Albumin synthesis in cultured hepatoma cells

90

Biochimica et Biophysica Acta, 475 (1977) 90--95 © Elsevier/North-Holland Biomedical Press

BBA 98869

ALBUMIN SYNTHESIS IN C U L T U R E D HEPATOMA CELLS R E G U L A T I O N BY ESSENTIAL AMINO ACIDS

BARRY E. LEDFORD, RICHARD W. WARNER and ROBERT A. COCHRAN Department o f Biochemistry, Medical University of South Carolina, 80 Barre Street, Charleston, S.C. 29401 (U.S.A.) (Received September 30th, 1976)

Summary The effects of essential amino acids on albumin synthesis b y a mouse hepat o m a cell line have been investigated. The amino acids tested were tryptophan, phenylalanine, histidine, isoleucine and leucine. Cellular rates of synthesis (molecules albumin/cell per min) were determined from rates o f [3H] leucine incorporation into immunoprecipitable albumin in the culture medium. The effects of amino acids on albumin synthesis fall into three distinct groups. The concentration of t r y p t o p h a n producing half-maximal synthesis is 4 pM. The corresponding concentration for leucine is 100 pM. Histidine, phenylalanine and isoleucine were very similar, the half-maximal concentrations being approximately 15/~M. The concentrations of amino acids producing half-maximal synthesis correlate directly with the amino acid composition of albumin. The levels o f these essential amino acids necessary to saturate albumin synthesis have been compared with amino acid levels in normal plasma.

Introduction Albumin is the major plasma protein in mammals. It appears to serve three distinct functions in the plasma. First, due to its size, charge and concentration, albumin contributes disproportionately to the colloid osmotic pressure of the plasma. Secondly, albumin binds a broad spectrum of metabolites and metabolic effectors and thereby serves a transport function. Finally, albumin appears to serve as a storage form for excess amino acids, perhaps analogous to glycogen and triglyceride reserves of glucose and fatty acids. Albumin metabolism, structure and function have been reviewed b y Peters [1], Rothschild et al. [2] and Munro [3]. A relationship b e t w e e n circulating levels of albumin and dietary intake of

91 protein has been clearly established in humans [4,5] and in experimental animals [6--12]. Protein
92

Albumin was immunoprecipitated from each sample by: (1) the addition of 0.7 ml 0.9% NaC1, 0.1 ml rabbit anti-mouse albumin and 20 pgm mouse albumin; (2) incubation ay 37°C for 2 h, then overnight at 4°C. Precipitates were washed 5 times in 2.0 ml 0.9% NaC1. The final pellet was dissolved in 1.0 ml 0.1 M NaOH, mixed with scintillation cocktail and counted. Results The rate of albumin secretion b y Hepa was determined from the rate of leucine incorporation into secreted albumin. Fig. 1 shows leucine incorporation into albumin at different concentrations of tryptophan. The data points were adjusted to compensate for differences in the fraction of the total volume sampled and for activity removed in previous samples. The slope of the regression fit was used to calculate the cellular rate of albumin secretion. The cell n u m b e r was determined at the end of the incorporation period. The specific activity of leucine in the culture medium was determined directly b y counting a sample of the medium which contains a known amount (30 mg/1) of leucine. Since albumin contains 64 leucine residues, it is then possible to calculate the rate of albumin secretion in terms of molecules of albumin/ceU per min. These numbers vary somewhat from experiment to experiment depending upon growth rate. F o r this reason the data from each experiment have been normalized to percentage of maximum secretion rate.

1400 1200

10001 3E 0

800 Q

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4 (hrs)

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Fig. 1. E f f e c t o f t r y p t o p h a n o n a l b u m i n s e c r e t i o n b y H e p a cells. I n c o r p o r a t i o n o f [ 3 H ] l e u c i n e i n t o i m m u n o p r e c i p i t a b l e a l b u m i n s e c r e t e d into t h e c u l t u r e m e d i u m . A l i q u o t s ( 2 5 /~I) o f t h e c u l t u r e m e d i u m ( 1 . 1 m l ) w e r e t a k e n at t h e i n d i c a t e d t i m e s a f t e r a d d i t i o n o f [ 3 H ] l e u c i n e . A l i q u o t s w e r e d i l u t e d w i t h 0.7 m l 0.9% NaCL A l b u m i n w a s i m m u n o p r e c i p i t a t e d b y a d d i t i o n o f 0.1 m l rabbit a n t i - m o u s e a l b u m i n a n d 2 0 Mgm m o u s e a l b u m i n . P r e c i p i t a t e s w e r e w a s h e d five t i m e s w i t h 2 . 0 m l 0 . 9 % NaCL T r y p t o p h a n c o n c e n trations: ~ e, 49 # M ; A •, 16.3 # M ; ~ . n, 5 . 0 / ~ M ; o o, 2.0 ~M.

93

i

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6 8 I0 40 6080100 AMINO ACID CONCENTRATION (/dM)

Fig. 2. E f f e c t s o f essential a m i n o acids o n t h e r e l a t i v e r a t e o f a l b u m i n s e c r e t i o n b y H e p a cells. T h e cellular rate of a l b u m i n secretion (molecules album/n/cell per rain) was d e t e r m i n e d f r o m rates of [3H]leucine i n c o r p o r a t i o n i n t o i m m u n o p r e c i p i t a h l e a l b u m i n in the culture m e d i u m as s h o w n in Fig. 1. All v a l u e s w e r e n o r m a l i z e d as a p e r c e n t a g e o f t h e m a x i m u m secretion rate o b s e r v e d w i t h e a c h a m i n o acid. The s t a n d a r d e r r o r o f each point w a s c a l c u l a t e d f r o m t h e r e g r e s s i o n fit o f t h e i n c o r p o r a t i o n d a t a . ~ o, tryptophan; = ~, p h e n y l a l a n i n e , ~ -A histidine; • •, isoleucine; o o, leucine.

The normalized rates of albumin synthesis as a function of amino acid concentrations are shown in Fig. 2. The concentration of t r y p t o p h a n producing a half-maximal rate of albumin secretion is approximately 4/~M. At the other extreme, the leucine concentration producing a half-maximal rate of albumin secretion is 100/~M. Results using three other essential amino acids (isoleucine, histidine and phenylalanine) are very similar, showing half-maximal concentrations around 15 pM. Leucine was used as the label in all of these experiments. The concentration of leucine in the culture media o f experiments involving amino acids other than leucine was 229/~M. This leucine concentration was chosen to maximize the rate of albumin synthesis and the specific activity of leucine in the culture media. Leucine dependence, however, was measured using media o f constant activity and varying specific activities. This proved to be particularly useful since the sensitivity of the method increased as the rate of synthesis decreased. This is reflected in the small standard errors associated with the points on t h e leucine dependence curve. Discussion The relationship b e t w e e n dietary intake of protein and the rate o f albumin synthesis b y the liver has been established in intact animals. This is consistent with the hypothesis that one of the physiological functions o f albumin is to serve as a reserve o f amino acids. The mechanism by which albumin synthesis responds to a variable supply of amino acids has been difficult to establish.

94 TABLE I AMINO ACID COMPOSITION OF MOUSE PLASMA AND MOUSE SERUM ALBUMIN Amino acid

P l a s m a c o n c e n t r a t i o n (#M) [ 2 1 ]

Residues/molecule albumin [22]

Tryptophan Histidine Isoleucine Phenylalanine Leucine

60 101 112 145 183

1 14 14 28 64

(54--69) (90--110) (92--153) (121--194) (168--214)

Some experiments have suggested that t r y p t o p h a n plays a specific role in regulating albumin synthesis [21--24]. The cell line Hepa, which retains a number of liver-specific functions, provided an o p p o r t u n i t y to study, in a well defined environment, the effects of amino acids on albumin synthesis. These experiments show that the five essential amino acids tested fall into three discrete groups. Tryptophan saturation of albumin synthesis occurs at the lowest concentration, whereas leucine saturation occurs at a much higher concentration. The remaining amino acids (histidine, isoleucine and phenylalaine) cluster together in an intermediate group. The physiological significance of these groupings can only be understood by comparison with measured plasma amino acid concentrations in the mouse. Table I shows normal mouse plasma levels of the tested amino acids. With the exception of t r y p t o p h a n these values lie very close to the saturation level. It is apparent that any decrease in the plasma concentrations of leucine, isoleucine, histidine or phenylalanine will reduce the rate of albumin synthesis. The plasma concentration of tryptophan, however, is approximately five-fold greater than the saturating level of tryptophan. Thus, t r y p t o p h a n would only limit albumin synthesis if its concentration decreased several fold. It is entirely possible that t r y p t o p h a n does become specifically limiting in the fasted animal. A direct measurement of plasma amino acid levels in fasted animals would then provide essential information. Table I also includes information regarding the amino acid composition of albumin. It is obvious that these five amino acids can be divided into three distinct groups. Albumin is rich in leucine, poor in t r y p t o p h a n and contains intermediate amounts of histidine, isoleucine and phenylalanine. This division correlates directly with the amino acid dependence of albumin synthesis shown in Fig. 2. In fact, the relative values are also in good agreement. This indicates that the relationship b e t w e e n amino acid supply and albumin synthesis is determined primarily b y the amino acid composition of albumin. Acknowledgements The assistance of Judith C. Rankin in the preparation of this manuscript is gratefully acknowledged. This w o r k was supported b y a grant from the NIH (CA 17037).

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