar to its putative regulatory f u n c t i o n i n t i s s u e s w i t h u n u s u a l l y h i g h GP s h u t t l e activity (15,16). In the liver of rats f e d t h e low p r o t e i n - h i g h carbohydrate (L8) d i e t , continuous glycolytic activity would require an a d e q u a t e c y t o p l a s m i c concentration of NAD m a i n t a i n e d by t h e GP s h u t t l e . The s u b s e q u e n t i n c r e a s e i n t h e c y t o p l a s m i c NAD/NADH r a t i o f a c i l i t a t e s carbohydrate degradation and prevents resynthesis of glycogen. In liver, glycogen depletion is a well documented characteristic of the hyperthyroid state. In addition, utilizing cytoplasmic glycerol-3-phosphate in the shuttle, would limit its availability as a substrate for triglyceride formation. The nonesterified fatty acyl-CoA collecting in the cytoplasm as a result of reduced triglyceide formation could negatively modulate lipogenesis by inhibiting acety-CoA carboxylase activity (17). In in vitro studies, Harter et al. (18) reported an i n c r e a s e in net protein degradation in epitrochlearis muscle isolated from partially n e p h r e c t o m i z e d r a t s w h i c h was g r e a t e s t i n t h e u r e m i c r a t s f e d a 10% c a s e i n diet. The a d d i t i o n o f i n s u l i n t o t h e i n c u b a t i o n m e d i a d i d n o t c h a n g e p r o t e i n degradation rates in tissue from nephrectomized rats to control rats. In our study, reducing the concentration of dietary protein f r o m 22 t o 8% w h i l e maintaining the energy content of the diets constant resulted in a slight but statistically insignificant decrease in atrial muscle protein degradation while protein synthesis was s l g n i f i c a n t l y increased in the uremic, low protein fed rats. However, t h i s p a t t e r n of cardiac muscle protein turnover may n o t n e c e s s a r i l y reflect what is occurring in skeletal muscles elsewhere i n he body ( 1 9 ) . F u r t h e r m o r e , o u r i n c u b a t i o n medium d i d n o t c o n t a i n i n s u l i n b u t was s u p p l e m e n t e d w i t h b o t h p h e n y l a l a n i n e and l e u c i n e , the latter amino a c i d w e l l known t o r e d u c e n e t p r o t e i n degradation under these in vitro conditions. Thus, differing experimental conditions, especially the absence of leucine i n t h e m e d i a , may p o s s i b l y account for the differences in net protein degradation b e t w e e n t h e two t i s s u e s . Diet composition, particularly lowering dietary protein content to a level sufficient t o c a u s e an i n c r e a s e i n s e r u m T3 l e v e l may b e a n o t h e r reason for the differences in protein

DIETARY PROTEIN IN UREMIA

77

degradation rates. A reduction in muscle protein breakdown, measured as reduced N-methylhistidine excretion, with increased plasma T 3 levels has recently been reported in rats fed a low protein diet but not in rats maintained on a moderate dietary energy deficiency (20). It is noteworthy that the 8% protein level is also the lowest concentration o f dietary protein which when fed to female rats will support reproductive capacity while insuring the survival of the offspring (21). One may speculate from an evolutionary viewpoint that the 8% protein-high carbohydrate diet may represent the critical macronutrient content capable of inducing alterations in thyroid hormone levels and hepatic intermediary metabolism to insure species survival in times of protein scarcity.

These r e s u l t s i n d i c a t e t h a t in young, m o d e r a t e l y uremic r a t s , r e d u c i n g d i e t a r y p r o t e i n from 22% t o 8%, m a i n t a i n i n g t h e f a t c o n t e n t a t 10~ and a d j u s t i n g the carbohydrate to maintain isoenergetic c o n s t a n c y does n o t s e r i o u s l y a f f e c t r e n a l f u n c t i o n , h e p a t i c m i t o c h o n d r i a l r e s p i r a t i o n or c a r d i a c muscle p r o t e i n t u r n o v e r . However, body w e i g h t g a i n was s l i g h t l y d e p r e s s e d in the low protein fed rats, regardless of renal status. Thus, if T3 stimulation of hepatic mitochondrial GP shuttle activity induced by feeding the 8% protein diet is significant in the amelioration of uremic symptoms, it occurs at an overall systemic metabolic cost which is probably unrelated to the partial renal insufficiency. Serum T 3 level and GP shuttle activity were not altered in uremic or sham-operated rats fed an 8% casein, 59% carbohydrate, 24% fat diet (Tyzbir and Kunin, unpublished data) indicating that the diet induced changes reported here (Figure 1) specifically resulted from feeding an 8% protein - high carbohydrate but not high fat diet. In summary, our results suggest that the diet-induced, thyroid hormone mediated alteration in hepatic mitochondrial intermediary metabolism may prove beneflcial in avoiding one potentially serious consequence of chronic renal sufficiency. ACKNOWLEDGEMENTS A preliminary report of part of this work was presented at the Second International Congress on Nutrition in Renal Disease, Bologna, Italy, June 13-15, 1979, and is briefly summarized Am J Clin Nutr 33:1451-1455, 1980. These studies were supported in part by an institutional grant from the Graduate College and by the Department of Medicine, University of Vermont, Burlington, ArT, and by a contract with the U.S. Army Medical Research and Development command. The views, opinions and/or findings contained herein are those of the authors and should not be construed as official or as reflecting the views of the Department of the Army of the Department of Defense. The authors acknowledge the helpful suggestions of Drs. Alfred L. Goldberg of Harvard Medical School, Eliot Danforth and William L. Meyer of the University of Vermont College of Medicine. REFERENCES I.

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for

protein

determination.

Scand

J

Clin

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plasma

Thyroid function in a hypothyroidism. J Clin

synthesis

of

saturated

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Accepted for publication October I, 1984