Summary
Supplementation of Ham's F12 culture medium with essential amino acids (EAA) up to the rat plasma levels increased the rates of synthesis of albumin and transferrin by cultured rat hepatocytes by 1.3 and 1.7, respectively. Fifty percent of this increase could be attributed to three of the EAA: the branched-chain amino acids (BCAA: Leu Ile and Val). Non-branched-chain essential amino acids (non-BC-EAA) stimulated only 25% of the increase produced by the whole EAA mixture. When each EAA was tested individually, none of them caused an appreciable increase in albumin and transferrin in culture medium. When the concentrations of all EAA were raised to rat postprandial portal levels, albumin and transferrin synthesis rates reached a maximum, increasing by 3.2 and 3.5, respectively. Supplementation with BCAA at postprandial portal concentrations increased albumin and transferrin synthesis rates by 2.2 and 2.0, respectively, and had no noteworthy effect on the synthesis of cellular proteins. Non-BC-EAA at their postprandial portal concentrations increased albumin and transferrin synthesis rates by 1.7 and 1.9, respectively. Supplementation with alanine to reach a nitrogen content equal to that of the modified EAA-enriched medium had no stimulatory effect. Our results show that EAA have a specific effect on the synthesis of plasma proteins by cultured hepatocytes, and that BCAA at physiologic concentrations account for the major part of this stimulatory effect. Consequently, EAA and particularly BCAA concentration should be elevated in serum-free nutrient media to sustain maximum plasma protein synthesis.
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Berry, M. N.; Friend, D. S. High yield preparation of isolated rat liver parenchymal cells. J. Cell Biol. 43:506–520; 1969.
Castell, J. V.; Gómez-Lechón, M. J.; Coloma, J., et al. Preservation of adult functionality of hepatocytes in serum-free cultures. In: Fischer, G.; Wieser, R. J., eds. Hormonally defined media. A tool in cell biology. Berlin: Springer Verlag; 1984:333–336.
Castell, J. V.; Montoya, A.; Larrauri, A., et al. Effects of benorylate and impacina on the metabolism of cultured hepatocytes. Xenobiotica 15:743–749; 1985.
Gómez-Lechón, M. J.; Castell, J. V. Efficiency of phylogenetically unrelated plasma fibronectins on hepatocyte attachment to substrata. Cienc. Biol. 8:49–56; 1983.
Gómez-Lechón, M. J.; López, M. P.; Castell, J. V. Biochemical functionality and recovery of hepatocytes after deep freezing storage. In Vitro 20:826–832; 1984.
Grinde, B.; Seglen, P. O. Leucine inhibition of autophagic vacuole formation in isolated rat hepatocytes. Exp. Cell Res. 134:33–39; 1981.
Harper, A. E.; Miller, R. H.; Block, K. P. Branched-chain amino acid metabolism. Annu. Rev. Nutr. 4:409–454; 1984.
Ichiara, A.; Nakamura, T.; Tanaka, K. Use of hepatocytes in primary culture for biochemical studies on liver functions. Mol. Cell. Biochem. 43:145–160; 1982.
Ledford, B. E.; Jacobs, D. F. Translation kinetics in cultured mouse hepatoma cells. Regulation of albumin synthesis by amino acids. Eur. J. Biochem. 152:611–618; 1985.
Lowry, P. H.; Rosebrough, N. J.; Farr, A. L., et al. Protein measurement with Folin phenol reagent. J. Biol. Chem. 193:265–275; 1951.
Maher, T. H. Plasma branched chain amino acids as regulators of brain neurotransmitters. In: Adibi, S. A.; Fekl, W.; Langenbeck, U., et al., eds. Branched chain amino and keto acids in health and disease. Basel: Karger; 1984:242–259.
Montoya, A.; Gómez-Lechón, M. J.; Castell, J. V. Plasma protein synthesis by serum-free cultured hepatocytes. Role of amino acid composition of the medium. In Vitro 21:26A; 1985.
Mortimore, G. E.; Pösö, A. R. Lysosomal pathways in hepatic protein degradation: regulatory role of amino acids. Fed. Proc. 43:1289–1294; 1984.
Rémésy, C.; Demigne, C.; Aufrére, J. Inter-organ relationships between glucose, lactate and amino acids in rats fed on highcarbohydrate or high-protein diet. Biochem. J. 170:321–329; 1978.
Schwarze, P. E.; Seglen, P. O. Protein metabolism and survival of rats hepatocytes in early culture. Exp. Cell Res. 130:185–190; 1980.
Schwarze, P. E.; Solheim, A. E.; Seglen, P. O. Amino acid and energy requirements for rat hepatocytes in primary culture. In Vitro 18:43–54; 1982.
Seglen, P. O. Protein-catabolic state of isolated rat hepatocytes. Biochim. Biophys. Acta. 496:182–191; 1977.
Seglen, P. O.; Gordon, P. B.; Poli, A. Amino acid inhibition of the autophagic/lysosomal pathway of protein degradation in isolated rat hepatocytes. Biochim. Biophys. Acta. 630:103–118; 1980.
Seglen, P. O.; Solheim, A. E. Effects of aminooxyacetate, alanine and other amino acids on protein synthesis in isolated rat hepatocytes. Biochim. Biophys. Acta. 520:630–641; 1978.
Shotwell, M. A.; Kilberg, M. S.; Oxender, D. L. The regulation of neutral amino acid transport in mammalian cells. Biochim. Biophys. Acta. 737:267–284; 1983.
Smith, J. E.; Lunn, P. G. Albumin-synthesizing capacity of hepatocytes isolated from rats fed diets differing in protein and energy content. Ann. Nutr. Metab. 28:281–287; 1984.
Tanaka, K.; Ichiara, A. Different effects of amino acid deprivation on synthesis of intracellular and extracellular proteins in rat hepatocytes in primary culture. J. Biochem. 94:1339–1348; 1983.
Tanaka, K.; Kishi, K.; Ichiara, A. Biochemical studies on liver functions in primary cultured hepatocytes of adult rats. II. Regulation of protein and amino acid metabolism. J. Biochem. 86:863–870; 1979.
Weigand, K.; Wernze, H.; Falge, C. Synthesis of angiotensinogen by isolated rat liver cells and its regulation in comparison to serum-albumin. Biochem. Biophys. Res. Com. 75:102–110; 1977.
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Montoya, A., Gómez-Lechón, M.J. & Castell, J.V. Influence of branched-chain amino acid composition of culture media on the synthesis of plasma proteins by serum-free cultured rat hepatocytes. In Vitro Cell Dev Biol 25, 358–364 (1989). https://doi.org/10.1007/BF02624599
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DOI: https://doi.org/10.1007/BF02624599