Molecular and functional analysis of glutamine uptake in human hepatoma and liver-derived cells

Human hepatoma cells take up glutamine at rates severalfold faster than the system N-mediated transport rates observed in normal human hepatocytes. Amino acid inhibition, kinetic, Northern blotting, RT-PCR, and restriction enzyme analyses collectively identified the transporter responsible in six human hepatoma cell lines as amino acid transporter B(0) (ATB(0)), the human ortholog of rodent ASCT2. The majority of glutamine uptake in liver fibroblasts and an immortalized human liver epithelial cell line (THLE-5B) was also mediated by ATB(0). The 2.9-kb ATB(0) mRNA was equally expressed in all cell lines, whereas expression of the system A transporters ATA2 and ATA3 was variable. In contrast, the system N isoforms (SN1 and SN2) were expressed only in well-differentiated hepatomas. ATB(0) mRNA was also expressed in cirrhotic liver and adult and pediatric liver cancer biopsies but was not detectable in isolated human hepatocytes or fetal liver. Although the growth of all hepatomas was glutamine dependent, competitive inhibition of ATB(0)-mediated glutamine uptake blocked proliferation only in poorly differentiated cells lacking SN1 or SN2 expression and exhibiting low glutamine synthetase mRNA levels.     

Epinephrine regulation of amino acid transport in rat hepatocytes isolated during development

The effect of epinephrine on the amino acid transport mediated by system A was investigated by determining the uptake of 2-amino [1-14C]isobutyric acid (AIB) in rat hepatocytes, freshly isolated at different stages of pre- and postnatal development. The data obtained show that the hormone increased AIB uptake, enhancing the Vmax, while Km was unchanged. This effect was evident in cells from adult, 18- to 20-day-old fetus, and neonate rat. Actinomycin D or cycloheximide abolished the hormone dependent increase. Experiments carried out with alpha- and beta-antagonists showed that the effect of epinephrine was beta-mediated in fetal life and alpha-mediated in adult life. Membrane binding experiments showed a higher value for epinephrine and beta-agonist dihydroalprenolol in the fetus versus the adult. The calcium depletion obtained after cell incubation with EGTA or calcium ionophore A23187 reduced the hormonal stimulation in the adult, and was ineffective in the prenatal period. An involvement of cAMP was present in the epinephrine modulation of AIB transport, both in adult and in fetal life.     

Different signal transduction by epidermal growth factor may be responsible for the difference in modulation of amino acid transport between fetal and adult hepatocytes

[1-¹⁴C]-2-aminoisobutyric acid (AIB) uptake and signal transduction pattern after epidermal growth factor (EGF) stimulation were examined in freshly isolated hepatocytes from 20-day-old fetuses and 3-month-old rats. EGF induced a transient increase of AIB transport after 10 min only in adult animals; the observed unresponsiveness of fetal liver is not dependent on a lack of EGF receptors which are present though to a lesser extent on the plasma membrane in this period. As far as the production of the second messengers, inositol trisphosphate (IP₃) and calcium, is concerned, substantial differences were found: EGF increased IP₃ production in adult hepatocytes, whereas it had no effect in fetal ones. Moreover, the addition of EGF induced a calcium transient in hepatocytes from adult animals, while there was no increase in fetal cells. The lack of EGF effect on amino acid transport in fetal cells could be due to its inability to produce both IP₃ and calcium transients, suggesting that this transduction pathway is not activated during fetal life.     

Comparison of system N in fetal hepatocytes and in related cell lines

In contrast to the changes seen in membrane transport systems for other neutral, anionic, and cationic amino acids, System N for glutamine, histidine, and asparagine in the rat hepatocytes shows nearly constant properties at the fetal, differentiated, and cultured hepatoma stages. These properties were tested by measuring the Na+-dependent transport of glutamine. This approximate constancy applies not only to the transport selectivity of the system among neutral amino acids, but also to its tolerance of Li+ as a substitute for Na+, its characteristic sensitivity to pH lowering, its relative sensitivity to N-ethylmaleimide, its stimulation by amino acid deprivation, and its failure to respond to insulin or glucagon. The properties of histidine as a substrate for System N were also examined. Inhibition studies with different cell types suggest that the Na+-dependent glutamine and histidine uptake is more restricted to System N in the hepatoma line H35 (H4-11-EC,3) and in the fetal hepatocyte than in hepatoma line HTC and the Ehrlich cells. The Na+-independent component of glutamine and histidine uptake was greater in the hepatoma cells in continuous culture than in fetal and adult hepatocytes in primary culture. Trans-stimulation of glutamine and histidine influx into H35 cells occurs predominantly by the Na+-independent route.     

The effects of bioregulators upon amino acid transport and protein synthesis in isolated rat hepatocytes

Isolated rat hepatocytes prepared by an enzyme perfusion technique possess a functional amino acid transport system and retain the capacity to synthesize protein. Amino acid transport was studied using the non-metabolizable amino acid analog alpha-aminoisobutyric acid. The transport process was time, temperature and concentration dependent. Similarly, leucine incorporation into protein was time and temperature dependent being optimal at 3m degrees C. Amino acid, fetal calf serum, growth hormone and glucose all produced small, reproducible increases in protein synthesis rates. Bovine serum albumin diminished the uptake of alpha-aminoisobutyric acid and leucine incorporation into protein. The amino acid content on either side of the cell membrane was found to affect transport into or out of the cellular compartment (transconcentration effects). High cell concentrations decreased transport and protein synthesis as a result of isotopic dilution of labelled amino acids with those released by the hepatocytes. This was consistent with the capacity of naturally occurring amino aicds to compete with alpha-aminoisobutyric acid for uptake into the hepatocyte. In order to define more precisely the effects of bioregulators on transport and protein synthesis it will be necessary to define and subfractionate cellular compartments and proteins which are the specific targets of cellular regulation.     

The effect of amiloride on hormonal regulation of amino acid transport in isolated and cultured adult rat hepatocytes

Insulin and glucagon stimulate amino acid transport in isolated rat hepatocytes. Amiloride, a specific Na+-influx inhibitor, completely inhibited the hormonal (glucagon or insulin) stimulation of alpha-aminoisobutyric acid influx by preventing the emergence of a high-affinity transport component. The drug also inhibited [14C]valine incorporation into hepatocyte protein. The half-maximal concentration of amiloride for inhibition of protein synthesis was similar to that required for inhibition of hormone-stimulated amino acid transport (approx. 0.1 mM). In primary cultured rat hepatocytes, amiloride markedly depressed the stimulation of alpha-aminoisobutyric acid transport by glucagon, or a mixture of glucagon, insulin and epidermal growth factor. These results suggest that amiloride inhibits the hormonal stimulation of hepatocyte amino acid transport by preventing the synthesis of high-affinity transport proteins. They also suggest that the hormonal stimulation of hepatocyte amino acid transport is dependent, at least partly, on Na+ influx.     

Amino acid transport in isolated rat hepatocytes

Summary Improvements in the collagenase perfusion techniques have made isolated rat hepatocytes a popular model in which to study hepatic function. Our knowledge of hepatic amino acid transport has been advanced as a result of this methodology. Translocation across the hepatocyte plasma membrane can, in some instances, represent the rate-limiting step in the overall metabolism of certain amino acids. Furthermore, regulation of amino acid uptake by hepatocytes appears to play a role in diabetes, and perhaps in malignant transformation. Comparisons between normal adult hepatocytes and several hepatoma cell lines show basic differences in amino acids transport. There are at least eight distinct systems in normal hepatocytes for transport of the amino acids. One of these, System A, transports the small neutral amino acids most efficiently and responds to a wide variety of hormones. Systems A and N exhibit enhanced uptake rates after the cells have been maintained in the absence of extracellular amino acids, a phenomenon termed adaptive control. Further studies using isolated hepatocytes will increase our basic understanding of membrane transport processes and their regulation.     

Regulation of neutral amino acid transport in hepatocytes isolated from adrenalectomized rats

The present report shows that System A-mediated 2-aminoisobutyric acid (AIB) uptake is elevated in hepatocytes isolated from adrenalectomized rats when they are compared to control cells. Although System ASC activity also shows this perturbation, Systems N, beta, L1, and L2 are unaffected. Transport of AIB in both cell types is stimulated by dexamethasone, insulin, and glucagon, yet the hepatocytes from the adrenalectomized rats are much less responsive to these hormones. This apparent decrease in competence is seen for adaptive regulation of System A as well. The in vitro addition of dexamethasone to the hepatocytes from the adrenalectomized animals does not restore fully their ability to respond to hormones or amino acid deprivation. These effects are observed even after the cells have been held in primary culture for 24 hr. The simultaneous addition of glucagon and dexamethasone to either cell type resulted in stimulation of transport to rates significantly greater than the sum of the increases produced by the two hormones when added separately. In contrast, insulin and dexamethasone were additive in their effects rather than synergistic. These results suggest that hepatocytes from adrenalectomized rats are less competent than control cells with respect to regulation of neutral amino acid transport, including stimulation by insulin or amino acid starvation, two processes which appear not to depend on glucocorticoid for maximal response.     

Inhibition of sodium-independent amino acid transport by dexamethasone in rat hepatoma cells

We studied the uptake of leucine, phenylalanine, and the amino acid analog, 2-aminonorborane-2-carboxylic acid, by rat hepatoma cells in tissue culture. The uptake of these amino acids was partially mediated by a plasma membrane transport system similar to the L agency described in other cell types in that it does not require extracellular sodium and is subject to trans-stimulation. Initial rates of sodium-independent transport of these amino acids were calculated using mathematical transformations of the uptake time course curves. The glucocorticoid dexamethasone inhibits the activity of this transport system; the initial rates of sodium-independent uptake of leucine, phenylalanine, and 2-aminonorborane-2-carboxylic acid are decreased by approximately one-third (average = 30%, n = 19) after incubation of HTC cells with 0.1 microM dexamethasone. This inhibition requires at least 15 h, reaching a maximum at 24 h of exposure of the cells to the hormone. Dexamethasone has an asymmetrical effect on sodium-independent amino acid transport in that exposure of the cells to the hormone does not inhibit the rates of outflow of leucine or phenylalanine from preloaded cells into medium without sodium. Inhibition of uptake is blocked by 0.1 mM cycloheximide and 4 microM actinomycin D, indicating the need for continuous protein synthesis for dexamethasone action. Insulin, which is known to partially reverse the inhibitory effect of dexamethasone on the A amino acid transport system in HTC cells, does not alter the action of dexamethasone on the L system. Previous investigations have demonstrated inhibition by dexamethasone of at least two distinct sodium-dependent amino acid transport activities in HTC cells. The data presented here, showing inhibition by the glucocorticoid of a sodium-independent transport activity, indicate that the effect of the hormone is independent of the energy source of the amino acid transport systems affected.     

Characterization of amino acid transport during erythroid cell differentiation

We have studied the changes in amino acid transport in fetal erythroid cells isolated from rat fetal liver at different gestation days. Our results show that System A transport as measured by the Na+-dependent uptake of 2-(methylamino)isobutyric acid (MeAIB) was conspicuous at day 13 but virtually disappeared between days 16 and 18. In contrast, the activity of System ASC measured by the Na+-dependent uptake of MeAIB-insensitive threonine uptake increased after day 14 and was optimal between days 16 and 18. This transport system regressed in activity with further maturation, but remained conspicuously saturable in the matured red blood cell. Interestingly, the newly discovered Na+-independent System asc (Vadgama, J. V., and Christensen, H.N. (1985) J. Biol. Chem. 260, 2912-2921), selective for the uptake of test substrates threonine, serine, and alanine, was present in these erythroid cells. Its activity increased during gestation days 16-18. System L transport was present simultaneously with the Na+-independent System asc. As we had previously demonstrated for the pigeon red blood cell, these two transport systems are kinetically independent as confirmed with inhibition studies and the special selectivity of System L to trans stimulation. Tryptophan uptake could be attributed predominantly to System L, as also observed for the nucleated pigeon red blood cells and certain other cells. Arginine showed its familiar Na+-independent mode of uptake as a cation throughout the interval of study. An exceptional Na+-dependent component of arginine uptake emerged after day 14, peaked at day 18, and then disappeared on further maturation of the erythroid cell.     

Glucagon regulation of amino acid transport in hepatocytes: Effect of cell enucleation

Glucagon and cAMP analogs stimulate amino acid transport in freshly isolated hepatocytes by inducing the synthesis of new transport proteins. The role of the cell nucleus in the glucagon regulation of amino acid transport has been studied in rat hepatocytes enucleated by centrifugation through a discontinuous Ficoll gradient in the presence of cytochalasin B. Enucleated hepatocytes take up alpha-aminoisobutyric acid (AIB) through a Na⁺-dependent transport component with kinetic properties similar to those found in intact hepatocytes. Cytoplasts prepared from glucagon-stimulated cells retain the increase AIB transport induced by the hormone in the intact cells. The direct addition of glucagon to cytoplasts has no effect on AIB transport, in spite of the fact that the cytoplasts exhibit a higher capacity to bind glucagon than their nucleated counterparts. These data indicate that the nucleus is required for the glucagon stimulation of amino acid transport in isolated hepatocytes.     

Factors regulating the induction of ornithine decarboxylase in fetal rat liver cells in culture.

Various hormonal and non-hormonal agents were tested for their ability to induce ornithine decarboxylase (EC 4.1.1.17) in primary cultures of fetal rat liver cells that retain many of the differentiated functions of hepatocytes. The only agents to induce ornithine decarboxylase in this cell type were fetal calf serum, prostaglandin E1 and cyclic AMP derivatives. Also, the amino acid arginine would induce ornithine decarboxylase in this cell type following arginine starvation for 24 h. These observations are in contrast to the wide range of hormones, e.g. insulin, hydrocortisone, glucagon and growth hormone, than can induce ornithine decarboxylase in vivo in the adult rat liver but which are all without effect on fetal rat liver cells.     

Inhibition of intracellular proteolysis in muscle cultures by multiplication-stimulating activity. Comparison of effects of multiplication-stimulating activity and insulin on proteolysis, protein synthesis, amino acid uptake, and sugar transport

The effects of the insulin-like growth factor, multiplication-stimulating activity (MSA), on chick myotube cultures were investigated. In serum-free media, MSA at levels reported to be present in fetal serum (5 ng/ml) significantly inhibited overall rates of protein degradation and stimulated protein synthesis and amino acid uptake. Half-maximal effects on protein degradation (-30%), synthesis (+25%), and amino acid uptake (+50%) occurred at approximately 0.05 micrograms/ml. In contrast, 10(2)-10(3)-fold higher concentrations (5 micrograms/ml) were required to stimulate transport of the glucose analog 2-deoxyglucose. The results indicate that MSA is an effective anabolic agent regulating protein metabolism and amino acid uptake, but not sugar transport in these cells. Parallel studies conducted with insulin demonstrated similar size effects on protein metabolism and amino acid uptake in serum-free media. However, unlike MSA, insulin levels (10(-2) units/ml) well in excess of its normal physiological range were required to produce significant effects. In addition, the relative sensitivity of sugar transport with respect to protein metabolic effects differed for insulin and MSA. Thus, 2-deoxyglucose transport was approximately 10 times more sensitive to insulin than protein synthesis, proteolysis, or amino acid uptake in contrast to MSA where the reverse was true. However, despite the relatively higher sensitivity of sugar transport to insulin, supraphysiological levels (10(-3) units/ml) of this hormone were still required for significant stimulation. These results suggest a generally low insulin sensitivity in cultured chick myotubes relative to adult tissues. In contrast, the effects of MSA are consistent with a possible role of this or similar factors in regulating growth and development of embryonic muscle.     

Paradoxical effects of protein synthesis inhibitors on uridine uptake in cultured cells: Possible role of uncharged tRNA in regulating metabolism

Previous studies (J. Biol. Chem, 253: 99–105, 1978) showed that thyrotropin-releasing hormone (TRH) acutely stimulated uridine uptake in pituitary cell (GH₄C₁) cultures. Studies on the role of protein synthesis in this response to TRH led to the finding that an inhibitor of ribosomal translation, cycloheximide, also stimulated uridine uptake acutely. Studies reported here attempt to determine the mechanism of cycloheximide action and whether cycloheximide and hormone stimulation of uridine uptake occurred by similar pathways. The experiments presented indicate that: (1) seven inhibitors of ribosomal translation stimulated uridine uptake; (2) in contrast, inhibition of protein synthesis at tRNA aminoacylation resulted in reduced rates of uridine uptake; (3) inhibition of tRNA aminoacylation blocked cycloheximide but not TRH stimulation of uptake; (4) cycloheximide stimulation of uptake was restricted to amino acid-depleted cultures; (5) amino acid supplementation stimulated uridine uptake with a time-course identical to that of cycloheximide; (6) cycloheximide and amino acid supplementation promoted reacylation of cellular tRNAs in amino acid-depleted cultures; and (7) cycloheximide stimulation of uridine uptake resulted from enhanced nucleoside phosphorylation rather than increased uridine transport. We conclude that cycloheximide and amino acid stimulation of uridine phosphorylation may be mediated through a common pathway involving the extent of amino-acylation of cellular tRNAs. Furthermore, cycloheximide and TRH stimulate uridine phosphorylation by pathways that are distinguishable. It is apparent that not all cellular effects of cycloheximde can be attributed solely to inhibition of the synthesis of proteins.     

System A transport activity in normal rat hepatocytes and transformed liver cells: substrate protection from inactivation by sulfhydryl-modifying reagents

The transport of amino acids by normal rat hepatocytes and several hepatoma cell lines has been examined for inactivation by various protein-modifying reagents, including the sulfhydryl-preferring reagents N-ethylmaleimide (NEM) and p-chloromercuribenzene sulfonate (PCMBS). Uptake of 2-aminoisobutyric acid (AIB), a specific probe for hepatic System A-mediated transport, was equally sensitive to inhibition by the organic mercurial PCMBS in each of the cell types tested. In contrast, the sensitivity of System A to inactivation by NEM was substantially different among the five cell types. Normal hepatocytes showed the greatest sensitivity, while the hepatoma cells varied in their responsiveness from moderate to no inhibition. PCMBS inactivated greater than 85% of the System A activity in rat H4 hepatoma cells within 10 min (t1/2 = 3 min). The inhibition by PCMBS was rapidly reversed by treatment of the cells with dithiothreitol. Amino acids showing a high affinity for System A protected the transport system from inactivation, whereas non-substrates produced little or no protection. Amino acid-dependent protection was stereospecific and system-specific. L-norleucine competitively inhibited AIB uptake (Ki = 1.9 +/- 0.1 mM) in H4 cells and also protected System A from PCMBS-dependent inactivation (half-maximal protection occurred at an amino acid concentration of 0.6 +/- 0.1 mM). N-bromosuccinimide was completely ineffective as an inhibitor of System A activity in hepatocytes, whereas treatment of H4 rat hepatoma cells with this reagent resulted in greater than 95% inhibition.     

Amino acid transport in a polyaromatic amino acid auxotroph of Saccharomyces cerevisiae

The initiation of growth of a polyaromatic auxotrophic mutant of Saccharomyces cerevisiae was inhibited by several amino acids, whereas growth of the parent prototroph was unaffected. A comparative investigation of amino acid transport in the two strains employing (14)C-labeled amino acids revealed that the transport of amino acids in S. cerevisiae was mediated by a general transport system responsible for the uptake of all neutral as well as basic amino acids. Both auxotrophic and prototrophic strains exhibited stereospecificity for l-amino acids and a K(m) ranging from 1.5 x 10(-5) to 5.0 x 10(-5) M. Optimal transport activity occurred at pH 5.7. Cycloheximide had no effect on amino acid uptake, indicating that protein synthesis was not a direct requirement for amino acid transport. Regulation of amino acid transport was subject to the concentration of amino acids in the free amino acid pool. Amino acid inhibition of the uptake of the aromatic amino acids by the aromatic auxotroph did not correlate directly with the effect of amino acids on the initiation of growth of the auxotroph but provides a partial explanation of this effect.     

Factors regulating the induction of ornithine decarboxylase in fetal rat liver cells in culture

Various hormonal and non-hormonal agents were tested for their ability to induce ornithine decarboxylase (EC 4.1.1.17) in primary cultures of fetal rat liver cells that retain many of the differentiated functions of hepatocytes. The only agents to induce ornithine decarboxylase in this cell type were fetal calf serum, prostaglandin E₁ and cyclic AMP derivatives. Also, the amino acid arginine would induce ornithine decarboxylase in this cell type following arginine starvation for 24 h. These observations are in contrast to the wide range of hormones, e.g. insulin, hydrocotisone, glucagon and growth hormone, that can induce ornithine decarboxylase in vivo in the adult rat liver but which are all without effect on fetal rat liver cells.     

Regulation of amino acid transport in L6 myoblasts. II. Different chemical properties of transport after amino acid deprivation

The mechanism of stimulation of amino acid transport system A caused by amino acid deprivation in L6 cells was investigated. In cells loaded with alpha-aminoisobutyric acid (AIB), amino acid deprivation increased the rate of proline uptake only after the intracellular [AIB] dropped below 7 mM. Efflux of proline was not sensitive to the presence of proline in the outer medium (with or without external Na+), suggesting that efflux through system A (and possibly uptake) is not susceptible to transinhibition. Transport (stimulated uptake) into amino acid-deprived cells and that into amino acid-supplemented cells differed in several chemical properties: 1) In the former group, transport was higher at lower pH values than in the latter, and the optimum pH values were 7.5 and 7.8, respectively. 2) Unlike proline uptake in supplemented cells, uptake in deprived cells was inhibited by 50% with N-ethylmaleimide (1 mM) or by 50 microM p-chloromercuribenzoate (PCMBS). Inhibition by PCMBS was not due to collapse of the Na+ gradient. The mercurial inhibited only the deprivation-induced stimulation of transport, bringing the rate of proline uptake to the "basal" uptake level observed in amino acid-supplemented cells. Proline uptake was not stimulated by a second deprivation following treatment with PCMBS and a supplementation-deprivation cycle. However, in untreated cells, or by reversing mercaptide formation with dithiotreitol, the second deprivation stimulated transport. Deprivation at 4 degrees C did not elicit stimulation of proline uptake. Cycloheximide prevented the stimulation and decreased the rate of proline uptake in deprived cells more efficiently than in supplemented cells. Actinomycin D prevented stimulation when added at the onset of deprivation. The above data indicate that stimulation of transport by deprivation is protein synthesis-dependent and that the stimulated transport had chemical properties distinct from the "basal" transport in supplemented cells. The evidence presented is consistent with a model of activation of a finite pool of transporters upon deprivation, the chemical characteristics of which differ from those of the "basal" transport system.     

Insulin-like growth factor II binding and action in human fetal fibroblasts

To investigate the role of insulin-like growth factor II (IGF-II) in human prenatal growth, IGF-II binding and biological action were studied in four lines of fetal and three lines of postnatal human fibroblasts. Specific binding of IGF-II was similar in both groups: 15.7% and 14.9% for fetal and postnatal fibroblasts, respectively. This was 5-10 times the amount of IGF-I binding found in these cells. IGF-I and IGF-II caused dose-dependent increases in [14C]aminoisobutyric acid (AIB) uptake. IGF-II was sevenfold less potent than IGF-I in stimulating this metabolic response in both fetal and postnatal fibroblasts. The maximal effect of IGF-II in stimulating [14C]AIB uptake approach that of IGF-I. Similar results were obtained when IGF-I and IGF-II stimulation of [3H]thymidine incorporation was compared in fetal and postnatal fibroblasts. Incubation in the presence of alpha IR-3, a monoclonal antibody to the type I IGF receptor, inhibited the ability of both IGF-I and IGF-II to stimulate [14C]AIB uptake and [3H]thymidine incorporation in fetal and postnatal cells. A monoclonal antibody to the insulin receptor did not affect IGF action. These data indicate that IGF-II is a potent metabolic and mitogenic stimulus for human fetal fibroblasts. However, despite the presence of abundant type II IGF receptors on both fetal and postnatal human fibroblasts, IGF-II stimulation of amino acid transport and DNA synthesis appears to be mediated through the type I rather than through its own type II IGF receptor.     

Regulation of amino acid transport and protein metabolism in myotubes derived from chicken muscle satellite cells by insulin-like growth factor-I

The effects of insulin and insulin-like growth factor-I (IGF-I) on amino acid transport and protein metabolism were compared in myotubes derived from chicken breast muscle satellite cells. Protein synthesis was assessed by continuous labelling with [³H]-tyrosine. Protein degradation was estimated by the release of trichloroacetic acid (TCA) soluble radioactivity by cells which had been previously labelled with [³H]-tyrosine for 3 days. Amino acid transport was measured in myotubes incubated in Dulbecco's modified Eagle's medium (DMEM) 0.5% bovine serum albumin (BSA) with or without insulin or IGF-I. Subsequent [³H]-aminoisobutyric acid (AIB) uptake was then measured in amino acid-free medium. IGF-I was more efficient than insulin at equimolar concentration (3.2 nmol/l) in stimulating protein synthesis (127 and 113% of basal, respectively) and inhibiting protein degradation (32% and 13% inhibition of protein degradation following 4 h incubation). Half maximal effective concentrations for stimulation of AIB uptake were 0.27 ± 0.03 nmol/l and 34.8 ± 3.1 nmol/l for IGF-I and insulin respectively, with maximal stimulation of about 340% of basal. Cycloheximide (3.6 μmol/l) diminished IGF-I-stimulated AIB uptake by 55%. Chicken growth hormone had no effect on basal AIB uptake in these cells and neither glucagon nor dexamethasone had an effect on basal or IGF-I-stimulated AIB uptake. This study demonstrates an anabolic effect for IGF-I in myotubes derived from primary chicken satellite cells which is mediated by the type I IGF receptor, since the cation-independent mannose 6-phosphate receptor does not bind IGF-II in chicken cells. © 1993 Wiley-Liss, Inc.