Amino Acid Transport In Muscle Research Articles

Skeletal muscle amino acid transporter and BCAT2 expression prior to and following interval running or resistance exercise in mode-specific trained males

Endurance (END)- and resistance (RES)-trained males performed interval running or resistance exercise during three consecutive days (bouts 1-3). Muscle biopsies were obtained at baseline, 2h post-bout 1, and 72h post-bout 3. Amino acid transporter SNAT2 mRNA was 75% greater in END (group p = 0.008), and increased ~ 70% 2h post in both groups (time p = 0.023). Amino acid transporter PAT1 mRNA was 2.7-fold greater in RES (group p = 0.002). Baseline protein levels of the mitochondrial aminotransferase BCAT2 were 79% greater in END (p = 0.015).

Intermittent Bolus Feeding Enhances Lean Tissue Accretion by Increasing Muscle Amino Acid Transport and Protein Turnover Compared with Continuous Feeding in Neonates

Orogastric tube feeding is recommended in neonates unable to ingest food normally, and can be administered by intermittent bolus (INT) or continuous (CON) infusion. Recently, we showed that INT compared to CON feeding enhances lean tissue accretion by increasing muscle protein synthesis in neonates. To determine the mechanism for this response, neonatal pigs (n=6, 6‐d‐old) were fed the same diet in equivalent amounts continuously or intermittently (meal every 4h) for 21d. Insulin receptor and insulin receptor substrate 1 expression and protein kinase B phosphorylation in skeletal muscle were unaffected by feeding modality. Formation of the active eukaryotic initiation factor (eIF) 4E‐eIF4G complex and phosphorylation of ribosomal protein S6 kinase were higher and phosphorylation of eIF2α was lower in INT compared to CON fed pigs indicating increased translation initiation (P<0.05). Sodium‐coupled neutral amino acid transporter 2, but not L‐type amino acid transporter 1, expression was higher in INT compared to CON pigs suggesting enhanced glutamine transport (P<0.05). Abundance of the ubiquitin ligases, muscle RING finger‐1 and F‐box protein atrogin‐1/MAFbx, was higher for INT compared to CON pigs (P<0.05). These results suggest that intermittent feeding enhances lean tissue accretion by increasing amino acid transport and protein turnover. (Supported by NIH AR444474 and USDA/ARS 6250–51000‐055)

Aging differentially affects human skeletal muscle amino acid transporter expression when essential amino acids are ingested after exercise

Amino acid transporters have been proposed as regulators of protein synthesis. The primary aim of this study was to determine whether amino acid transporter expression is increased in human muscle following resistance exercise (RE) coupled with essential amino acid (EAA) ingestion, and whether a differential response occurs with aging. Secondly, we aimed to compare this response to a previous study examining RE alone. Young (n=7, 30±2yr) and older men (n=6, 70±2yr) ingested EAA 1h after RE. Muscle biopsies were obtained at rest and 3 and 6h post exercise to examine amino acid transporter mRNA and protein expression. In both age groups, RE+EAA increased mRNA of L-type amino acid transporter 1 (LAT1)/solute linked carrier (SLC)7A5, sodium-coupled neutral amino acid transporter 2 (SNAT2)/SLC38A2, and cationic amino acid transporter 1/SLC7A1 (p<0.05). SNAT2 protein increased in young at 3 and 6h (p<0.05), whereas old maintained higher LAT1 protein (p<0.05). Compared to RE alone, RE+EAA enhanced amino acid transporter expression only in young (p<0.05). RE increases muscle amino acid transporter expression in young and older adults, however, post exercise EAA ingestion enhances amino acid transporter expression only in young indicating that aging may influence the function of specific amino acid transporters.

Bed rest impairs skeletal muscle amino acid transporter expression, mTORC1 signaling, and protein synthesis in response to essential amino acids in older adults

Skeletal muscle atrophy during bed rest is attributed, at least in part, to slower basal muscle protein synthesis (MPS). Essential amino acids (EAA) stimulate mammalian target of rapamycin (mTORC1) signaling, amino acid transporter expression, and MPS and are necessary for muscle mass maintenance, but there are no data on the effect of inactivity on this anabolic mechanism. We hypothesized that bed rest decreases muscle mass in older adults by blunting the EAA stimulation of MPS through reduced mTORC1 signaling and amino acid transporter expression in older adults. Six healthy older adults (67 ± 2 yr) participated in a 7-day bed rest study. We used stable isotope tracers, Western blotting, and real-time qPCR to determine the effect of bed rest on MPS, muscle mTORC1 signaling, and amino acid transporter expression and content in the postabsorptive state and after acute EAA ingestion. Bed rest decreased leg lean mass by ∼4% (P < 0.05) and increased postabsorptive mTOR protein (P < 0.05) levels while postabsorptive MPS was unchanged (P > 0.05). Before bed rest acute EAA ingestion increased MPS, mTOR (Ser(2448)), S6 kinase 1 (Thr(389), Thr(421)/Ser(424)), and ribosomal protein S6 (Ser(240/244)) phosphorylation, activating transcription factor 4, L-type amino acid transporter 1 and sodium-coupled amino acid transporter 2 protein content (P < 0.05). However, bed rest blunted the EAA-induced increase in MPS, mTORC1 signaling, and amino acid transporter protein content. We conclude that bed rest in older adults significantly attenuated the EAA-induced increase in MPS with a mechanism involving reduced mTORC1 signaling and amino acid transporter protein content. Together, our data suggest that a blunted EAA stimulation of MPS may contribute to muscle loss with inactivity in older persons.

Skeletal muscle amino acid transporter expression is increased in young and older adults following resistance exercise

Amino acid transporters and mammalian target of rapamycin complex 1 (mTORC1) signaling are important contributors to muscle protein anabolism. Aging is associated with reduced mTORC1 signaling following resistance exercise, but the role of amino acid transporters is unknown. Young (n = 13; 28 ± 2 yr) and older (n = 13; 68 ± 2 yr) subjects performed a bout of resistance exercise. Skeletal muscle biopsies (vastus lateralis) were obtained at basal and 3, 6, and 24 h postexercise and were analyzed for amino acid transporter mRNA and protein expression and regulators of amino acid transporter transcription utilizing real-time PCR and Western blotting. We found that basal amino acid transporter expression was similar in young and older adults (P > 0.05). Exercise increased L-type amino acid transporter 1/solute-linked carrier (SLC) 7A5, CD98/SLC3A2, sodium-coupled neutral amino acid transporter 2/SLC38A2, proton-assisted amino acid transporter 1/SLC36A1, and cationic amino acid transporter 1/SLC7A1 mRNA expression in both young and older adults (P < 0.05). L-type amino acid transporter 1 and CD98 protein increased only in younger adults (P < 0.05). eukaryotic initiation factor 2 α-subunit (S52) increased similarly in young and older adults postexercise (P < 0.05). Ribosomal protein S6 (S240/244) and activating transcription factor 4 nuclear protein expression tended to be higher in the young, while nuclear signal transducer and activator of transcription 3 (STAT3) (Y705) was higher in the older subjects postexercise (P < 0.05). These results suggest that the rapid upregulation of amino acid transporter expression following resistance exercise may be regulated differently between the age groups, but involves a combination of mTORC1, activating transcription factor 4, eukaryotic initiation factor 2 α-subunit, and STAT3. We propose an increase in amino acid transporter expression may contribute to enhanced amino acid sensitivity following exercise in young and older adults. In older adults, the increased nuclear STAT3 phosphorylation may be indicative of an exercise-induced stress response, perhaps to export amino acids from muscle cells.

Skeletal Muscle Amino Acid Transporter mRNA Expression Is Increased In Young And Older Humans Following Resistance Exercise

INTRODUCTION: Amino acid availability is important for increasing muscle protein synthesis. However, for amino acids to be taken up and utilized by the cell, adequate transport of amino acids must occur. Interestingly, amino acid transporters have been linked to mTOR cellular signaling- a major regulator of muscle protein synthesis. PURPOSE: Therefore, we evaluated the expression levels of select amino acid transporters in young and older humans following a single bout of heavy resistance exercise. METHODS: Young (n=5) and Older (n=5) male subjects participated in a single bout of resistance exercise. Muscle biopsies were sampled at Basal, 6 and 24h post exercise. Muscle biopsies were analyzed for LAT1/SLC7A5, CD98/SLC3A2, SNAT2/SLC38A2 and PAT1/SLC36A1 mRNA expression utilizing real-time PCR. RESULTS: LAT1, CD98 and PAT1 mRNA expression were significantly elevated at 6h post in young and older subjects. However, LAT1 response was greater and was prolonged (24h) for the older subjects. Additionally, SNAT2 mRNA expression was elevated at 6 and 24h post in the older while unchanged for the younger subjects. CONCLUSIONS: These data support a rapid upregulation of amino acid transporters gene expression which may support enhanced protein synthesis following a bout of exercise. However, the robust and sustained transporter expression in the old may reflect a greater export of amino acids due to accelerated protein breakdown and muscle remodeling. Supported by ACSM Research Endowment Grant (MJD), P30 AG-024832 (MJD) and NIH/NIAMS grant R01 AR049877 (BBR)

Amino Acid Transporter Expression is Increased in Human Skeletal Muscle Following Essential Amino Acid Ingestion

Essential amino acids (EAA) stimulate skeletal muscle mTORC1 signaling and protein synthesis in humans. It has recently been reported that amino acid transporters may play a role in the regulation of mTORC1 signaling and translation initiation. The purpose of this study was to profile the expression of several amino acid transporters in human skeletal muscle before and after the ingestion of EAA. Muscle biopsies were obtained from the vastus lateralis of 7 young adult subjects (3 male; 4 female) before and 1–3h after ingesting 10 grams of EAA. Blood and muscle samples were analyzed for leucine kinetics using stable isotopic techniques and qRT‐PCR was used to determine mRNA expression for L‐type (LAT1/SLC7A5, CD98/SLC34A2, LAT3/SLC7A7), sodium‐coupled neutral (SNAT2/SLC38A2 and SNAT4/SLC38A4), proton‐assisted (PAT1/SLC36A1 and PAT2/SLC36A2), cationic (CAT2/SLC7A2), and excitatory (EAAT1/SLC1A3) amino acid transporters. EAA ingestion increased blood leucine concentration, delivery to muscle, transport into muscle, muscle concentration and muscle protein synthesis in association with an increase in LAT1, CD98, SNAT2, and PAT1 expression during the first hour post‐ingestion (P&lt;0.05). We conclude that an increase in EAA availability upregulates human skeletal muscle amino acid transporter expression which likely plays a key role in stimulating anabolic signaling and muscle protein synthesis.Supported by NIA P30 AG024832 (MJD) &amp; NIAMS R01 AR049877 (BBR)

Biochemistry of Exercise

Biochemistry of Exercise Editors: HARGREAVES, Mark, PhD (Deakin Univ); THOMPSON, Martin, PhD (Univ of Sydney) Bibliographic Data: (ISBN: 0–88011-758–3, Human Kinetics Inc, 1999, $60.00) 337 pages, Contributors, hard cover Audiences: Exercise Biochemists, Exercise Physiologists Subjects: Sports Medicine, Biochemistry Other Info: The book contains black-and-white illustrations. DESCRIPTION: This book, dedicated to the memory of Dr. John R. Sutton, contains four main subheadings related to the conference theme of membranes, muscle, and exercise. PURPOSE: The purpose is to present a number of invited presentations at the 10th International Conference on the Biochemistry of Exercise. AUDIENCE: While not designed as a textbook, it will make a useful handbook for scientists and students alike. FEATURES: The first section on signaling contains four chapters on topics ranging from intracellular insulin signaling in skeletal muscle and sympathoadrenergic regulation of substrate metabolism during exercise to regional blood flow and functional vascular shunts. Also included in this section is a chapter addressing the role of diet and exercise on muscle membranes. The next major section (seven chapters) is entitled “Excitation-Contraction.” Several of the chapters are focused on the possible mechanisms associated with muscle fatigue including factors such as calcium kinetics, muscle acidity, inorganic phosphate accumulation, nitric oxide derivatives, ATPase activity, and oxidative stress. Other chapters pertain to the role of the Na+ K+ pump in muscle during exercise as well as energy conversion and efficiency during locomotion. The following section covers various aspects of metabolism (eight chapters). Carbohydrate, fat, and protein metabolism are discussed. For carbohydrate metabolism, special attention is given to the mechanisms of glucose transport, skeletal muscle transporters, lactate exchange, and overall regulation during exercise. In regard to fat metabolism, two chapters pertaining to the regulation of fat oxidation during exercise as well as fatty acid transport and uptake by muscle are included. Single chapters exist addressing muscle amino acid transport and metabolism, and the metabolic basis of fatigue in muscle during exercise. The final section (four chapters) relates to adaptation. How exercise training alters gene expression in skeletal muscle including fiber type specificity and potential signaling is discussed in the initial chapter of this section. Next, a chapter dealing with the training-induced alterations related to vascular remodeling is presented. This is followed by a chapter addressing the role of contractile activity and neural factors on the regulation of glucose transporters in skeletal muscle. The final chapter concerns the fiber specific adaptive response of stress proteins to exercise stimulation in muscle. ASSESSMENT: This book is impressive in both its breath of content related to skeletal muscle and exercise as well as in the quality of authors contributing chapters. SCORE: Weighted Numerical Score: 68-**

Counter-regulatory hormones and mechanisms in amino acid metabolism with special reference to the catabolic response in skeletal muscle.

There is evidence that both counter-regulatory hormones, in particular glucocorticoids, and cytokines influence amino acid and protein metabolism in skeletal muscle, and that these two groups of regulators interact in the development of muscle catabolism. Glucocorticoids stimulate muscle proteolysis during sepsis and also in other catabolic conditions. In addition, glucocorticoids regulate muscle glutamine metabolism, resulting in increased glutamine release and reduced glutamine concentrations in skeletal muscle. Glucocorticoids inhibit the glutamine transporter in skeletal muscle and stimulate glutamine synthetase activity. Proinflammatory cytokines, in particular tumor necrosis factor and interleukin-1, inhibit muscle amino acid transport by system A, and these cytokine effects are probably indirect. Most of the catabolic effects of tumor necrosis factor in skeletal muscle, including stimulated protein degradation and inhibited amino acid uptake, are mediated by glucocorticoids.

Evidence that inhibition of muscle amino acid uptake during endotoxemia is not mediated by glucocorticoids

Sepsis and endotoxemia are associated with increased muscle protein breakdown and inhibited amino acid uptake. Glucocorticoids are important for the regulation of muscle protein breakdown in catabolic conditions; in contrast, the role of glucocorticoids in the regulation of muscle amino acid transport during sepsis or endotoxemia is not known. The present study was designed to test the role of glucocorticoids in the regulation of muscle amino acid uptake during endotoxemia. Amino acid transport, determined as uptake of 3H-α-aminoisobutyric acid (AIB) by incubated soleus muscles in vitro, was reduced by approximately 40% 2 hours after intraperitoneal (IP) injection of 10 μg/kg endotoxin in rats. Administration of 5 mg/kg of the glucocorticoid receptor antagonist RU 38486 2 hours before endotoxin injection did not affect the inhibition of amino acid uptake. In vitro addition of plasma from endotoxemic rats to incubated rat soleus muscles inhibited amino acid uptake by approximately 30%. This effect of endotoxic plasma also was noted when muscles were from rats that had been treated with RU 38486 and when RU 38486 was present in the incubation medium. Results confirm previous reports of reduced muscle amino acid transport during endotoxemia and of the presence of a circulating factor that inhibits muscle amino acid uptake in this condition. Data suggest that inhibited muscle amino acid transport during endotoxemia is not regulated by glucocorticoids.

Okadaic acid, insulin, and denervation effects on glucose and amino acid transport and glycogen synthesis in muscle.

Effects of okadaic acid (OKA) and calyculin A, cell-permeating specific inhibitors of phosphoprotein phosphatases-1 and -2A, were studied in isolated rat hemidiaphragms. OKA stimulated glucose transport (half-maximum = approximately 0.1 microM; maximum = approximately 1 microM) but was less effective than 6 nM insulin. Insulin and OKA effects were not additive. OKA diminished or abolished glucose transport-stimulation by insulin. System A amino acid transport was also stimulated by OKA, insulin was more effective, and preexposure to OKA inhibited insulin stimulation. Calyculin A affected both transport systems similarly to OKA. OKA did not affect basal glycogen synthesis but abolished its stimulation by insulin. Denervated muscles develop post-receptor insulin resistance. Glucose transport and glycogen synthesis were essentially unresponsive to insulin 3 days postdenervation; however, glucose transport was stimulated by OKA similarly to controls. OKA did not affect glycogen synthesis in denervated muscle except for abolishing a small insulin effect. The data suggest similar acute regulation of glucose and system A amino acid transport in muscle. Enhanced Ser/Thr phosphorylation of unidentified protein(s) stimulates both processes but inhibits their full stimulation by insulin. Postdenervation insulin resistance likely reflects impaired signal transduction.

Differential effects of okadaic acid on insulin-stimulated glucose and amino acid uptake and phosphatidylinositol 3-kinase activity

The effect of okadaic acid, a serine/threonine phosphatase inhibitor, was analyzed in two insulin-responsive systems, the isolated mouse soleus muscle and 3T3-L1 adipocytes. While okadaic acid alone was a potent stimulator of glucose transport in both systems, it prevented transport stimulation by insulin. To gain insight into this inhibitory action, the activation of phosphatidylinositol 3-kinase (PI3-kinase), one of the earliest postreceptor steps identified so far, was studied. In 3T3-L1 adipocytes and muscle, insulin increased PI3-kinase activity in immunoprecipitates obtained with antibodies to phosphotyrosine. Okadaic acid alone had no effect but strongly inhibited this hormonal action. Okadaic acid treatment did not interfere with insulin-induced receptor autophosphorylation or with its tyrosine kinase activity toward artificial substrates. In contrast, in the presence of the phosphatase inhibitor, we did not observe tyrosine phosphorylation of the insulin receptor cellular substrate p185 (IRS-1) or immunoprecipitation of PI3-kinase by antibodies to phosphotyrosine. These results suggest that okadaic acid interferes with insulin's stimulation of glucose transport by inhibiting IRS-1 phosphorylation and its association with PI3-kinase and/or other signaling molecules. However, okadaic acid did not block the insulin stimulation of aminoisobutyric acid uptake in muscle. This would indicate that IRS-1 phosphorylation and PI3-kinase activation are not required for all the effects of insulin and that the serine/threonine phosphorylation events implicated in the translocation of glucose transporters are not controlling amino acid transport in muscle.

Protein kinase C activators selectively inhibit insulin-stimulated system A transport activity in skeletal muscle at a post-receptor level

We have investigated the role of phorbol esters on different biological effects induced by insulin in muscle, such as activation of system A transport activity, glucose utilization and insulin receptor function. System A transport activity was measured by monitoring the uptake of the system A-specific analogue alpha-(methyl)aminoisobutyric acid (MeAIB), by intact rat extensor digitorum longus muscle. The addition of 12-O-tetradecanoylphorbol 13-acetate (TPA, 0.5 microM) for 60 or 180 min did not modify basal MeAIB uptake by muscle, suggesting that insulin signalling required to stimulate MeAIB transport does not involve protein kinase C activation. However, TPA added 30 min before insulin (100 nM) markedly inhibited insulin-stimulated MeAIB uptake. The addition of polymyxin B (0.1 mM) or H-7 (1 mM), protein kinase C inhibitors, alone or in combination with TPA leads to impairment of insulin-stimulated MeAIB uptake. This paradoxical pattern is incompatible with a unique action of Polymyxin B or H-7 on protein kinase C activity. Therefore these agents are not suitable tools with which to investigate whether a certain insulin effect is mediated by protein kinase C. TPA did not cause a generalized inhibition of insulin action. Thus both TPA and insulin increased 3-O-methylglucose uptake by muscle, and their effects were not additive. Furthermore, TPA did not modify insulin-stimulated lactate production by muscle. In keeping with this selective modification of insulin action, treatment of muscles with TPA did not modify insulin receptor binding or kinase activities. In conclusion, phorbol esters do not mimic insulin action on system A transport activity; however, they markedly inhibit insulin-stimulated amino acid transport, with no modification of insulin receptor function in rat skeletal muscle. It is suggested that protein kinase C activation causes a selective post-receptor modification on the biochemical pathway by which insulin activates system A amino acid transport in muscle.

Kinetics of system A amino acid uptake by muscle: effects of insulin and acute uremia

We found that acute renal failure (ARF) depresses maximal responsiveness to insulin of system A amino acid transport in muscle [Maroni, Karapanos, and Mitch. Am. J. Physiol. 251 (Renal Fluid Electrolyte Physiol. 20): F74-F80, 1986]. To examine mechanisms for this change, we measured the kinetics of system A in response to insulin and ARF in epitrochlearis muscle. Insulin stimulation increased the diffusion constant (KD) approximately twofold in muscles from both ARF and control rats, suggesting that insulin can modulate nonsaturable alpha-(methylamino)isobutyrate uptake. Compared with basal values, insulin did not significantly change maximal transport velocity (Vmax) in ARF or control rats; however, insulin decreased Michaelis constant (Km) by 79 and 63%, respectively. Inhibition of protein synthesis with cycloheximide did not prevent stimulation of system A by insulin. Acute uremia had no effect on nonsaturable uptake or basal system A kinetics. However, ARF produced opposing effects on insulin-stimulated system A kinetics. There was a approximately 47% decrease in Vmax, which was partially offset by a 66% reduction in Km. In summary, both insulin and ARF modulate system A kinetics by mechanisms that are independent of protein synthesis.

Effects of adrenaline and prior exercise on the release of alanine, glutamine and glutamate from incubated rat skeletal muscle

Catecholamines have been proposed as important regulators of the rate of amino acid release from skeletal muscle. In the present study, we have investigated the influence of adrenergic action and its possible interaction with exercise on muscle release and tissue content of alanine, glutamine and glutamate. For this purpose epitrochlearis muscles were dissected from resting and exercised (1 and 2 h) rats and incubated for 1 h in the presence or absence of adrenaline. In addition, muscles from water-immersed resting rats were included to separate the influence of the stress involved in the swimming exercise from that of muscle contractile activity per se. In muscles from untreated resting rats, the release, tissue content and total amount (released amount + tissue content) of the three amino acids were not influenced by 10(-7) M adrenaline; when the adrenaline concentration was raised to 10(-5) M only the tissue content of glutamate was significantly changed (-50%, P less than 0.001). However, in muscles of rats subjected to 2 h prior exercise or water immersion, 10(-7) M adrenaline significantly increased the release of glutamine (+ 48% and +34%, P less than 0.05) and glutamate (+38% and +27%, P less than 0.05). Moreover, 1 h of water immersion resulted in a significant increase in muscle glutamine and glutamate compared to values from the exercised and control rats. The data suggest that adrenergic action is involved in the regulation of muscle amino acid transport during exercise and that the stress involved in exercise may mask the influence of contractile activity per se on formation of amino acids in skeletal muscle.

Superoxide dismutase in rats with sepsis. Effect on survival rate and amino acid transport.

In a recent study, administration of the free radical scavenger superoxide dismutase (SOD) improved survival in rats with sepsis when administered two hours before induction of sepsis. The present study was designed to determine whether free radical-induced membrane damage is involved in the pathogenesis of decreased muscle amino acid uptake, noted in sepsis. Additionally, the effect on survival rate of SOD given after the onset of sepsis was studied. Sepsis was induced by cecal ligation and puncture in rats. Amino acid transport in incubated soleus muscles was studied using tritiated alpha-aminoisobutyric acid. Amino acid uptake was significantly reduced in muscle from rats with sepsis. Administration of SOD before induction of sepsis or added in vitro to incubated muscles with sepsis had no effect on alpha-aminoisobutyric acid uptake. Survival rate was not increased when SOD was given two hours after cecal ligation and puncture. The results suggest that free radical-induced membrane damage is not the mechanism of inhibited muscle amino acid transport in sepsis. Since survival was not improved by SOD administered after induction of sepsis, the role of the enzyme in the treatment of sepsis may be questioned.

System ASC and sodium-independent neutral amino acid transport in muscle of uremic rats.

Neutral amino acids are transported by systems A, ASC, and L. In the previous companion study we demonstrated that 2-(methylamino) isobutyrate (MeAIB) is a specific substrate for system A in muscle and that stimulation of system A by physiological concentrations of insulin is preserved in acute uremia (ARF). Insulin-stimulated uptake of the nonspecific probes cycloleucine and alpha-aminoisobutyrate (AIB) is reportedly blunted by uremia; the cause of this and whether transport by systems ASC and L is defective are unknown. In this study we examined these questions using incubated epitrochlearis muscles from normal fed, ARF, and sham-operated control (SO) rats. System ASC was studied by measuring AIB and cycloleucine uptake in the presence of inhibitors of systems A and L, MeAIB and 2-amino-2-norbornane carboxylic acid (BCH), respectively. System L was defined as sodium-independent uptake suppressible by BCH. Excess MeAIB completely inhibited insulin-stimulated AIB and cycloleucine uptake, indicating that system A is the only insulin-responsive neutral amino acid carrier in muscle. In ARF and SO mucles both AIB and cycloleucine uptake were indistinguishable in the absence or presence of insulin. Moreover, ARF caused no detectable abnormality in transport by systems ASC and L.

Inhibited muscle amino acid uptake in sepsis.

Amino acid uptake in vivo was determined in soleus (SOL) muscle, diaphragm, heart, and liver following intravenous injection of [3H]-alpha-amino-isobutyric acid ([3H]-AIB) in rats made septic by cecal ligation and puncture (CLP) and in sham-operated controls. Muscle amino acid transport was also measured in vitro by determining uptake of [3H]-AIB in incubated extensor digitorum longus (EDL) and SOL muscles. Results were expressed as distribution ratio between [3H]-AIB in intracellular and extracellular fluid. AIB uptake in vivo was reduced by 90% in SOL and cardiac muscle and by 45% in diaphragm 16 hours after CLP. In contrast, AIB uptake by liver was almost four times higher in septic than in control animals. AIB uptake in vitro was reduced by 18% in EDL 8 hours after CLP but was not significantly altered in SOL at the same time point. Sixteen hours after CLP, AIB uptake was significantly reduced in both muscles, i.e., by 17% in EDL and by 65% in SOL. When muscles from untreated rats were incubated in the presence of plasma from septic animals (16 hours CLP) or from animals injected with endotoxin (2 mg/kg body weight), AIB uptake was reduced. Addition of endotoxin in vitro (2-200 micrograms/ml) to incubated muscles did not affect AIB uptake. The results suggest that sepsis leads to marked impairment of amino acid transport system A in muscle and that this impairment is mediated by a circulating factor that is not endotoxin. Reduced uptake of amino acids by skeletal muscle during sepsis may divert amino acids to the liver for increased gluconeogenesis and protein synthesis.

The Actions of Insulin, Trypsin, and Electrical Stimulation on Amino Acid Transport in Muscle

Abstract The manner in which intact skeletal muscle cells regulate amino acid transport across the cell membrane was examined with the use of α-aminoisobutyrate in isolated frog sartorius muscles, and the results were compared with the behavior of sugar transport in this tissue. α-Aminoisobutyrate entered the muscle cells through two mechanisms, one involving active transport by a readily saturable system, the other resembling passive diffusion. Glucose did not compete with α-aminoisobutyrate for entry. Insulin enhanced the rate of penetration of α-aminoisobutyrate by increasing the Vmax of the saturable system without significantly changing its apparent Km for the amino acid; the rate of the diffusion-like process was not affected. A steady state of active accumulation of α-aminoisobutyrate was attained in muscles incubated for several days. The intracellular steady state concentration was increased by insulin. In contrast, 3-O-methylglucose equilibrated passively across the cell membrane, both in the presence and absence of insulin. Electrical stimulation of muscles and treatment with a low concentration, 2 µg per ml, of trypsin augmented the initial rate of entry of α-aminoisobutyrate into muscle cells; trypsin also raised the steady state level of accumulation. The response of this amino acid transport system to the stimulatory agents resembled that of the sugar transport system in the following particular ways. Insulin acted on both systems at 19° but not at 0° in a 3½-hour exposure. Electrical stimulation affected both systems at 0 and 19°. The effects of electrical stimulation on the transport of the amino acid or sugar persisted essentially undiminished for 5 to 6 hours after cessation of the treatment. Insulin increased the Vmax of entry without altering the apparent Km for either type of substrate. The amino acid transport system differed from the sugar transport system in its ability to accomplish an active accumulation of substrate, in its dependence on Na+ for optimal rates of transport, and in its notably smaller responses to insulin, electrical stimulation, and trypsin. Observations are cited which support the hypothesis that insulin, trypsin, and electrical stimulation enhance the penetration of sugar into the cells of frog sartorius muscles by altering the organization of the cell membrane so that a sugar carrier can move more rapidly within the lipid barrier. Our results suggest that a similar hypothesis is applicable to the transport of amino acids, provided that the additional assumption is made that energy coupling, accomplished through an interaction of the amino acid carrier with Na+, introduces steps that can become rate-limiting when transport is stimulated by the various agents.

Effect of puromycin on insulin-stimulated amino-acid transport in muscle.

INSULIN stimulates both the transport1,2 of amino-acids and their incorporation3 into protein in skeletal muscle. Previous attempts to associate these two effects have failed4–7. That is, inhibition of new protein synthesis by puromycin did not impair the stimulatory effect of insulin on amino-acid transport. It was therefore concluded that the stimulation of amino-acid transport by insulin did not depend on the synthesis of new protein. Recent studies have shown that the transport of neutral amino-acids in embryonic bone8, foetal calvarium (G. A. Finerman, S. J. Downing and L. E. Rosenberg, unpublished results), diaphragm9 and kidney cortex10 was impaired by puromycin or cycloheximide when 2 h of incubation8 or preincubation9,10 with these antibiotics preceded the uptake study. Because previous experiments with insulin and puromycin in diaphragm muscle did not measure amino-acid uptake after such intervals of preincubation with puromycin, we carried out experiments to reinvestigate the effect of prolonged inhibition of protein synthesis on the stimulation by insulin of amino-acid transport in muscle.