Amino acid transport was studied in primary cultures of parenchymal cells isolated from adult rat liver by a collagenase perfusion technique and maintained as a monolayer in a serum-free culture medium. These cells carried out gluconeogenesis from three carbon precursors (alanine, pyruvate, and lactate) in response to glucagon addition. Amino acid transport was assayed by measuring the uptake of the nonmetabolizable amino acid, alpha-aminoisobutyric acid (AIB). Addition of insulin or glucagon to culture rat liver parenchymal cells resulted in an increased influx of AIB transport. The glucocorticoid, dexamethasone, when added alone to cultures did not affect AIB transport. However, prior or simultaneous addition of dexamethasone to glucagon-treated cells caused a strong potentiation of the glucagon induction of AIB transport. Kinetic analysis of the effects of insulin and glucagon demonstrated that insulin increased the Vmax for transport without changing the Km while glucagon primarily decreased the Km for AIB transport. The effect of dexamethasone was to increase the Vmax of the low Km system.
Liver parenchymal cells were isolated from adult rats and cultured in collagen-coated plastic petri dishes in serum-free medium. Glucagon induced 4 to 5-fold increases in a-aminoisobutyric acid (AIB) transport within 6 hr. Dexamethasone had no direct effect on AIB transport but greatly potentiated the induction by glucagon ("permissive effect"). Levels of 3':5'-cyclic AMP increased 30-to 100-fold within 30 min after glucagon addition to cultures that had been treated with dexamethasone, and dibutyryl cyclic AMP mimicked the glucagon induction of AIB transport. Additionally, dexamethasone exerted a "permissive" effect on induction of AIB transport by dibutyryl cyclic AMP, whereas the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine induced AIB transport only in cultures that had been treated with dexamethasone. Induction of AI transport was not dependent upon the continued presence of glucagon, but induced AIB transport activity decayed to uninduced levels within 3-4 hr after glucagon removal. The protein synthesis inhibitors puromycin and cycloheximide inhibited both induction and decay of glucagon-induced AIB transport, but had a stabilizing effect if added once induction or decay had commenced. Unlike cycloheximide, the inhibitory effect of puromycin on the glucagon induction of AIB transport was reversible.Previous reports from this (1-4) and other (5-7) laboratories have established methodology for the isolation and primary culture of liver parenchymal cells from adult rats. These cells can be cultured in serum-free medium (2-4) and do not divide even in medium supplemented with serum (1, 2). However, various biochemical activities are inducible under appropriate conditions. Ornithine decarboxylase activity (EC 4.1.1.17) is induced by insulin but not by hydrocortisone (2), whereas dexamethasone (DEX), a synthetic glucocorticoid, will induce tyrosine aminotransferase (EC 2.6.1.5) activity (1, 2). While DEX does not by itself affect amino acid transport in this system (8), in contrast to results with liver in vivo (ref. 9; and see refs. 8 and 10), DEX greatly potentiates the induction of a-aminoisobutyric acid (AIB) transport by glucagon. This action was recognized as a "permissive" effect (8). Glucagon lowered the Km for transport, whereas DEX exerted the "permissive" effect by raising the Vmax (10). Thus, glucagon may induce the synthesis of a new amino acid transport system or effect an alter- (125-250 g) were used in this investigation. The methods for maintaining the rats on a controlled feeding and lighting schedule (2 + 22 schedule) as well as the diet (60% protein) have been described in detail elsewhere (12).Protocol. The specific protocol used in this investigation was as follows:(i) Cells were isolated by a modification of the collagenase perfusion technique of Berry and Friend (1-3, 6, 13) (yield: 300 to 600 X 106 cells per liver).(ii) Cells were suspended at 1 X 106 cells per ml in a modified Waymouth's MB 752/1 medium (WO/BA-M2, see refs. 3 and 4). WO/BA-M2 is serum-free, and was...
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