Differences between resting and insulin-stimulated amino acid transport in frog skeletal muscle

Summary We have compared some features of the resting and the insulin-stimulated uptake of -aminoisobutyrate (AIB) in frog skeletal muscle. We found a substantial difference between the two processes, namely, that resting AIB uptake is Na-independent while the insulin-stimulated fraction of the AIB uptake is Na-dependent.Since the amino acid transport systems in frog skeletal muscle are poorly characterized, we have also surveyed some of their properties. One of the most interesting findings of this survey is that both the uptake and efflux of AIB are inhibited by low concentrations of PCMBS (parachloro-mercury-benzene sulfonic acid 5×10^–5 m). In contrast, the carrier mediated transport of basic amino acids is neither inhibited by this mercurial agent nor accelerated by insulin.The action of PCMBS strongly suggests the presence of a critical sulfhydryl group in the amino acid carrier system utilized by AIB. This group is exposed to the outside solution since PCMBS penetrates cell membranes poorly, and in addition its inhibitory actions were reverted by agents that do not penetrate the cell membrane like albumin or glutathione.     

Hormonal regulation of amino acid transport and cAMP production in monolayer cultures of rat hepatocytes

The effects of insulin, glucagon or Dexamethasone (DEX) and of glucagon with insulin or DEX were examined on the uptake of 2-amino [1-¹⁴C]isobutyric acid (AIB) and N-Methyl-2-amino [1-¹⁴C]isobutyric acid (NMe AIB) in monolayer cultures of rat hepatocytes. Insulin and glucagon stimulated the uptake of both the amino acids and DEX inhibited it, showing that all three of these hormones regulate the A system (the sodium-dependent system that permits the transport of NMe AIB) for amino acid transport in these cultures. Experiments investigating the transport of aminocyclopentane-1-carboxylic acid, 1- [carboxyl-¹⁴C] in the presence of excess AIB or in the absence of sodium showed that insulin had no effect on the activity of the L system (the sodium-independent system that prefers leucine). Experiments on the uptake of AIB in the presence of excess NMe AIB showed insulin had no effect on the transport activity of the ASC system (the sodium-dependent system that does not transport NEe AIB). Insulin concentrations ranging from 0.1 nM to 100 nM did not antagonize the stimulatory effect of optimum or suboptimum concentrations of glucagon on the uptake of either AIB or NMe AIB. Similarly, glucagon did not antagonize the stimulatory effect of optimum or suboptimum concentrations of insulin on the uptake of both the amino acids. The combined effect of insulin and glucagon was additive on the rate as well as the cumulative uptake of both AIB and NMe AIB. DEX alone inhibited the transport of both AIB and NMe AIB by about 25%, while glucagon caused a 2–3-fold increase; however, the addition of glucagon to cultures containing DEX caused a 7–8-fold increase in the uptake of both AIB and NMe AIB when compared to cultures containing DEX alone. The effect of insulin on the levels of cAMP was also investigated. Insulin had no effect on the cAMP levels in cultures treated or untreated with optimum or suboptimum concentrations of glucagon.     

Role of microtubules in insulin and glucagon stimulation of amino acid transport in isolated rat hepatocytes

The effects of the microtubule inhibitor, colchicine, on insulin or glucagon stimulation of alpha-amino[1-14C]-isobutyric acid (AIB) transport were investigated in isolated hepatocytes from normal fed rats. Under all conditions tested, AIB uptake appeared to occur through two components of transport: a low affinity (Km approximately 50 mM) component and a high affinity (Km approximately 1 mM) component. Within 2 h of incubation, insulin and glucagon, at maximal concentrations, increase AIB (0.1 mM) uptake by 2- to 3-fold and 4- to 6-fold, respectively. Colchicine, at the low concentration of 5 X 10(-7) M, slightly reduces basal AIB transport, decreases by 80% the simulatory effect of insulin, and diminishes by 40% the stimulatory effect of either glucagon or dibutyryl cAMP. Kinetic analysis of AIB influx indicates that the drug inhibits the increase in Vmax of a high affinity (Km approximately 1 mM) component of transport stimulated by insulin or glucagon, without affecting the kinetic parameters of a low affinity component of transport (Km approximately 50 mM). Various short term hormonal effects of insulin and glucagon (changes in glucose, urea, and lactate production) were found not to be modified by the drug. Vinblastine elicits similar changes as colchicine on AIB uptake. Lumicolchicine, a colchicine analogue that does not bind to tubulin, has no effect. The concentration of colchicine (10(-7) M) required for half-maximal inhibition of hormone-stimulated AIB transport is in the appropriate range for specific microtubule disruption. These data suggest that microtubules are involved in the regulation of the insulin or glucagon stimulation of AIB transport in isolated rat hepatocytes.     

Hormonal regulation of hepatic amino acid transport

The transport of 2-aminoisobutyric acid (AIB) into liver tissue was increased by both insulin and glucagon. We have now shown that these hormones do not stimulate the same transport system. Glucagon, possibly via cAMP, increased the hepatic uptake of AIB by a mechanism which resembled system A. This glucagon-sensitive system could be monitored by the use of the model amino acid MeAIB. In contrast, the insulin-stimulated system exhibited little or no affinity for MeAIB and will be referred to as system B. On the basis of other reports that the hepatic transport of AIB is almost entirely Na⁺ dependent and the present finding that the uptake of 2-aminobicyclo [2,2,1] heptane-2-carboxylic acid (BCH) was not stimulated by either hormone, we conclude that system B is Na⁺ dependent. Furthermore, insulin added to the perfusate of livers from glucagon-pretreated donors suppressed the increase in AIB or MeAIB uptake. Depending upon the specificities of systems A and B, both of which are unknown for liver tissue, the insulin/glucagon ratio may alter the composition of the intracellular pool of amino acids.     

Hormonal regulation of amino acid transport and gluconeogenesis in primary cultures of adult rat liver parenchymal cells.

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.     

Regulation of Membrane Transport Sites for Amino Acids in Myogenic Cells

Abstract. Myoblasts from 12-day chick embryos in cell culture transport the nonmetabolizable amino acid α-aminoisobutyric acid (AIB) two to three-fold more rapidly than multinucleated myotubes which form from them. This decrease in transport is due to a relative decrease in the number of transport sites per unit area of cell surface suggesting a compositional change in the plasma membrane during myogenesis. In studies reported here, AIB transport was monitored throughout myogenesis and correlated with other aspects of differentiation. During myogenesis the number of amino acid transport sites remains constant per myotube nucleus. As myogenesis proceeds, there is a marked increase in cellular protein and cell surface without a commensurate increase in amino acid transport sites. The net consequence of the surface area change is fewer amino acid transport sites per unit area of myotube membrane surface. The decrease in membrane transport sites for AIB per unit area of membrane is not a result of length of time in culture per se, medium depletion, or cell density, but is a result of differentiation, since blocking myoblast fusion by deprivation of calcium delays the decrease in AIB transport sites per unit cell surface area while reversal of the calcium deprivation block is accompanied by a rapid decrease in the number of AIB transport sites per unit cell surface area. Thus, the decrease in AIB transport sites is an aspect of differentiation which accompanies the marked elaboration of surface membrane during myogenesis.     

Regulation of membrane transport sites for amino acids in myogenic cells. A differentiation dependent phenomenon

Myoblasts from 12-day chick embryos in cell culture transport the nonmetabolizable amino acid alpha-aminoisobutyric acid (AIB) two to three-fold more rapidly than multinucleated myotubes which form from them. This decrease in transport is due to a relative decrease in the number of transport sites per unit area of cell surface suggesting a compositional change in the plasma membrane during myogenesis. In studies reported here, AIB transport was monitored throughout myogenesis and correlated with other aspects of differentiation. During myogenesis the number of amino acid transport sites remains constant per myotube nucleus. As myogenesis proceeds, there is a marked increase in cellular protein and cell surface without a commensurate increase in amino acid transport sites. The net consequence of the surface area change is fewer amino acid transport sites per unit area of myotube membrane surface. The decrease in membrane transport sites for AIB per unit area of membrane is not a result of length of time in culture per se, medium depletion, or cell density, but is a result of differentiation, since blocking myoblast fusion by deprivation of calcium delays the decrease in AIB transport sites per unit cell surface area while reversal of the calcium deprivation block is accompanied by a rapid decrease in the number of AIB transport sites per unit cell surface area. Thus, the decrease in AIB transport sites is an aspect of differentiation which accompanies the marked elaboration of surface membrane during myogenesis.     

Hormonal regulation of amino acid uptake by cultured 3T3-L1 adipocytes

Incubation of the adipocytes for 20 hours with insulin or with Bt2cAMP plus the theophylline stimulated adipocyte uptake of AIB and MeAIB but did not stimulate the uptake of glutamine or cycloleucine. MeAIB uptake by both 3T3-L1 preadipocytes and 3T3-C2 cells was relatively unresponsive to insulin. However, MeAIB uptake by 3T3-C2 cells was stimulated by treatment with Bt2cAMP plus theophylline. Incubation of 3T3 adipocytes for 60 min with insulin yielded maximal stimulation of 2-deoxyglucose uptake but no stimulation of the uptake of AIB, MeAIB or glutamine. Responsiveness of transport to Bt2cAMP does not appear to require adipocyte differentiation. By contrast, adipocyte differentiation may be required for the development of the insulin-responsive transport systems.     

Insulin-like growth factor-1 stimulates amino acid uptake by the cultured human placental trophoblast

Amino acid uptake by the human placenta is known to occur via several transport mechanisms. However, regulation by extracellular factors has received relatively little attention. A recent report by this laboratory characterized the uptake of α-aminoisobutyric acid (AIB) stimulated by insulin in the cultured human placental trophoblast The current study evaluated the effect of insulin-like growth factor-1 (IGF-1) on AIB uptake in cultured human placental trophoblasts. Na⁺-dependent AIB uptake was significantly stimulated by IGF-l in a time-dependent manner, as early as 30 min after hormone exposure. The maximum effect was at 2–4 hr of continuous exposure to IGF-l and the stimulation was dependent upon IGF-1 concentration approaching maximal stimulation at 50 ng.ml^?1. AIB uptake was inhibited by increasing concentrations of α-(methylamino)isobtyric acid (MeAIB). Approximately 75% of basal (unstimulated) Na⁺-dependent AIB uptake was inhibited by MeAIB. The IGF-1-stimulated increment above basal AIB uptake was completely inhibited by MeAIB. IGF-1 increased the maximum uptake yelocity but not Km. Using equimolar concentrations, stimulation was greater with IGF-1 then with IGF-2. Stimulation by IGF-1, but not insulin, was inhibited by anit-IGF-1 receptor antibody, indicating mediation via the IGF-1 receptor. H7, a nonspecific inhibitor of serine-threonine kinase, inhibited IGF-1-dependent stimulation of AIB uptake. In addition, calphostin C (a specific inhibitor of protein kinase C), but not H89 (a specific inhibitor of protein kinase A), inhibited the IGF-1 action. This study further characterizes regulated amino acid uptake by the human placental trophoblasts and demonstrates that the Na⁺-dependent component of AIB uptake is stimulated by physiologic concentrations of IGF-1. © 1995 Wiley-Liss Inc.     

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.     

Insulin and exercise stimulate muscle alpha-aminoisobutyric acid transport by a Na+-K+-ATPase independent pathway

Sodium ions are required for the active transport of amino acids such as alpha-aminoisobutyric acid (AIB) into skeletal muscle. To examine the role of Na+-K+-ATPase in this phenomenon, studies were carried out using the isolated perfused rat hindquarter preparation. Perfusion for 30 min with ouabain at a dose sufficient to inhibit the Na+-K+ pump (10(-4) M) inhibited the basal rate of AIB uptake in all muscles studied by up to 80%. However, it failed to inhibit the stimulation of AIB uptake, either by insulin (200 microU/ml) or electrically-induced muscle contractions. The increase in K+ release by the hindquarter in the presence of ouabain was the same under all conditions suggesting comparable inhibition of the Na+-K+ pump. These studies suggest that the basal, but not insulin or exercise-stimulated AIB transport into muscle is acutely dependent on a functional Na+-K+ pump. They also suggest that stimulated and basal uptake of AIB involve different mechanisms.     

Membrane function in differentiating skeletal muscle cells: I. Kinetic analysis of amino acid transport

Primary cultures of mononucleated myoblasts from 12-day-old chick embryos have a twofold higher rate of α-aminoisobutyric acid (AIB) transport before fusion occurs to form multinucleated myotubes. Several lines of evidence indicate that the uptake of AIB observed in both myoblasts and myotubes is primarily carrier-mediated by a membrane transport system. Increasing the temperature from 24 to 37°C results in a threefold increase in the rate of AIB uptake; both methionine and glycine inhibit AIB uptake by more than 85%; and 2,4-dinitrophenol inhibits AIB uptake by approximately 50%. In addition, the energies of activation (14.5 and 14.0 kcal/mole for myoblasts and myotubes, respectively) are characteristic of carrier-mediated transport. Resolution of AIB uptake into a saturable, carrier-mediated component and a nonsaturable, diffusion component shows that at concentrations of AIB≤1.5 mM over 97% of total AIB uptake is carriermediated in both myoblasts and myotubes. Kinetic analysis of carrier-mediated AIB uptake indicates that myoblasts and myotube membrane carriers have the same affinity for AIB (K_m values = 1.73 and 1.31 mM, respectively). However, the V_max for myoblasts is 23.7 nmole/mg/min while myotubes have a V_max of 12.6 nmole/mg/min. The twofold difference in V_max is shown to be due to a twofold difference in the quantity of membrane transport sites per milligram of protein.     

Effects of tumor necrosis factor-alpha on insulin stimulated amino acid transport in cultured rat hepatocytes

It has been suggested that tumor necrosis factor alpha (TNF-alpha) plays a pivotal role in the pathogenesis of insulin resistance. It could act directly or indirectly in liver. The aim of this study was to determine direct short time (4 h) and long time (24 h) action of TNF-alpha on amino acid transport in cultured rat hepatocytes and possible role of protein kinase C (PKC) in insulin signal pathway and insulin resistance. Hepatocytes were isolated by a modified collagenase perfusion technique and cultured for 24 h in M 199 medium. In the presence of insulin basal alpha-amino isobutyric acid (AIB) uptake was increased 55%. TNF-alpha in short time action did not change basal AIB transport, but significantly (25%) increased insulin stimulated uptake. Short time action of TNF-alpha was ameliorated by phorbol ester treatment. These results indicated that PKC activation is important in insulin signaling and TNF-alpha action. TNF-alpha acting directly did not cause insulin resistance in cultured hepatocytes.     

Okadaic acid inhibits insulin stimulation of both ornithine decarboxylase and spermidine transport in hepatocyte cultures

• 1.1. Okadaic acid inhibited basal ODC activity in rat hepatocytes in culture and prevented any increase in ODC activity and in the rate of spermidine uptake promoted by both insulin and hypotonicity.
• 2.2. The increase promoted by AIB was not counteracted by okadaic acid.

     

Induction of amino acid transport by L-triiodothyronine in cultured growth hormone-producing rat pituitary tumor cells (GC cells)

The action of L-triiodothyronine (T3) on amino acid transport in the GC clonal strain of rat pituitary cells was investigated by measurement of the uptake of the nonmetabolizable amino acid, alpha-aminoisobutyric acid (AIB). The uptake of AIB by GC cells appeared to require energy and Na+ and displayed Michaelis-Menten kinetics. In comparison to cultures maintained in the absence of T3, T3 addition resulted in an increase in AIB uptake which seemed due to an increase in the initial rate of AIB transport. T3 addition resulted in increased AIB accumulation at later time points as well. T3 induction of AIB transport did not occur until 3.5 h after addition of T3, and this effect was blocked by cycloheximide. Maximal induction occurred 48 to 72 h later. One-half maximal induction occurred 24 to 48 h after addition of T3. No detectable changes either in AIB uptake or intracellular water space, measured by uptake of the nonmetabolizable sugar, 3-O-methyl-D-glucose, were noted for the first 120 min after addition of T3. Induction of AIB transport occurred at 0.05 nM T3 (total medium concentration) and one-half maximal induction occurred at 0.17 nM T3. The relative potencies of four iodothyronine analogues for AIB transport were in accord with their reported activities in nuclear T3 receptor binding assays. These data suggest that induction of AIB transport by T3 may be mediated by the nuclear T3 receptor and may reflect the pleiotrophic response of GC cells to thyroid hormone.     

Insulin and somatomedins A and B: comparisons of biological activities in cultured human skin-derived fibroblasts

The responsiveness of cultured human skin-derived fibroblasts to insulin and to somatomedins-A and -B (stimulation of thymidine incorporation and alpha-aminoisobutyrate (AIB) uptake) was examined. Under selected conditions, insulin stimulates both AIB uptake and thymidine incorporation, whereas somatomedin-A stimulates only AIB uptake; under the same conditions, somatomedin-B stimulates neither process. On the basis of the different spectra of biological activities observed for the three polypeptides it is suggested that the receptor for insulin in cultured human fibroblasts is independent from the ones for the somatomedins.     

Stimulation of Amino Acid Uptake and Na+, K+-ATPase Activity by Norepinephrine in Superior Cervical Sympathetic Ganglia Excised from Adult Rats

Active uptake of a labelled nonmetabolizable amino acid, alpha-aminoisobutyric acid (AIB), into isolated superior cervical sympathetic ganglia (SCG) excised from adult rats was considerably stimulated by the addition of either norepinephrine (NE, 50 microM) or 3,4-dihydroxyphenylethylamine (dopamine, DA, 100 microM) to the medium during aerobic incubation for 2 h at 37 degrees C. The NE-induced increase in AIB uptake was significantly antagonized by the addition of an alpha 1-adrenoceptor antagonist (prazosin, 10 microM) in SCG axotomized 1 week prior to the examination, in which most of the ganglionic neurons had degenerated and reactive proliferation of the satellite glial components was in progress. The addition of neither acetylcholine (ACh, 1 mM) plus eserine (0.1 mM) nor cyclic nucleotides (1 mM) changed the AIB uptake by the SCG. In the axotomized SCG, the NE-evoked increase in AIB uptake was much more pronounced than that of intact or denervated SCG. A kinetic study of the active AIB uptake in the SCG showed that NE produced a decrease of the Km value and an increase in the Vmax, especially in the axotomized SCG. Ganglionic Na+, K+-ATPase activity was greatly stimulated in the presence of NE, but not by ACh. These results strongly suggest that the NE-induced enhancement of active AIB uptake in the isolated SCG is occurring in glial cells rather than in neuronal cells, with a possible alteration of membrane properties for amino acid uptake and with an apparent regulation by the stimulated transport enzyme Na+, K+-ATPase.     

Effects of multiplication stimulating activity (MSA) on AIB transport into myoblast and myotube cultures

The effects of a somatomedian analog, Temin's multiplication stimulating activity (MSA), on amino acid transport into muscle cells have been characterized in a series of experiments on myoblasts and myotubes in culture. Addition of MSA to serum-starved L6 myoblasts increased the rate of aminoisobutyrate (AIB) uptake 50-150% within five hours. This early effect on transport was followed by increases in cell number, protein content and 3H-thymidine incorporation. Kinetic analyses indicated that MSA increased the maximal velocity of AIB uptake but had no effect on the KM for AIB. When myoblasts were allowed to fuse (and dividing cells eliminated by addition of 10(-4) M cytosine arabinoside) the AIB transport system(s) remained similarly responsive to MSA. In myoblasts and in myotubes, both the basal and MSA-stimulated rate of AIB uptake were sodium-dependent processes; little stimrulation occurred if sodium was absent from the labeling medium. Further suggesting the involvement of cations in response to hormone, MSA stimulated uptake of the potassium analog, 86Rb+, and increase net intracellular potassium in both myoblasts and myotubes. MSA was active at concentrations equivalent to in vivo levels of somatomedins; neither insulin nor growth hormone had any effect at or near physiological concentrations.     

Aminoisobutyric acid transport in primary cultures of normal adult rat hepatocytes.

In contrast to suspensions of freshly isolated hepatic parenchymal cells (HPC), short-term monolayer cultures of HPC displayed properties of active transport for the amino acid analog aminoisobutyric acid (AIB). The uptake of AIB was inhibited by KCN and iodoacetate, failed to occur at 4 degrees, and was stimulated by glucagon. The apparent Km for AIB uptake by cultured HPC was approximately 19 mM. Glucagon did not alter the apparent Km but did increase V.     

Inhibition of alpha-aminoisobutyric acid uptake by mastoparan in Madin-Darby Canine Kidney cells

Mastoparan, a wasp venom, was found to inhibit Na(+)-dependent net alpha-aminoisobutyric acid (AIB) uptake in Madin Darby Canine Kidney (MDCK) cells. Mastoparan also produced a significant increase in AIB efflux when compared to controls. Pretreatment of MDCK cells with 2 mM neomycin attenuated mastoparan's inhibition of net AIB uptake and completely suppressed mastoparan-mediated increases in AIB efflux when compared to controls. These data suggest that mastoparan's inhibition of net AIB uptake involves more than a single basic mechanism.