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Characteristics of acidic, basic and neutral amino acid transport in the perfused rat hindlimb
Abstract
1. We have employed a paired-tracer isotope dilution technique in a perfused rat hindlimb preparation to obtain information on the kinetics of transport across the sarcolemmal membranes of acidic, neutral and basic amino acids. 2. We have defined the characteristics of the saturable transport of amino acids normally regarded as paradigm substrates for the A, ASC, L, y+(basic) and the dicarboxylic amino acid transport systems. Their maximal transport capacities (Vmax, nmol min-1 (g muscle)-1 and substrate concentrations for half-maximal transport (Km, mM) of representative amino acid substrates are as follows: 2-aminoisobutyrate (AIB), Vmax = 15 +/- 7, Km = 1.26 +/- 0.6; alanine, Vmax = 332 +/- 53, Km = 3.9 +/- 0.9; serine, Vmax = 410 +/- 61, Km 3.4 +/- 0.5; leucine, Vmax = 2800 +/- 420, Km = 20 +/- 2; lysine, Vmax = 136 +/- 46, Km = 2.1 +/- 1.3; glutamate, Vmax = 86 +/- 6, Km = 1.05 +/- 0.05; proline, Vmax = 196 +/- 48, Km = 4.1 +/- 0.6. 3. Glycine uptake was faster than expected on the basis of diffusion but was not saturable and showed uptake that could be best described by a first-order rate constant of 0.07 +/- 0.003 min-1. 4. We have attempted to discriminate kinetically between possible routes of entry for an amino acid on the basis of competitive and non-competitive interaction between substrates potentially sharing common routes. On this basis, the major routes of alanine entry appear to be via the ASC and L systems with the A system playing a quantitatively minor role. Glutamate and aspartate appear to be transported exclusively by a dicarboxylate amino acid carrier. The branched-chain amino acids (BCAA) and the aromatic amino acid, phenylalanine, are almost equivalent substrates for an L-like system. 5. Insulin had no detectable effect on the uptake of paradigm substrates for ASC, L, y+, the dicarboxylic amino acid or glycine transport systems. 6. Transport of serine and lysine was Na+ dependent. Lysine transport apparently occurred with a stoichiometry of 2 Na+: 1 lysine. With the exception of alanine, whose transport was partially Na+ dependent, all other amino acids examined in the present study were transported in a Na+-independent manner.
... was largely confined to descriptions of tracer uptake of a handful of amino acids, including a number of non-metabolizable amino acid analogues such as aminoisobutyric acid and methylaminoisobutyric acid. We set to work to define, in a systematic fashion, the characteristics of the transport of a variety of amino acids, using perfused skeletal muscle [4,5], muscle plasmamembrane vesicle preparations [6,7] and muscle culture systems [8]. We were particularly interested in relating the characteristics of the transporters to the involvement of amino acids in the whole body fuel and nitrogen economy. ...
... In contrast, branchedchain amino acids (principally leucine) activate Na+-dependent System A amino acid transport in L6 rat skeletal-muscle cells [14] by a mechanism involving transcriptional regulation. This observation is consistent with the known anabolic effects of leucine on protein turnover in skeletal muscle [15,16] but must reflect the operation of a mechanism distinct from the 'adaptive regulation' described above for glutamine (not least because transport by System A represents a minor component of leucine entry into muscle [5]). These contrasting effects of leucine and glutamine on different amino acid-transport systems highlight the importance of particular nutrients as modulators of specific cellular processes and metabolic pathways through regulation of gene transcription. ...
... This feature indicates that LPG is not the major cardiac glutamate transporter. In skeletal muscle the major gluta-mate transporter appears to be Na+-independent [5,13], except in circumstances of extracellular glutamate or glutamine deprivation, when the Na+-dependent variety shows substantial up-regulation [13]. Another odd feature of the cardiac glutamate transporter is that it is strongly inhibited by glutamine (at 20-fold inhibitor excess, glutamine inhibits glutamate transport by about 50%). ...
... There is also evidence for the operation in muscle of the transport of the -amino acid, taurine, by a Na + -dependent carrier [245]. Cationic amino acids are transported in muscle by the Na + -independent system y + [246] and aspartate and glutamate are taken up in muscle cells and in perfused hindquarter via Na + -dependent and Na + -independent mechanisms (Table 1) [246,247]. Because of the limitations imposed in the studies using skeletal muscle, it is likely that there are more amino acid agencies in this tissue than the ones identified by kinetic means. ...
... There is also evidence for the operation in muscle of the transport of the -amino acid, taurine, by a Na + -dependent carrier [245]. Cationic amino acids are transported in muscle by the Na + -independent system y + [246] and aspartate and glutamate are taken up in muscle cells and in perfused hindquarter via Na + -dependent and Na + -independent mechanisms (Table 1) [246,247]. Because of the limitations imposed in the studies using skeletal muscle, it is likely that there are more amino acid agencies in this tissue than the ones identified by kinetic means. ...
... The precise nature of the mechanisms that regulate system A is unknown since identification of the system A gene has not been reported. The activity of system N m is responsible for the transport of glutamine and asparagine in a sodium-and pH-dependent manner, with stoichiometry of 1 Na + :1 glutamine [243,246]. N m is responsible for both the influx and the efflux of glutamine in muscle and is subject to regulation. Glutamine transport in muscle is up-regulated in response to incubation in a glutamine-free medium [260], i.e. it undergoes adaptive regulation. ...
... There is also evidence for the operation in muscle of the transport of the -amino acid, taurine, by a Na + -dependent carrier [245]. Cationic amino acids are transported in muscle by the Na + -independent system y + [246] and aspartate and glutamate are taken up in muscle cells and in perfused hindquarter via Na + -dependent and Na + -independent mechanisms (Table 1) [246, 247]. Because of the limitations imposed in the studies using skeletal muscle, it is likely that there are more amino acid agencies in this tissue than the ones identified by kinetic means. ...
... There is also evidence for the operation in muscle of the transport of the -amino acid, taurine, by a Na + -dependent carrier [245]. Cationic amino acids are transported in muscle by the Na + -independent system y + [246] and aspartate and glutamate are taken up in muscle cells and in perfused hindquarter via Na + -dependent and Na + -independent mechanisms (Table 1) [246, 247]. Because of the limitations imposed in the studies using skeletal muscle, it is likely that there are more amino acid agencies in this tissue than the ones identified by kinetic means. ...
... The precise nature of the mechanisms that regulate system A is unknown since identification of the system A gene has not been reported. The activity of system N m is responsible for the transport of glutamine and asparagine in a sodium-and pH-dependent manner, with stoichiometry of 1 Na + :1 glutamine [243, 246]. N m is responsible for both the influx and the efflux of glutamine in muscle and is subject to regulation. ...
El múscul té un paper central en el metabolisme. Així, el múscul utilitza quantitats substancials de glucosa durant l'estat absortiu, i els canvis en la captació muscular de la glucosa provoquen alteracions en la utilització global de la glucosa per l'organisme sencer. El múscul constitueix també el principal reservori corporal d'aminoàcids i de proteïnes. A més, el metabolisme muscular és mantingut mitjantçant l'activitat de molts diferents transportadors localitzats a la membrana plasmàtica, com són els transportadors de glucosa, carnitina, creatina o aminoàcids; aquests transportadors capten o alliberen, a través de la membrana plasmàtica de la cèl·lula muscular, diferents substrats o metabòlits. L'objectiu d'aquesta revisió consisteix en la caracterització molecular de les principals proteïnes transportadores presents a la membrana plasmàtica de les cèl·lules musculars, així com l'anàlisi de les seves propietats reguladores. Muscle is a major player in metabolism. It uses large amounts of glucose in the absorptive state and changes in muscle insulin-stimulated glucose uptake alter whole-body glucose disposal. Lipid substrates such as fatty acids or ketone bodies are preferentially used by muscle in certain physiological conditions. Muscle is also the main reservoir of amino acids and protein. The activity of many different plasma membrane transporters such as glucose carriers, carnitine, creatine or amino acid transporters maintain muscle metabolism by taking up or releasing substrates or metabolites across the cell surface. The goal of this review is the molecular characterization of muscle membrane transporter proteins and the analysis of their regulatory roles.
... We utilized a dual oral tracer model, as previous studies measuring dietary amino acid incorporation after feeding and resistance exercise have only traced phenylalanine into skeletal muscle [5][6][7], which would represent only one substrate (leucine being another) for LAT1 [27]. Furthermore, there is evidence to suggest that leucine and phenylalanine are equivalent substrates for LAT1 [28] but that there may be two pools of muscle intracellular leucine whereby exogenous leucine is primarily directed toward oxidation, and intracellular leucine from protein breakdown is preferentially reutilized for protein synthesis [29]. This influenced our decision to include two metabolic tracers of different essential amino acids, one of which (leucine) is generally considered to be the preeminent anabolic amino acid [11,30,31]. ...
... In contrast, the basal expression of LAT1 did not influence dietary leucine incorporation, but was negatively correlated with phenylalanine incorporation into myofibrillar protein. These disparate findings between presumably equivalent LAT1 substrates [28] may be explained in part by the potential for separate intracellular leucine pools that favor the oxidation of externally derived amino acids [29]. In contrast, phenylalanine is oxidized in the liver [43] and would only be utilized for protein synthesis within skeletal muscle, which may explain our ability to delineate a correlation (albeit negative) between dietary phenylalanine incorporation and LAT1 expression. ...
The influx of essential amino acids into skeletal muscle is primarily mediated by the large neutral amino acid transporter 1 (LAT1), which is dependent on the glutamine gradient generated by the sodium-dependent neutral amino acid transporter 2 (SNAT2). The protein expression and membrane localization of LAT1 may be influenced by amino acid ingestion and/or resistance exercise, although its acute influence on dietary amino acid incorporation into skeletal muscle protein has not been investigated. In a group design, healthy males consumed a mixed carbohydrate (0.75 g·kg−1) crystalline amino acid (0.25 g·kg−1) beverage enriched to 25% and 30% with LAT1 substrates L-[1-13C]leucine (LEU) and L-[ring-2H5]phenylalanine (PHE), respectively, at rest (FED: n = 7, 23 ± 5 y, 77 ± 4 kg) or after a bout of resistance exercise (EXFED: n = 7, 22 ± 2 y, 78 ± 11 kg). Postprandial muscle biopsies were collected at 0, 120, and 300 min to measure transporter protein expression (immunoblot), LAT1 membrane localization (immunofluorescence), and dietary amino acid incorporation into myofibrillar protein (ΔLEU and ΔPHE). Basal LAT1 and SNAT2 protein contents were correlated with each other (r = 0.55, p = 0.04) but their expression did not change across time in FED or EXFED (all, p > 0.05). Membrane localization of LAT1 did not change across time in FED or EXFED whether measured as outer 1.5 µm intensity or membrane-to-fiber ratio (all, p > 0.05). Basal SNAT2 protein expression was not correlated with ΔLEU or ΔPHE (all, p ≥ 0.05) whereas basal LAT1 expression was negatively correlated with ΔPHE in FED (r = −0.76, p = 0.04) and EXFED (r = −0.81, p = 0.03) but not ΔLEU (p > 0.05). Basal LAT1 membrane localization was not correlated with ΔLEU or ΔPHE (all, p > 0.05). Our results suggest that LAT1/SNAT2 protein expression and LAT1 membrane localization are not influenced by acute anabolic stimuli and do not positively influence the incorporation of dietary amino acids for de novo myofibrillar protein synthesis in healthy young males.
... The basic characteristics of glutamine and gluta mate transport in skeletal muscle are as follows (see Table 1): glutamate transport appears to be modulated by a low activity high affinity carrier that in perfused skeletal muscle, at least, appears to be H+-dependent rather than Na+-dependent (Hundal et al. 1989). In pri mary culture of rat skeletal muscle cells, the^^de pendent glutamate transporter can be substantially in duced by culturing the cells in a medium in which glutamine concentration is low (Low et al. 1994) (see later). ...
... In pri mary culture of rat skeletal muscle cells, the^^de pendent glutamate transporter can be substantially in duced by culturing the cells in a medium in which glutamine concentration is low (Low et al. 1994) (see later). Glutamate transport appears to be difficult to accelerate acutely in perfused skeletal muscle and to be unaffected by alterations in membrane potential or concentrations of other amino acids (apart from aspartate) or hormones (Hundal et al. 1989). Glutamine transport, on the other hand, is mediated by a very high activity transporter that in perfused skeletal muscle, and probably in vivo also, has a Kmabove the prevailing plasma concentration (Hundal et al. 1987). ...
The glutamine and glutamate transport ers in skeletal muscle and heart appear to play a role in control of the steady-state concentration of amino acids in the intracellular space and, in the case of skel etal muscle at least, in the rate of loss of glutamine to the plasma and to other organs and tissues. This arti cle reviews what is currently known about transporter characteristics and mechanisms in skeletal muscle and heart, the alterations in transport activity in patho- physiological conditions and the implications for ana bolic processes and cardiac function of altering the availability of glutamine. The possibilities that gluta mine pool size is part of an osmotic signaling mecha nism to regulate whole body protein metabolism is dis cussed and evidence is shown from work on cultured muscle cells. The possible uses of glutamine in main taining cardiac function perioperatively and in promot ing glycogen metabolism are discussed. J. Nutr. 126: 1142S-1149S, 1996.
... In industrial fermentation media, aliphatic amino acids form aqueous biphasic systems with ionic liquids that effectively separate aliphatic and aromatic amino acid mixtures (Capela et al. 2017). The role and rate of acidic, basic, and neutral amino acids in transfer across the muscle membrane are used to develop biotherapeutics with different transfer rates (Hundal et al. 1989). ...
Peptides are short linear molecules consisting of amino acids that play an essential role in most biological processes. They can treat diseases by working as a vaccine or antimicrobial agent and serves as a cancer molecule to deliver the drug to the target site for the treatment of cancer. They have the potential to solve the drawbacks of current medications and can be industrially produced in large quantities at low cost. However, poor chemical and physical stability, short circulating plasma half-life, and solubility are some issues that need solutions before they can be used as therapeutics. PepAnalyzer tool is a user-friendly tool that predicts 15 different properties such as binding potential, half-life, transmembrane patterns, test tube stability, charge, isoelectric point, molecular weights, and molar extinction coefficients only using the sequence. The tool is designed using BioPython utility and has even results with standard tools, such as Expasy, EBI, Genecorner, and Geneinfinity. The tool assists students, researchers, and the pharmaceutical sector. The PepAnalyzer tool's online platform is accessible at the link: http://www.iksmbrlabdu.in/peptool
... Last four amino acids in c-region of signal peptides were consensus sequence 'RSWA'. The amino acid on the cleavage site was Alanine (A) which is a nonpolar and uncharged or neutral amino acid (Hundal et al., 1989). This also supports our results of signal peptide prediction because the amino acid in cleavage region/site must be small and neutral for ease of correct cleavage (von Heijne, 1985). ...
Lepidoptera is the second most diverse insect order outnumbered only by the Coeleptera. Acetylcholinesterase (AChE) is the major target site for insecticides. Extensive use of insecticides, to inhibit the function of this enzyme, have resulted in the development of insecticide resistance. Complete knowledge of the target proteins is very important to know the cause of resistance. Computational annotation of insect acetylcholinesterase can be helpful for the characterization of this important protein. Acetylcholinesterase of fourteen lepidopteran insect pest species was annotated by using different bioinformatics tools. AChE in all the species was hydrophilic and thermostable. All the species showed lower values for instability index except L. orbonalis, S. exigua and T. absoluta. Highest percentage of Arg, Asp, Asn, Gln and Cys were recorded in P. rapae. High percentage of Cys and Gln might be reason for insecticide resistance development in P. rapae. Phylogenetic analysis revealed the AChE in T. absoluta, L. orbonalis and S. exigua are closely related and emerged from same primary branch. Three functional motifs were predicted in eleven species while only two were found in L. orbonalis, S. exigua and T. absoluta. AChEin eleven species followed secretory pathway and have signal peptides. No signal peptides were predicted for S. exigua, L. orbonalis and T. absoluta and follow non secretory pathway. Arginine methylation and cysteine palmotylation was found in all species except S. exigua, L. orbonalis and T. absoluta. Glycosylphosphatidylinositol (GPI) anchor was predicted in only nine species. 3D structures of proteins were predicted and refined.
... Although the widespread metabolic importance of glutamine for the immune system and gut had been identified, the control and functional importance of the large muscle free glutamine pool in health and disease was not understood. Mike's team identified a stereospecific Na + -linked insulin-sensitive glutamine transporter (Nm), which was also sensitive to those factors known to be associated with loss of muscle glutamine, e.g., corticosteroids, endotoxins, and denervation (26)(27)(28)(29)(30)). The team was also able to reproduce the relation between muscle glutamine concentration and MPS in their hind-limb preparation (31), which had been observed in vivo in healthy, malnourished, and endotoxin-treated rats (32). ...
... As proposed in Nolan and Lee [32], exchanges of metabolites between intracellular and extracellular volumes were not considered to be controlled by membrane protein transporters but by intracellular enzymes. This model simplification is also supported considering that the extracellular concentration of the metabolites is higher than their associated membrane transporter half-saturation Michaelis-Menten constant [57]. Thus, the uptake or secretion of extracellular metabolites is controlled by several intracellular reactions rather than their transporter. ...
Because of their unique ability to modulate the immune system, mesenchymal stromal cells (MSCs) are widely studied to develop cell therapies for detrimental immune and inflammatory disorders. However, controlling the final cell phenotype and determining immunosuppressive function following cell amplification in vitro often requires prolonged cell culture assays, all of which contribute to major bottlenecks, limiting the clinical emergence of cell therapies. For instance, the multipotent Wharton’s Jelly mesenchymal stem/stromal cells (WJMSC), extracted from human umbilical cord, exhibit immunosuppressive traits under pro-inflammatory conditions, in the presence of interferon-γ (IFNγ), and tumor necrosis factor-α (TNFα). However, WJMSCs require co-culture bioassays with immune cells, which can take days, to confirm their immunomodulatory function. Therefore, the establishment of robust cell therapies would benefit from fast and reliable characterization assays. To this end, we have explored the metabolic behaviour of WJMSCs in in vitro culture, to identify biomarkers that are specific to the cell passage effect and the loss of their immunosuppressive phenotype. We clearly show distinct metabolic behaviours comparing WJMSCs at the fourth (P4) and the late ninth (P9) passages, although both P4 and P9 cells do not exhibit significant differences in their low immunosuppressive capacity. Metabolomics data were analysed using an in silico modelling platform specifically adapted to WJMSCs. Of interest, P4 cells exhibit a glycolytic metabolism compared to late passage (P9) cells, which show a phosphorylation oxidative metabolism, while P4 cells show a doubling time of 29 h representing almost half of that for P9 cells (46 h). We also clearly show that fourth passage WJMSCs still express known immunosuppressive biomarkers, although, this behaviour shows overlapping with a senescence phenotype.
... • Except for the amino acids alanine, asparagine, aspartate, glutamine and glutamate, all other extracellular metabolite concentrations are considered higher that the affinity constants of associated cell membrane transporters 19 . Therefore, intra-and extracellular metabolites that are consumed or secreted were taken as being at the same concentration across the cell membrane; a reliable assumption we have demonstrated in past works dealing with similar models 10,11,[16][17][18] . ...
Recent years have witnessed an increasing interest at understanding the role of myeloid-derived suppressor cells (MDSCs) in cancer-induced immunosuppression, with efforts to inhibit their maturation and/or their activity. We have thus modelled MDSCs central carbon metabolism and bioenergetics dynamic, calibrating the model using experimental data on in vitro matured mice bone marrow cells into MDSCs. The model was then used to probe the cells metabolic state and dynamics, performing a dynamic metabolic flux analysis (dMFA) study. Indeed, MDSCs maturation correlates with a high glycolytic flux contributing to up to 95% of the global ATP turnover rate, while most of the glucose-derived carbon enters the TCA cycle. Model simulations also reveal that pentose phosphate pathway and oxidative phosphorylation activities were kept at minimal levels to ensure NADPH production and anabolic precursors synthesis. Surprisingly, MDSCs immunosuppressive activity, i.e. L-arginine uptake, metabolism and endogenous synthesis, only consumes sparse quantities of energy-rich nucleotides (ATP and NADPH). Therefore, model simulations suggest that MDSCs exhibit a heterogeous metabolic profile similar to tumour cells. This behavior is probably an indirect immunosuppressive mechanism where MDSCs reduce the availability of carbon sources in the tumour periphery microenvironment, which could explain the dysfuntion and death of immune effector cells.
... Skeletal muscles most abundantly require glutamic acid [24]. Glutamic acid is the only amino acid that is actively taken up from the circulation by the muscles in the post-absorptive state [25]. Intracellular degradation of myofibrillar proteins also release glutamic acid with large amounts contributing to the muscle glutamic acid pool [26]. ...
Background:
Like Duchenne muscular dystrophy (DMD), the Golden Retriever Muscular Dystrophy (GRMD) dog model of DMD is characterized by muscle necrosis, progressive paralysis, and pseudohypertrophy in specific skeletal muscles. This severe GRMD phenotype includes atrophy of the biceps femoris (BF) as compared to unaffected normal dogs, while the long digital extensor (LDE), which functions to flex the tibiotarsal joint and serves as a digital extensor, undergoes the most pronounced atrophy. A recent microarray analysis of GRMD identified alterations in genes associated with lipid metabolism and energy production.
Methods:
We, therefore, undertook a non-targeted metabolomics analysis of the GRMD BF (affected) and LDE (unaffected) using GC-MS to identify underlying metabolic defects specific for affected GRMD skeletal muscle.
Results:
Untargeted metabolomics analysis of severely-affected GRMD muscle (BF) identified eight significantly altered metabolites in affected GRMD muscle (BF), including significantly decreased stearamide (0.23-fold of controls, p = 2.89 × 10(-3)), carnosine (0.40-fold of controls, p = 1.88 × 10(-2)), fumaric acid (0.40-fold of controls, p = 7.40 × 10(-4)), lactamide (0.33-fold of controls, p = 4.84 × 10(-2)), myoinositol-2-phosphate (0.45-fold of controls, p = 3.66 × 10(-2)), and significantly increased oleic acid (1.77-fold of controls, p = 9.27 × 10(-2)), glutamic acid (2.48-fold of controls, p = 2.63 × 10(-2)), and proline (1.73-fold of controls, p = 3.01 × 10(-2)). Pathway enrichment analysis identified significant enrichment for arginine/proline metabolism (p = 5.88 × 10(-4), FDR 4.7 × 10(-2)), where alterations in L-glutamic acid, proline, and carnosine were found. Additionally, multiple Krebs cycle intermediates were significantly decreased (e.g., malic acid, fumaric acid, citric/isocitric acid, and succinic acid), suggesting that altered energy metabolism may be underlying the observed GRMD BF muscle dysfunction.
Conclusions:
The identification of elevated BF oleic acid (a long-chain fatty acid) is consistent with recent microarray studies identifying altered lipid metabolism genes, while alterations in arginine and proline metabolism are consistent with recent studies identifying elevated L-arginine in DMD patient sera as a biomarker of disease. Together, these studies demonstrate muscle-specific alterations in GRMD-affected muscle, which illustrate previously unidentified metabolic changes.
... For example, there is a high affinity of glucose for its transporters, which results in the proportional uptake of glucose from extracellular sources 44 . However, the affinity for several AAs to their transporters is lower, which results in the less efficient uptake of AAs into the cells 45,46 . Thus, despite increased leakiness in the tissue, a constant uptake of glucose will occur, whereas the lower efficiency of the AA transporters will result in higher extracellular AA levels. ...
Metabolic reprogramming is a hallmark of cancer. Nutrient availability in the tissue microenvironment determines cellular events and may play a role in breast carcinogenesis. High mammographic density is an independent risk factor for breast cancer. Whether nutrient availability differs in normal breast tissues with various densities is unknown. Therefore we investigated whether breast tissues with various densities exhibited differences in nutrient availability. Healthy postmenopausal women from the regular mammographic screening program who had either predominantly fatty breast tissue (nondense), n = 18, or extremely dense breast tissue (dense), n = 20, were included. Microdialysis was performed for the in vivo sampling of amino acids (AAs), analyzed by ultra-high performance liquid chromatography with tandem mass spectroscopy, glucose, lactate and vascular endothelial growth factor (VEGF) in breast tissues and, as a control, in abdominal subcutaneous (s.c.) fat. We found that dense breast tissue exhibited significantly increased levels of 20 proteinogenic AAs and that 18 of these AAs correlated significantly with VEGF. No differences were found in the s.c. fat, except for one AA, suggesting tissue-specific alterations in the breast. Glucose and lactate were unaltered. Our findings provide novel insights into the biology of dense breast tissue that may be explored for breast cancer prevention strategies.
... If similar constraints existed for AA then this would provide a focus for genetic selection based on increased transporter number or activity. Direct measurement of AA transport is difficult to assess in vivo, except for small laboratory species (Banos et al. 1973;Hundal et al. 1989), but indirect measures, based on kinetic transfers, can be applied to larger mammals (Biolo et al. 1992). This approach has shown that retention by ovine hind-quarter tissue (mainly muscle) is only 9-39% of inward transport (Hoskin et al. 2001(Hoskin et al. , 2003Fig. ...
The dynamics of protein turnover confer great advantages for homeothermy, plasticity and metabolic function in mammals. The different roles played by the various organs have led to aspects of protein synthesis and degradation that aid the various functions performed. The so-called "non-productive" organs such as the gastro-intestinal tract and liver produce large quantities of export proteins that perform vital functions. Not all these proteins are recovered, however, and thus function can result in lowered net conversion of plant protein to animal products. The splanchnic tissues also oxidize essential amino acids (AA). For example, the gut catabolizes leucine, lysine and methionine, but not threonine and phenylalanine, as part of a complex interaction between AA supply and tissue metabolic activity. Losses by oxidation and endogenous secretions can markedly alter the pattern of absorbed AA. The fractional rates of extraction of total AA inflow to the liver are low and this allows short-term flexibility in controlling supply to peripheral tissues. Recent evidence suggests that the role of the liver in AA catabolism is more a response to non-use by other tissues rather than an immediate regulation of supply to the periphery. Neither arterial supply of AA nor the rate of transport into peripheral tissues limits protein gain, except when supply is very limited. Rather, control is probably exerted via hormone-nutrient interactions.
... If similar constraints existed for AA then this would provide a focus for genetic selection based on increased transporter number or activity. Direct measurement of AA transport is difficult to assess in vivo, except for small laboratory species (Banos et al. 1973;Hundal et al. 1989), but indirect measures, based on kinetic transfers, can be applied to larger mammals (Biolo et al. 1992). This approach has shown that retention by ovine hind-quarter tissue (mainly muscle) is only 9-39% of inward transport (Hoskin et al. 2001(Hoskin et al. , 2003Fig. ...
The dynamics of protein turnover confer great advantages for homeothermy, plasticity and metabolic function in mammals. The different roles played by the various organs have led to aspects of protein synthesis and degradation that aid the various functions performed. The so-called “non-productive” organs such as the gastro-intestinal tract and liver produce large quantities of export proteins that perform vital functions. Not all these proteins are recovered, however, and thus function can result in lowered net conversion of plant protein to animal products. The splanchnic tissues also oxidize essential amino acids (AA). For example, the gut catabolizes leucine, lysine and methionine, but not threonine and phenylalanine, as part of a complex interaction between AA supply and tissue metabolic activity. Losses by oxidation and endogenous secretions can markedly alter the pattern of absorbed AA. The fractional rates of extraction of total AA inflow to the liver are low and this allows short-term flexibility ...
... 66 In vitro and in vivo experiments have shown that insulin stimulates sodium-dependent system A amino acid transporter, in which alanine is the major substrate and has a large transmembrane concentration gradient. [67][68][69][70][71][72] A recent study indicated that long-term high-dose insulin infusion combined with enteral feeding markedly stimulates transmembrane amino acid transport in skeletal muscle, resulting in the improvement of amino acid and protein balance in skeletal muscle in severely burned patients (Fig. 2). 53 These results have provided an insight into the mechanism involved in the regulation of the balance of amino acid and protein kinetics in critically ill patients. ...
The response to critical illness involves alterations in all aspects of metabolic control, favoring catabolism of body protein. In particular, body protein loss occurring as a result of the alteration of protein metabolism has been reported to be inversely correlated with the survival of critically ill patients. Despite the availability of various therapeutic modalities aiming to prevent loss of the body protein pool, such as total parenteral nutrition, enteral nutrition designed to provide excessive calories as a form of energy substrate, and protein itself, the loss of body protein cannot be prevented by any of these. Loss of the boyd protein store occurs as a consequence of the alteration of the intermediate metabolism that works for the production of energy substrate. This alteration of substrate metabolism may be linked to the alteration of protein metabolism. However, no specific factors regulating amino acid and protein metabolism have been identified. Thus, further investigations evaluating amino acid and protein metabolism are required to obtain better understanding of metabolic regulation in the body, which may lead to the development of novel and more effective therapeutic modalities for nutrition in the future.
... Moreover, the uptake rates of extracellular metabolites (glucose, glutamine, amino acids) are the dominant factors driving changes in the metabolic system. However, the Km values for most metabolite transporters are low relative to the extracellular concentration of the metabolites (Tables S6-S7) [52]. This suggests that the transport of a metabolite into a cell may not be mainly controlled by the transporter, but rather from the intracellular enzymatic reactions and regulation. ...
Monoclonal antibody producing Chinese hamster ovary (CHO) cells have been shown to undergo metabolic changes when engineered to produce high titers of recombinant proteins. In this work, we have studied the distinct metabolism of CHO cell clones harboring an efficient inducible expression system, based on the cumate gene switch, and displaying different expression levels, high and low productivities, compared to that of the parental cells from which they were derived. A kinetic model for CHO cell metabolism was further developed to include metabolic regulation. Model calibration was performed using intracellular and extracellular metabolite profiles obtained from shake flask batch cultures. Model simulations of intracellular fluxes and ratios known as biomarkers revealed significant changes correlated with clonal variation but not to the recombinant protein expression level. Metabolic flux distribution mostly differs in the reactions involving pyruvate metabolism, with an increased net flux of pyruvate into the tricarboxylic acid (TCA) cycle in the high-producer clone, either being induced or non-induced with cumate. More specifically, CHO cell metabolism in this clone was characterized by an efficient utilization of glucose and a high pyruvate dehydrogenase flux. Moreover, the high-producer clone shows a high rate of anaplerosis from pyruvate to oxaloacetate, through pyruvate carboxylase and from glutamate to α-ketoglutarate, through glutamate dehydrogenase, and a reduced rate of cataplerosis from malate to pyruvate, through malic enzyme. Indeed, the increase of flux through pyruvate carboxylase was not driven by an increased anabolic demand. It is in fact linked to an increase of the TCA cycle global flux, which allows better regulation of higher redox and more efficient metabolic states. To the best of our knowledge, this is the first time a dynamic in silico platform is proposed to analyze and compare the metabolomic behavior of different CHO clones.
... It has been reported that insulin stimulates peripheral BCAA uptake in fasted men (14). Interestingly, however, the AA transport system L, which largely mediates BCAA transport (8), appears to be insulin insensitive (18). Alternatively, the insulin-sensitive AA transport system A (25) has been reported to have some affinity for cysteine, methionine, and Leu (44). ...
Protein and certain amino acids (AA) have been found to lower blood glucose. Although these glucose-lowering AA are important modulators of skeletal muscle metabolism, their impact on muscle glucose uptake remains unclear. We therefore examined how an AA mixture consisting of 2 mM isoleucine, 0.012 mM cysteine, 0.006 mM methionine, 0.0016 mM valine, and 0.014 mM leucine impacts skeletal muscle glucose uptake in the absence or presence of a submaximal (sINS) or maximal insulin (mINS) concentration. The AA mixture, sINS, and mINS significantly increased 2-[(3)H]deoxyglucose (2-DG) uptake by 63, 79, and 298% above basal, respectively. When the AA mixture was combined with sINS and mINS, 2-DG uptake was further increased significantly by 26% (P = 0.028) and 14% (P = 0.032), respectively. Western blotting analysis revealed that the AA mixture increased basal and sINS Akt substrate of 160 kDa (AS160) phosphorylation, while AA mixture did not change phosphorylation of Akt or mammalian target of rapamycin (mTOR) under these conditions. Interestingly, addition of the AA mixture to mINS increased phosphorylation of mTOR, Akt as well as AS160, compared with mINS alone. These data suggest that certain AA increase glucose uptake in the absence of insulin and augment insulin-stimulated glucose uptake in an additive manner. Furthermore, these effects appear to be mediated via a pathway that is independent from the canonical insulin cascade and therefore may prove effective as an alternative therapeutic treatment for insulin resistance.
... The branched-chain (leucine, valine, and isoleucine) and the aromatic (phenylalanine and tyrosine) amino acids are preferably transported through system L (15-17, 19, 20). This sodium-independent system is unable to generate high transmembrane gradients for its substrates (151617 19, 20). It has been shown that the kinetic characteristics of system L are not influenced by insulin (15-17, 19, 20). ...
We have investigated the mechanisms of the anabolic effect of insulin on muscle protein metabolism in healthy volunteers, using stable isotopic tracers of amino acids. Calculations of muscle protein synthesis, breakdown, and amino acid transport were based on data obtained with the leg arteriovenous catheterization and muscle biopsy. Insulin was infused (0.15 mU/min per 100 ml leg) into the femoral artery to increase femoral venous insulin concentration (from 10 +/- 2 to 77 +/- 9 microU/ml) with minimal systemic perturbations. Tissue concentrations of free essential amino acids decreased (P < 0.05) after insulin. The fractional synthesis rate of muscle protein (precursor-product approach) increased (P < 0.01) after insulin from 0.0401 +/- 0.0072 to 0.0677 +/- 0.0101%/h. Consistent with this observation, rates of utilization for protein synthesis of intracellular phenylalanine and lysine (arteriovenous balance approach) also increased from 40 +/- 8 to 59 +/- 8 (P < 0.05) and from 219 +/- 21 to 298 +/- 37 (P < 0.08) nmol/min per 100 ml leg, respectively. Release from protein breakdown of phenylalanine, leucine, and lysine was not significantly modified by insulin. Local hyperinsulinemia increased (P < 0.05) the rates of inward transport of leucine, lysine, and alanine, from 164 +/- 22 to 200 +/- 25, from 126 +/- 11 to 221 +/- 30, and from 403 +/- 64 to 595 +/- 106 nmol/min per 100 ml leg, respectively. Transport of phenylalanine did not change significantly. We conclude that insulin promoted muscle anabolism, primarily by stimulating protein synthesis independently of any effect on transmembrane transport.
Carrier-mediated transport of blood-borne substrates at the blood-tissue interface in the microcirculation is a very rapid process and influx of labelled molecules can often be measured within the time of a single circulation through an organ. The plasma membranes involved in the tracer uptake may be those of (i) the endothelial cell, (ii) parenchymal cell and (iii) neuronal terminals.
Techniques for studying amino acid transport in various cells and organelles have been well established for a number of years. Detailed protocols for the measurement of amino acid transport in various systems have recently appeared in a series of articles in Methods in Enzymology (Kilberg, 1989; Eddy and Johnson, 1989; Harvey and Ellory, 1989; Murer et al., 1989; Hopf er, 1989; McGivan, 1989). The purpose of this article is not to recapitulate this information in detail but rather to focus on points of critical importance in various procedures. The discussion will concentrate on aspects of the measurement of plasma membrane transport. Some details of the measurement of transport in subcellular organelles will be found elsewhere in this volume. There is at present increasing interest in the reconstitution of plasma membrane transport systems into artificial phospholipid membrane vesicles. The methodology used for this has not been reviewed previously, and some account of these techniques will also be given here.
The title of this article implies that we understand the nature of the regulation of the size of the muscle mass in normal man and how this is disrupted by disease and injury. In fact, most of what we know is phenomenologieal rather than mechanistic. Nevertheless, we are beginning to understand the gross effects of some of the more obvious candidates as regulators, such as amino acid availability and hormone concentration. The phenomenology is important: there can be no relief for a while yet from the dogged application of presently available techniques until we have a complete knowledge of what actually happens to protein metabolism in skeletal muscle when we feed and fast, or suffer from injury or acute and chronic disease. There are still methodological problems outstanding, but except for a few cases which I will discuss, I do not think that they qualitatively affect (or will in future affect) our interpretation of most experimental data.
In mammals, including man, skeletal muscle is the largest part of the lean body mass and contains most of the protein in the body; it is responsible for 40–60% of whole-body protein turnover and in addition it has important functions in the diurnal flow of fuel and nitrogen between the periphery and the viscera and in the supply of amino acids during starvation, injury and disease (Daniel et al, 1977; Rennie, 1985; Rennie and Harrison, 1984).
Exercise raises brain serotonin release and is postulated to cause fatigue in athletes; ingestion of branched-chain amino acids (BCAA), by competitively inhibiting tryptophan transport into brain, lowers brain tryptophan uptake and serotonin synthesis and release in rats, and reputedly in humans prevents exercise-induced increases in serotonin and fatigue. This latter effect in humans is disputed. But BCAA also competitively inhibit tyrosine uptake into brain, and thus catecholamine synthesis and release. Since increasing brain catecholamines enhances physical performance, BCAA ingestion could lower catecholamines, reduce performance and thus negate any serotonin-linked benefit. We therefore examined in rats whether BCAA would reduce both brain tryptophan and tyrosine concentrations and serotonin and catecholamine synthesis. Sedentary and exercising rats received BCAA or vehicle orally; tryptophan and tyrosine concentrations and serotonin and catecholamine synthesis rates were measured 1 h later in brain. BCAA reduced brain tryptophan and tyrosine concentrations, and serotonin and catecholamine synthesis. These reductions in tyrosine concentrations and catecholamine synthesis, but not tryptophan or serotonin synthesis, could be prevented by co-administering tyrosine with BCAA. Complete essential amino acid mixtures, used to maintain or build muscle mass, were also studied, and produced different effects on brain tryptophan and tyrosine concentrations and serotonin and catecholamine synthesis. Since pharmacologically increasing brain catecholamine function improves physical performance, the finding that BCAA reduce catecholamine synthesis may explain why this treatment does not enhance physical performance in humans, despite reducing serotonin synthesis. If so, adding tyrosine to BCAA supplements might allow a positive action on performance to emerge.
The sections in this article are:
General Aspects of Amino Acid and Protein Metabolism in Muscle
Amino Acid Transport and the Free Amino Acid Pool
Outline of the Metabolism of Amino Acids in Muscle in Relation to Exercise
Interrelationship of BCAA , Alanine, and Glutamine Metabolism
The Purine Nucleotide Cycle in Muscle
Amino Acid Catabolism and Gluconeogenesis from Amino Acids in Liver and Kidney
Training Effects on the Capacities of Muscle Enzymes of Amino Acid Metabolism
The Effects of Contractile Activity on Ammonia Production and its Relation to the Free Amino Acid Pool
Alterations of Muscle and Blood Amino Acid Concentrations during Exercise
Production and Consumption of Amino Acids by Muscle during Exercise
Effects of Training on Amino Acid Metabolism during Exercise
Interrelationships between Working Muscle and the Viscera during Exercise
Protein Turnover During Exercise
Effects of Acute Contractile Activity on Protein Turnover
Postexercise Alterations in Muscle Protein Synthesis
Effects of Habitual Exercise on Whole‐Body Protein Turnover
Effects of Immobilization and Disuse
Collagen Turnover in Muscle
Physical Activity and Protein Requirements
Outstanding Questions
The goal of this chapter is to highlight some critical physiological relationships that exist among glutamate transport systems (biochemically defined activities), glutamate trans porters (molecularly identified proteins capable of the biochemically defined activities), and the metabolism of glutamate by several peripheral (noncentral nervous system) tissues. Collectively, these processes account for much of the whole-body flux of nitrogen and car bon. Presented is (a) an overview of the importance of glutamate metabolism to the function of peripheral tissues, (b) molecular and functional characteristics, and expression patterns, of transport proteins capable of glutamate transport, (c) a detailed examination of how glutamate transport activities and proteins support the function of hepatic (mature and fetal), placental, white adipose, and muscle tissues, and (d) a listing of underexplored areas of research that this author thinks are important to more fully understand the integrated role of glutamate transport capacity and peripheral tissue function. This chapter does not review the vastly greater body of literature that exists about how the expression and function of glutamate transporters support central nervous system or renal tissue metabolism, nor about research regarding the importance of glutamate transport capac ity to pulmonary tissue metabolism. For convenience, the "L" form of amino acids is implied, unless indicated otherwise.
Fed-batch cultures are extensively used for the production of therapeutic proteins. However, process optimization is hampered by lack of quantitative models of mammalian cellular metabolism in these cultures. This paper presents a new kinetic model of CHO cell metabolism and a novel framework for simulating the dynamics of metabolic and biosynthetic pathways of these cells grown in fed-batch culture. The model defines a subset of the intracellular reactions with kinetic rate expressions based on extracellular metabolite concentrations and temperature- and redox-dependent regulatory variables. The simulation uses the rate expressions to calculate pseudo-steady state flux distributions and extracellular metabolite concentrations at discrete time points. Experimental data collected in this study for several different CHO cell fed-batch cultures are used to derive the rate expressions, fit the parameters, and validate the model. The simulations accurately predicted the effects of process variables, including temperature shift, seed density, specific productivity, and nutrient concentrations.
Thesis (Ph. D.)--Texas Tech University, 1993. Includes bibliographical references (leaves 69-74).
TO THE EDITOR: We were interested in the contribution by Tesch and colleagues to the evidence base concerning the events subsequent to acute immobilization in human muscle. Tesch and colleagues (14) used a microdialysis technique applied to human muscle in an attempt to measure an index of myofibril- lar proteolysis 72 h after immobilization using the unilateral leg suspension technique. The authors discuss their results as though they had good evidence that the technique they used produces results that are sufficiently firm to be the basis of conjecture about mechanisms of protein balance in muscle after immobilization. We demur and propose that they are on very shaky ground. First, we wonder about the size of the effect reported. The authors claim it to be 44%, but calculation of the change shows it to be actually 29% with the SEs sufficiently large that they almost span the difference; SDs must overlap. Significance at the 5% level can only barely have been reached. Are the authors convinced that they have not ignored a type I error? The authors (14) begin their explanation of the advantages of the dialysis technique using 3-methylhistidine (3MeHis) by describing the gold standard method, i.e., the stable isotope- labeled amino acid tracer dilution method, as cumbersome and possibly unjustified in study of otherwise healthy subjects. We find this notion strange since Ferrando and colleagues (6, 12) have used the technique in many studies of how immobiliza- tion affects muscle protein turnover subsequent to disuse. We have also used the techniques in a wide variety of studies of proteolysis in healthy and infirm subjects (1, 2, 10, 11, 15) with no problems and, we would propose, producing results with a greater degree of certainty and biological appropriateness than those obtained by the 3MeHis dialysis technique.
The effect of varying cellular oxygenation on L6 muscle cell 2-deoxy-D-glucose transport, glucose utilization, lactate production, and expression of GLUT1 and GLUT4 transport proteins was investigated. Incubation of L6 myotubes in 3% O2 (mimicking a state of hypoxia) elevated glucose uptake by 6.5-fold over 48 h relative to cells incubated in 21% O2 (normoxia). Incubation of L6 cells in hyperoxic conditions (50% O2) significantly depressed glucose uptake by 0.4-fold. These effects were fully reversible. Incubation in 3% O2 also caused lactate accumulation and enhanced glucose consumption from the medium. Hypoxia elevated 2-deoxy-D-glucose transport even when the concentration of glucose in the medium was kept constant, suggesting that glucose deprivation alone was not responsible for increased cellular glucose uptake. Incubation in 3% O2 also elevated 3-O-methylglucose uptake but not amino acid uptake. Cycloheximide prevented the hypoxia-induced increase in glucose uptake, indicating that de novo synthesis of glucose transport-related proteins was the major means by which cells increased glucose uptake. The content of GLUT1 glucose transporter was significantly elevated in total membranes of cells incubated in 3% O2 and depressed in membranes from cells incubated in hyperoxic conditions, whereas GLUT4 expression was not affected. These results indicate that hypoxia induces an adaptive response of increasing cellular glucose uptake through elevated expression of GLUT1 in an attempt to maintain supply of glucose for utilization by nonoxidative pathways.
Incorporation of L-[1-13C]leucine into muscle protein and leg exchange of L-[15N]phenylalanine were used to assess the effects over 240 min of amino acid supply on leg protein turnover in anesthetized, overnight-fasted (Landrace x Great White) female pigs. In all pigs, plasma insulin and glucagon stability was ensured by infusion of somatostatin (8 micrograms.kg-1.h-1), insulin (6 mU.kg-1.h-1), and glucagon (72 ng.kg-1.h-1). Mixed amino acid infusion (260 mg.kg-1.h-1) caused a 2- to 2.5-fold elevation of arterial plasma phenylalanine and leucine; in a control group (no amino acid infusion), an increase in phenylalanine and leucine concentration was observed as a result of the hormone clamp. Plasma insulin and glucagon concentrations were steady and not significantly different between control and amino acid-infused groups during the final 240 min, but plasma glucose fell (P less than 0.05) in both groups (4.57 +/- 0.17 to 3.15 +/- 0.73 mM). Muscle protein synthetic rate (estimated from the change in L-[1-13C]leucine incorporation compared with labeling of [13C]leucyl-tRNA) was greater in amino acid-infused (0.076%/h) than in control (0.053%/h) pigs. In the control group, leg amino acid balance was negative (Phe alone, -10.2 +/- 9.4 nmol Phe.100 g-1.min-1; total amino acids, -0.27 +/- 1.04 micrograms amino N.100 g-1.min-1), but during amino acid infusion, balance was positive (Phe alone, +33.6 +/- 8.8 nmol Phe.100 g-1.min-1; total amino acids, +58.2 +/- 4.9 micrograms amino N.100 g-1.min-1).(ABSTRACT TRUNCATED AT 250 WORDS)
Transport of the neutral amino acids, 2-(methylamino)isobutyrate (MeAIB) and Phe, was examined in isolated rat hearts perfused by the Langendorff method. Hearts were perfused by recirculating for various time periods buffer containing [14C]-MeAIB or [14C]-Phe plus desired additions. Uptake of MeAIB was linear for approximately 30 minutes; Phe uptake was linear for a maximum of 5 minutes, and reached a steady state after 15 minutes. Km and Vmax for MeAIB were 1.1 +/- 0.03 mmol/L and 37.7 +/- 0.4 pmol/microL intracellular fluid (ICF)/min; values for Phe were 1.8 +/- 0.02 mmol/L and 364 +/- 5 pmol/microL ICF/minute. Uptake of MeAIB (0.2 mmol/L) was reduced 95% in the presence of Ser (10 mmol/L), and less severely by large neutral amino acids ([LNAA], 10 mmol/L) such as Phe and Leu (by 46% and 54%, respectively). Uptake of Phe (0.2 mmol/L) was reduced by LNAA such as Val, Leu, and Ile (by 51%, 78%, and 81%, respectively), or by commercial preparations used in parenteral nutrition, eg, Travasol or Travasol plus extra branched-chain amino acids (BCAA) (Branchamin); Ser had little effect (8% reduction). Insulin in the perfusion medium increased the fractional rate of protein synthesis. Individual BCAA at physiological concentrations (0.2 mmol/L) did not alter the rate of protein synthesis. Branchamin or Travasol plus Branchamin also had no effect on the rate of protein synthesis in heart, but did depress the rate of degradation. These studies suggest that amino acid transport into heart may be affected by normal levels of plasma amino acids, whereas protein synthesis is not.
The bidirectional transmembrane transport rates of leucine (Leu), valine (Val), phenylalanine (Phe), lysine (Lys), and alanine (Ala) were measured in vivo in the hindlimb muscle of five dogs and related to the rates of protein synthesis and degradation. The compartmental model was based on the systemic continuous infusion of stable isotopic tracers of the amino acids, and the measurement of the enrichment and concentration in the arterial and femoral vein plasma and the intracellular free water in muscle (obtained by biopsy). The transport rate from plasma to tissue (in micromoles per minute) was: Leu, 18.1 +/- 1.8; Val, 26.9 +/- 3.5; Phe, 10.5 +/- 1.6 Lys; 12.2 +/- 1.8; and Ala, 10.7 +/- 3.4. The transport rate from tissue to plasma (in micromoles per minute) was: Leu, 25.5 +/- 2.5; Val, 32.4 +/- 2.8; Phe, 17.0 +/- 2.8; Lys, 24.9 +/- 3.4; Ala, 34.4 +/- 9.0. When the transmembrane transport rate was normalized per unit of amino acid concentration in the source pool, we found that the transport of Leu, Val, and Phe was significantly faster (p less than .05) than the transport of Lys and Ala. The calculated rates of incorporation into hindlimb muscle protein of Phe and Lys (in micromoles per minute) were 4.2 +/- 1.3 and 19.4 +/- 5.3, respectively, and the rates of intracellular appearance from breakdown were 10.7 +/- 1.9 and 32.1 +/- 6.6, respectively. We concluded, therefore, that (1) the transmembrane amino acid transport rate can be measured in vivo in muscle with a relatively noninvasive technique, (2) in the dog hindlimb the equilibration between tissue and plasma free amino acid pool is different for each amino acid depending on the kinetics of the transmembrane transport systems, and (3) the transport rates of amino acids and their rate of appearance from protein breakdown are roughly comparable, suggesting that variations in transport rates could play a role in controlling the rate of protein synthesis.
Objectives were to characterize mechanisms and biochemical properties of transport systems responsible for the uptake of branched-chain amino acids (BCAAs) in muscle cells. Rat omega myoblasts (RMo) were grown to confluency and allowed to differentiate prior to conduct of transport assays. Myotubes concentrated cycloleucine (cLeu) in a sodium (Na)-free medium. The Na gradient-independent transporter possessed high affinity (Km = 0.12 mM) and high capacity (Vmax = 6.4 nmol cLeu/mg protein per min). Cycloleucine transport was strongly inhibited by nonpolar neutral amino acids but not by alpha-aminoisobutyric acid or lysine. Myotubes possessed a Na gradient-independent trans-exchange mechanism. Hence, myotubes possess a System L-like transporter. In the second part of the study we determined that various inhibitors (KCN, oligomycin, iodoacetamide and cycloheximide) increased leucine transport. Their actions were not mediated by reductions in ATP concentration but were instead associated with changes in protein synthesis. Hence, regulation of muscle protein synthesis may also influence transporter activity.
Intramuscular glutamine falls with injury and disease in circumstances associated with increases in blood corticosteroids. We have investigated the effects of corticosteroid administration (0.44 mg/kg dexamethasone daily for 8 days, 200 g female rats) on intramuscular glutamine and Na+, muscle glutamine metabolism and sarcolemmal glutamine transport in the perfused hindlimb. After dexamethasone treatment intramuscular glutamine fell by 45% and Na+ rose by 25% (the respective muscle/plasma distribution ratios changed from 8.6 to 4.5 and 0.12 to 0.15); glutamine synthetase and glutaminase activities were unchanged at 475 ± 75 and 60 ± 19 nmol/g muscle per min. Glutamine output by the hindlimb of anaesthetized rats was increased from 31 to 85 nmol/g per min. Sarcolemmal glutamine transport was studied by paired-tracer dilution in the perfused hindlimb: the maximal capacity (Vmax) for glutamine transport into muscle (by Na+-glutamine symport) fell from 1058 ± 310 to 395 ± 110 nmol/g muscle per min after dexamethasone treatment, accompanied by a decrease in the Km (from 8.1 ± 1.9 to 2.1 ± 0.4 mM glutamine). At physiological plasma glutamine concentration (0.75 mM) dexamethasone appeared to cause a proportional increase in sarcolemmal glutamine efflux over influx. Addition of dexamethasone (200 nM) to the perfusate of control rat hindlimbs caused acute changes in Vmax and Km of glutamine transport similar to those resulting from 8-day dexamethasone treatment. The reduction in muscle glutamine concentration after dexamethasone treatment may be primarily due to a reduction in the driving force for intramuscular glutamine accumulation, i.e., in the Na+ electrochemical gradient. The prolonged increase in muscle glutamine output after dexamethasone treatment (which occurs despite a reduction in the size of the intramuscular glutamine pool) appears to be due to a combination of (a) accelerated sarcolemmal glutamine efflux and (b) increased intramuscular synthesis of glutamine.
Amino acid transport activity from bovine renal brush-border membrane vesicles (BBMV) was reconstituted into phospholipid vesicles composed of phosphatidylcholine/5% stearylamine. Reconstitutable transport activity was enhanced in protein fractions binding to various lectins. When solubilized BBMV were fractionated on peanut lectin, a single protein band of average molecular mass 132 kDa was obtained. When this protein fraction was reconstituted into phospholipid membrane vesicles, amino acid transport activity was obtained with properties similar to those in native BBMV with regard to amino acid specificity, although the cation specificity was different. A monoclonal antibody which reacted with the same protein removed reconstitutable amino acid transport activity from solubilized BBMV. These findings may provide the first identification of a renal amino acid-transporting protein, although confirmation of this identification by other approaches will be required.
Transport of cationic amino acids in the isolated perfused rat pancreas was studied using dual-isotope dilution techniques. At 50 microM substrate concentration, unidirectional tracer uptakes for L-arginine (56 +/- 1%), L-lysine (49 +/- 2%), and L-ornithine (44 +/- 3%) were followed by rapid tracer efflux. In the presence of Na+, influx of L-arginine [Michaelis constant (Km) = 1.74 +/- 0.15 mM, maximum velocity (Vmax) = 1.97 +/- 0.07 mumol.min-1.g-1] and L-lysine (Km = 2.48 +/- 0.17 mM, Vmax = 2.42 +/- 0.08 mumol.min-1.g-1) was mediated by a common transport system, sensitive to cis-inhibition by L-ornithine, 2,4-L-diaminobutyric acid, D-lysine, and D-arginine. Substrates for system A [alpha-(methylamino)isobutyric acid] and an anionic carrier (L-aspartate) were poor cis-inhibitors of L-arginine entry. Removal of Na+ resulted in a 40% reduction in cationic amino acid influx. After cell loading (20 min), L-[3H]-lysine cleared predominantly from a slowly exchanging pool with a rate constant of 5.97 +/- 0.67 min. An influx/efflux permeability ratio of 14.5 +/- 1.6 was determined, and efflux of L-lysine was trans-stimulated by vascular challenges with cationic or large neutral amino acids. The specificity, relative Na+ independence, and exchange properties of this saturable cationic amino acid transporter in the pancreatic epithelium resemble those reported for system y+ in cultured fibroblasts and hepatocytes.
Amino acid (AA) transport systems A and L, which transfer preferentially small neutral AA (SNAA) and large neutral AA (LNAA), respectively, were studied in the isolated soleus muscle with the specific models, 2-(methylamino)isobutyrate (MeAIB) and 2-aminobicyclo[2,2,1]heptane-2-carboxylate (BCH). Affinity for MeAIB was greater than for BCH (Km = 3.2 +/- 0.2 and 8.7 +/- 0.2 mm, respectively). Rate of transport of MeAIB (Vmax = 104 +/- 3 pmol/microL/min) was slower than for BCH (970 +/- 12 pmol/microL/min), but accumulation was far more concentrative; transport of BCH, but not MeAIB, rapidly reached a steady-state level. MeAIB transport was reduced in the presence of SNAA; BCH transport was reduced to a lesser extent only by LNAA. Mixtures of AA at concentrations resembling those in plasmas of rats fed either a 6% or 50% casein diet reduced transport of MeAIB, whereas BCH transport was low only with the latter mixture. Only MeAIB transport was stimulated by insulin. Preincubation of muscles for 5 hours in a AA-free medium stimulated subsequent MeAIB uptake by about twofold to fourfold; this effect was suppressed by inhibitors of protein synthesis. Selective differences were thus observed in transport by skeletal muscle of model AA for the A and L systems: increased transport resulting from various stimuli was limited to the model for the A system, and transport of either model was depressed with mixtures containing physiological levels of AA. Changes in dietary protein or AA intake may thus alter transport of certain neutral AA into skeletal muscle via changes in plasma AA pools.
A continuous intravenous infusion of L-leucine (300 mumols min-1) was given to 12 healthy females over a 2 1/2 h period. Arterial plasma concentrations of amino acids and the keto acids of the branched-chain amino acids (BCAA) were measured. In six subjects muscle biopsies were taken before and at the end of the infusion for determination of intracellular (i.c.) free amino acid concentrations, and leg exchange of amino acids was measured. During infusion the plasma level of leucine rose sixfold. Approximately 40% of the infused amount was taken up by muscle. Of this, half was accumulated intracellularly, where the free leucine concentration increased from basal 190 +/- 22 to 580 +/- 110 mumols l-1 ICW (intracellular water) at the end of infusion. The concentrations of most other amino acids, above all the other BCAA and the aromatic amino acids, decreased, by 17-48% in the i.c. pool and by 17-79% in plasma. The plasma level of ketoisocaproic acid (KIC), the keto acid of leucine, increased in parallel with that of leucine. The concentration of keto valine, ketoisovaleric acid (KIV), decreased by 75%, whereas the keto acid of isoleucine, ketomethylvaleric acid (KMV), was unchanged. Leg release of alanine decreased significantly, whereas the exchange of other amino acids were unchanged. Taken together, decreased i.c. and plasma concentrations but unchanged leg exchange of tyrosine and phenylalanine suggest i.c. accumulation of protein. It can be calculated that approximately 40% of the leucine taken up by muscle was accumulated in the intracellular free pool, some 20% could have been incorporated into protein and 40% was probably oxidized.
Amino acid transport was measured in rat sarcolemmal vesicles (approximately 0.5 microliters/mg protein). Initial (45 s) uptake of glutamine tracer was stereospecific and saturable [Km 90 +/- 14 microM; maximum velocity (Vmax) 60 +/- 3 pmol.mg protein-1.min-1], it was Na+ dependent (but tolerated Li+ instead), and was stimulated by inside negative membrane potential. Transport of glutamine (5 microM) was inhibited by asparagine, histidine, alanine, serine, and phenylalanine at 1 mM (25-74%), but leucine and N-methylaminoisobutyric acid (MeAIB) did not significantly inhibit glutamine uptake. Glutamine efflux was accelerated by an outwardly directed Na+ concentration gradient. L-[14C]asparagine uptake was Na+ dependent and strongly inhibited by glutamine. L-[3H]serine uptake was Na+ dependent but did not tolerate Li(+)-for-Na+ substitution. L-[3H]phenylalanine uptake was Na+ independent. Differences between the ion dependence of glutamine, serine, and phenylalanine uptake and the lack of glutamine transport inhibition by MeAIB indicated that glutamine is not transported by systems ASC, L, or A. The properties of the glutamine transporter in sarcolemmal vesicles resemble those of the system Nm previously characterized in perfused skeletal muscle.
Rat skeletal muscle glutamine fell by 40% from 4.18 to 2.5 mumols/g wet weight (P less than 0.01) after 4 days of denervation. Over the same period net glutamine efflux from denervated hindlimbs [i.e., arteriovenous (a-v) concentration differences x blood flow] increased 3.5-fold (from -6.72 +/- 1.73 to -26 +/- 4.81 nmol.min-1.g-1, P less than 0.001). Gastrocnemius glutamine synthetase activity fell 48% after denervation (from 475 +/- 81 to 248 +/- 39 nmol.min-1.g-1, P less than 0.001), but glutaminase activity was not significantly altered (17 nmol.min-1.g-1). The maximal activity (Vmax) of the unidirectional Na(+)-dependent glutamine transporter (system Nm) was depressed by 45% from 1,020 +/- 104 to 571 +/- 9 nmol.min-1.g-1 (P less than 0.01), but the concentration at which transport was half maximal (Km) was not significantly altered (control 8.1 +/- 0.6 mM; denervated 6.52 +/- 0.12). Hindlimb denervation resulted in an increase of intramuscular Na+ by 17% and a fall of K+ by 12%, and the resting membrane potential in isolated muscles decreased from -75 +/- 10 to -59.5 +/- 5.5 mV. Membrane potential of perfused denervated muscle, isolated after acute addition of the Na+ channel blocker tetrodotoxin (TTX, 3 microM), repolarized to -66.4 +/- 3.2 mV. In perfused denervated preparations TTX caused an acute recovery of Vmax of unidirectional glutamine transport to 848 +/- 75 nmol.min-1.g-1; Km was unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)
Despite its anabolic effects on protein balance, acute administration of insulin has been reported to have no effect on skeletal muscle or whole body protein synthesis in man. However, insulin also reduces plasma and intramuscular amino acid availability, which may limit protein synthesis. We have therefore measured the acute effects of insulin on skeletal muscle (anterior tibialis) protein synthesis and whole body leucine turnover in eight insulin-withdrawn Type 1 (insulin-dependent) diabetic patients. They were studied initially when insulin deficient, but during infusion of mixed amino acids at a rate sufficient to raise plasma amino acids by 30% i.e. to 4 mmol/l in total; measurements were continued when insulin was infused together with an increased rate of amino acids to maintain insulinopoenic plasma amino acid concentrations. Using 13C-alpha-ketoisocaproate in plasma as an index of the intracellular precursor labelling, incorporation of [1-13C]leucine into skeletal muscle protein was 0.068 +/- 0.007%/h during insulin withdrawal and was unaltered during insulin infusion. The value is higher than observed in muscle of healthy man, possibly because of a stimulatory effect of endogenous intramuscular amino acids. Also, calculated on the basis of alpha-ketoisocaproate labelling, non-oxidised whole body leucine disappearance (i.e. whole body protein synthesis) was 110 +/- 4 mumol.kg-1.h-1 during insulin withdrawal; this also was unchanged during insulin infusion. Despite stable or increased plasma concentrations of most amino acids, the intramuscular concentrations of a number of amino acids decreased during insulin infusion.(ABSTRACT TRUNCATED AT 250 WORDS)
In this study, we present evidence showing that leucine is involved in the upregulation of system A amino acid transport activity in the L6 rat skeletal muscle cell line. At leucine concentrations of > or = 0.05 mM, the uptake of N-methylamino-alpha-isobutyric acid (MeAIB), a paradigm system A substrate, was stimulated by up to 50%. Kinetic analysis revealed that this stimulation was a result of an increase in the maximal transport rate of MeAIB uptake, from 327 +/- 26 to 450 +/- 8 pmol.min-1.mg protein-1 after incubation of cells with leucine. No significant change in the concentration at which MeAIB transport was half maximal was observed. System A activation was biphasic, reaching an initial plateau after 3 h, with a second phase of activation being observed after 5 h. The initial activation of system A transport occurred by a mechanism distinct from that activated by insulin-like growth factor-I (IGF-I) (3 nM), since the effects of leucine and IGF-I were additive. This activation was not due to transstimulation, since 2-amino-2-norbornane-carboxylic acid, a specific system L substrate, did not stimulate system A. Leucine's keto acid, ketoisocaproic acid, prevented the activation of system A transport, whereas aminooxyacetate, a transaminase inhibitor, augmented the increase in system A activity by leucine. Both cycloheximide and actinomycin D inhibited the leucine-induced increase in MeAIB uptake. The present results indicate that leucine, or some cellular component regulated by it, is capable of stimulating system A transport through control of DNA transcription, possibly of a gene encoding either a repressor or enhancer molecule of system A or perhaps of the gene encoding system A itself.
This study was conducted to determine the effects of different concentrations of leucine on the transport, transamination and oxidation of valine and on incorporation of valine into heart proteins in the isolated perfused rat heart. Valine metabolism was studied in rat hearts perfused with medium containing glucose and graded levels of L-leucine. In transport studies L-phenylalanine was also tested. Uptake of L-[1-14C]valine (0.2 mmol/L) was significantly reduced (-50%) by inclusion of 0.2 mmol/L phenylalanine or leucine, and by -70% by inclusion of 1.0 mmol/L phenylalanine or leucine in the perfusate. Transamination of valine decreased by 37 and 48%, and oxidation of valine by 53 and 71%, respectively, when 0.2 or 1.0 mmol/L leucine was included in the perfusate. Tissue concentrations of valine decreased by 43, 48 and 62% in the presence of 0.2, 0.5 and 1.0 mmol/L leucine, respectively; tissue concentrations of leucine, glutamate and alanine increased approximately 11-fold, 1.2-fold and 0.5-fold, respectively, when 1.0 mmol/L leucine was present in the perfusate. Addition of 0.2-1.0 mmol/L leucine did not affect incorporation of valine into heart proteins. We conclude that 1) competition among large neutral amino acids for transport into heart occurs at physiological concentrations of these amino acids in plasma; 2) inhibition of valine uptake by leucine can limit the rate of valine catabolism in heart; and 3) depletion of tissue valine concentration by an excess of leucine did not affect the rate of protein synthesis.
Effects of gut regulatory peptides and growth factors on the uptake of 2-aminoisobutyric acid (AIB) and 2-deoxy-D-glucose (2-DOG) were examined in differentiated ovine satellite cell cultures. Insulin and insulin-like growth factor I (IGF-I) gave maximal increases of 160-180% of controls for AIB and over 190% for 2-DOG. IGF-I showed half-maximal effects at 0.1-1 nM, and insulin at 1-10 nM. Bovine growth hormone (0.01-100 nM) had no effect. Gastrin, gastric inhibitory polypeptide (GIP), bombesin and somatostatin had no action in either the absence or presence of insulin. In primary cultures epidermal growth factor (EGF) increased the uptake of AIB (133-137%) and 2-DOG (171-176%). In clonal lines, EGF had little effect on nutrient uptake but still simulated protein synthesis.
We have used stable isotopic tracers of amino acids to measure in vivo transmembrane transport of phenylalanine, leucine, lysine, alanine, and glutamine as well as the rates of intracellular amino acid appearance from proteolysis, de novo synthesis, and disappearance to protein synthesis in human skeletal muscle. Calculations were based on data obtained by the arteriovenous catheterization of the femoral vessels and muscle biopsy. We found that the fractional contribution of transport from the bloodstream to the total intracellular amino acid appearance depends on the individual amino acid, varying between 0.63 +/- 0.02 for phenylalanine and 0.22 +/- 0.02 for alanine. Rates of alanine and glutamine de novo synthesis were approximately eight and five times their rate of appearance from protein breakdown, respectively. The model-derived rate of protein synthesis was highly correlated with the same value calculated by means of the tracer incorporation technique. Furthermore, amino acid transport rates were in the range expected from literature values. Consequently, we conclude that our new model provides a valid means of quantifying the important aspects of protein synthesis, breakdown, and amino acid transport in human subjects.
Glutamine produced and stored in skeletal muscle is an important source of nitrogen and energy for the whole body in health and disease and, unsurprisingly, glutamine turnover in muscle is subject to substantial metabolic control. L-Glutamate, a necessary substrate for glutamine synthetase, is transported into muscle cells by Na(+)-dependent and -independent transport systems. In primary cultures of rat skeletal muscle myotubes (a useful model system for studies of muscle metabolism and membrane transport), Na(+)-dependent glutamate transport (Km approximately 0.7 mM glutamate) shows adaptive upregulation (65% increase in transport Vmax from 2.7 to 4.4 nmol.min-1 x mg protein-1) in cells within 24 h of glutamine depletion (t1/2 for increase of approximately 4 h), whereas Na(+)-independent glutamate uptake remains unaltered. Up-regulation of transport is suppressed by inhibitors of gene transcription (actinomycin-D) and translation (cycloheximide) and is reversed by glutamine supplementation. Increased glutamate transport capacity should provide extra substrate for glutamine synthesis in muscle cells. Thus, in concert with previously discovered increases in cell glutamine transport capacity and glutamine synthetase activity, it may represent part of a co-ordinated response to decreased glutamine availability (e.g., under circumstances of increased glutamine utilization by other tissues in vivo.
A study has been made of the ability of insulin to stimulate the transport of l-[14C]alanine into paired "intact" rat hemidiaphragms in vitro, and the results have been compared with those obtained with α-amino[14C]isobutyrate and l-[14C]leucine. A variety of agents and conditions have been tested for their relative abilities to alter total saturable uptake
of the amino acids on the one hand, and the insulin stimulation of uptake on the other.
Transport of alanine (0.5 mm) was increased about 20% by the presence of insulin (0.1 unit per ml) under the conditions of the experiment. A 10 to 20
mm level of α-aminoisobutyrate eliminated the insulin stimulation of alanine uptake while decreasing its total saturable uptake
by only about 10%. Leucine, in contrast, could inhibit at least three-fourths of the total saturable alanine uptake without
decreasing the increment in uptake produced by insulin. Omitting Na+ from the incubation medium decreased total alanine uptake by about 60%, but abolished insulin stimulation. Decreasing the
pH of the incubation medium progressively from 7.4 to 5.4 brought a regular reduction in the insulin stimulation, but at the
same time the total saturable alanine uptake was increased. Other agents and conditions gave less sharp distinction between
the transport systems used by alanine.
α-Aminoisobutyrate transport was more than doubled by 0.1 unit of insulin per ml in 1 hour. The pattern of its inhibition
by different conditions suggests that this amino acid is transported in diaphragm nearly completely by one system, which is
Na+-dependent as well as sensitive to insulin. Leucine transport, on the other hand, showed relatively little response to insulin,
and was essentially unaltered by changes in buffer [Na+] or pH, or by the presence of competitive inhibitors other than leucine and isoleucine.
The results suggest that l-alanine enters rat diaphragm by at least three saturable routes. One is Na+-independent; another is Na+-dependent and sensitive to insulin; while the third is Na+-dependent but not affected by insulin. The insulin-sensitive route accounts for no more than about 10 to 20% of the total
saturable uptake at the alanine levels used. These facts support the hypothesis that insulin acts on only one neutral amino
acid transport system in diaphragm; and that the hormone does not increase alanine transport greatly in this tissue because
alanine does not use the insulin-sensitive system extensively.
To study glutamate and glutamine kinetics, 4-h unprimed intravenous infusions of L-[15N]glutamate, L-[2-15N]glutamine, and L-[5-15N]-glutamine were administered to healthy young adult male subjects in the postabsorptive state. Arterialized-venous blood samples were drawn and analyzed for glutamate and glutamine 15N enrichments. The fractional turnover rates of the tracer-miscible glutamate and glutamine pools were fast, 8.0 and 2.8% min-1, respectively. The glutamate tracer-miscible pool accounted for less than one-tenth the estimated free glutamate pool in the body. The plasma glutamate amino N, glutamine amino N and glutamine amide N rates of appearance were 83 +/- 22 (means +/- SD), 348 +/- 33, and 283 +/- 31 mumol X kg-1 X h-1, respectively. The glutamine amide N appearance rate was 20% slower than the amino N appearance rate, indicating that glutamine transaminase is an active pathway in human glutamine metabolism. From measurement of transfer of tracer 15N, we found that only 5% of the glutamine synthesized in cells and released into plasma was derived from intracellular glutamate that had mixed with plasma. These data demonstrate that intravenously administered tracers of glutamate or glutamine do not mix thoroughly with the intracellular pools, and their measured kinetics reflect transport rates through plasma rather than whole-body fluxes.
Flux rates of amino acids were measured across the leg after an overnight fast in resting human volunteers. A balanced amino acid solution was, after a primed infusion, continuously infused for 2 h at each of three step-wise and increasing rates corresponding to 8.3, 16.7, 33.2 mg N/kg per h that were equivalent to 0.2, 0.4, 0.8 g N/kg per d. Flux of amino acids across the leg was compared with the flux of glucose, glycerol, lactate, free fatty acids, and oxygen. The size of the muscular tissue pool of amino acids was measured. Whole body amino acid oxidation was estimated by means of the continuous infusion of a 14C-labeled mixture of amino acids. Arterial steady state levels were obtained for most amino acids within 30 to 45 min after the primed constant infusion. Leg flux of amino acids switched from a net efflux after an overnight fast to a balanced flux between infusion rates corresponding to 0.2-0.4 g N/kg per d. At 0.8 g N/kg per d essentially all amino acids showed uptake. The infusion of amino acids stimulated leg uptake of glucose and lactate production and decreased FFA release. Oxygen uptake and leg blood flow increased significantly with increased infusion of amino acids. There was significant variability in transport rate among individual amino acids. Branched chain amino acids showed rapid transport and methionine slow transport rate. Only small changes in the muscle tissue concentration of certain amino acids were registered after 6 h of amino acid infusion despite uptake for several hours. When amino acids were infused at a rate corresponding to 0.8 g N/kg per d, the leg uptake of amino acids was 6% and the simultaneous whole body oxidation of infused amino acids was approximately 10%. Net uptake of leucine across the leg per hour was 62% of the muscle pool of free leucine when amino acids were infused at a rate corresponding to 0.4 g N/kg per d. Multiple regression analysis showed that the arterial concentration of an amino acid was the most important factor for uptake, more so than insulin concentration and blood flow. It is concluded that leg exchange of amino acids is large enough to rapidly change the pool size of the amino acids in skeletal muscle, if not counter-regulated by changes in rates of protein synthesis and degradation. Estimates of the capacity for protein synthesis and transfer RNA acceptor sites in muscles agree in order of magnitude with the net uptake of amino acids at high infusion rates of amino acids. Therefore, measurements of the balance of tyrosine, phenylalanine, and particularly methionine at steady state may reflect net balance of proteins across skeletal muscles even in short-time experiments.
On the basis of inhibition analysis two bicyclic amino acid analogs appear to enter human red blood cells by much the same Na+-independent mediation, whereas striking differences are apparent in the routes for tryptophan and leucine, confirming a role for System T, but also suggesting the participation of a third system of low affinity somewhat selective for weakly basic amino acids. System T of the human cell is specifically inhibited by 4-azidophenylalanine, and is highly sensitive, relative to System L, to N-ethylmaleimide inhibition. Uptake by System T approaches its steady state much more slowly than does System L, and its participation in trans-stimulation is questionable, whereas that of System L is as usual strong. A different added transport system became apparent in the slow approach of the Na+-independent mediation of uptake of 3- and 4-carbon dipolar amino acids by the nucleated pigeon red cell to its steady state. In that cell System T makes at most a minor contribution. The patterns of trans-stimulation of fluxes among selected pairs of amino acids in the pigeon cell correspond to a usual participation in transmembrane exchange by System L, and also by the new transport system. An important but not the sole source of the heterogeneity in the pigeon cell is the participation of the system conspicuously involved in the transport of alanine, serine, and threonine, among other amino acids. This route of transport of these amino acids is made conspicuous by their small transport by other Na+-independent agencies, notably System L. Reactivity with this system is enhanced by a side change hydroxyl or sulfhydryl group. Uptake by this route as tested by threonine showed little inhibition by cysteinesulfinate under conditions inhibitory to System asc; also a sensitivity to lowering of pH unlike that seen with System asc. The new Na+-dependent transport system appears to be a species variant of quite similar Na+-independent systems discovered by Young et al. (Young, J. D., Ellory, J. C., and Tucker, E. M. (1975) Nature (Lond.) 254, 156-157; Fincham, D. A., Mason, D. K., and Young, J. D. (1982) Biochem. Soc. Trans. 11, 776-777) in sheep and horse erythrocytes on the basis of their absence in phenotypes. These authors have emphasized several similarities in these two cases to Na+-dependent System asc, and they propose that Na+ dependence has specifically been lost on maturation of the red cells without major changes in amino acid selectivity.(ABSTRACT TRUNCATED AT 400 WORDS)
The transport of the aromatic amino acids into isolated rat liver cells was studied. There was a rapid and substantial binding of the aromatic amino acids, L-alanine and L-leucine to the plasma membrane. This has important consequences for the determination of rates of transport and intracellular concentrations of the amino acids. Inhibition studies with a variety of substrates of various transport systems gave results consistent with aromatic amino acid transport being catalysed by two systems: a 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (BCH)-insensitive aromatic D- and L-amino acid-specific system, and the L-type system (BCH-sensitive). The BCH-insensitive component of transport was Na+-independent and facilitated non-concentrative transport of the aromatic amino acids; it was unaffected by culture of liver cells for 24 h, by 48 h starvation, dexamethasone phosphate or glucagon. Kinetic properties of the BCH-inhibitable component were similar to those previously reported for the L2-system in liver cells. The BCH-insensitive component was a comparatively low-Km low-Vmax. transport system that we suggest is similar to the T-transport system previously seen only in human red blood cells. The results are discussed with reference to the importance of the T- and L-systems in the control of aromatic L-amino acid degradation in the liver.
The fiber type profiles of hindlimb muscles in the Hartley guinea pig, Sprague-Dawley rat, cat, Galago senegalensis (lesser bushbaby) and Nycticebus coucang (slow loris) were estimated histochemically. Fibers were classified as fast oxidative glycolytic, fast glycolytic or slow oxidative according to their myosin adenosine triphosphatase, α-glycerophosphate dehydrogenase and reduced nicotinamide adenine dinucleotide diaphorase activities. It was found that the soleus and vastus intermedius muscles had the highest proportion of slow oxidative fibers in all five species, demonstrating the constancy of muscle fiber profiles dependent upon anatomical position and functional utilization. The tensor fascia latae and white vastus lateralis of the guinea pig were mostly fast glycolytic, while the red vastus lateralis of the guinea pig consisted of predominantly fast oxidative glycolytic fibers. The majority of muscles investigated in these five mammals were heterogeneous, having a wide range of percentages of the three fiber types.
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 V max 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 V max 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.
For decades, investigators concerned with protein metabolism in man have performed detailed amino acid analyses of human plasma obtained under a wide range of experimental situations. A large body of information has been used to calculated rates of protein synthesis and proteolysis. During the course of an investigation of the effect of intrabrachial artery infusion of insulin (70 muU/min per kg body weight) on glutamate uptake by human forearm muscle, it was discovered that plasma arterio-deep venous glutamate difference analysis failed to document any increase in the uptake of this amino acid, suggesting that insulin had little influence on glutamate uptake by muscle. However, whole blood glutamate analyses, performed on the same blood samples, revealed that (a) the resting muscle uptake of glutamate is smaller than previously reported and (b) insulin is capable of markedly increasing glutamate uptake by muscle from whole blood. Since the hematocrit was obtained on all samples, detailed analyses of the various compartments in which glutamate could be found were performed. It was determined that circulating blood cells have a dynamic role in glutamate transport. These data underscore the need for both whole blood and plasma amino acid analysis in investigations concerned with protein synthesis and/or amino acid flux, for analysis of plasma samples alone could be misleading as illustrated in the present study.
Glutamate and aspartate influxes across the brush border of rabbit intestine are saturable processes that are subject to competitive inhibition and are markedly influenced by the Na concentration in the mucosal solution. Lowering the Na concentration increases the amino acid concentration needed to elicit a half-maximal influx but does not significantly affect the maximal influx. The interaction between Na and anionic amino acid influx can be described by the same kinetic model that has been applied to the influxes of neutral amino acids and lysine. Comparison of the kinetic parameters for anionic, neutral, and cationic amino acids suggests that amino acid charge influences (a) the stability of the binary (amino acid-site) complex and (b) the affinity of this binary complex for the subsequent binding of Na. A mechanistic interpretation of these interactions is proposed.
The Ehrlich cell has a component of transport, for which alanine, serine, and cysteine are preferred substrates, which is not reactive with α-(methylamino)isobutyric acid or N-methylalanine and is only weakly reactive with other methylamino acids and glycine. Although the new component is similar to one described before (the so-called A system) as to its rate, its dependence on the presence of Na⁺, its low sensitivity to irradiation, and other properties, nevertheless it shows a distinctly lower pH sensitivity, a somewhat lower sensitivity to metabolic inhibitors, and a much higher stereo-specificity than the A system. Furthermore, part of the exodus of alanine shows a stereospecificity corresponding to the new system, suggesting that the new system operates more reversibly than the A system. Systems with similar properties and substrate specificities occur in the rabbit reticulocyte and the pigeon erythrocyte, in the absence of any system reactive with N-methylalanine or α-(methylamino)isobutyric acid. From these considerations, we conclude that the new system and the A system very probably operate independently and not as a single complex transport system.
Both of the amino acids that serve to suppress the A system selectively, namely α-(methylamino)isobutyric acid and N-methyl-l-alanine, show peculiarly low Vmax values for uptake by the Ehrlich cell.
We have applied the paired-tracer dilution method to the study of transport processes in a mixed mammalian muscle preparation, the perfused rat hindlimb. The method is suitable for the characterization of the kinetic parameters of sugar and amino acid transport and its regulation by hormones, contractile activity, hypoxia, etc. Insulin stimulates sugar transport by increasing the Vmax. of the process 2-3 fold, but its affinity is unchanged. Starvation increases the affinity of sugar transport in perfused skeletal muscle.
Amino acids enter rabbit jejunal brush border membrane vesicles via three major transport systems: (1) simple passive diffusion; (2) Na-independent carriers; and (3) Na-dependent carriers. The passive permeability sequence of amino acids is very similar to that observed in other studies involving natural and artificial membranes. Based on uptake kinetics and cross-inhibition profiles, at least two Na-independent and three Na-dependent carrier-mediated pathways exist. One Na-independent pathway, similar to the classical L system, favors neutral amino acids, while the other pathway favors dibasic amino acids such as lysine. One Na-dependent pathway primarily serves neutral L-amino acids including 2-amino-2-norbornanecarboxylic acid hemihydrate (BCH), but not beta-alanine or alpha-methylaminoisobutyric acid (MeAIB). Another Na-dependent route favors phenylalanine and methionine, while the third pathway is selective for imino acids and MeAIB. Li is unable to substitute for Na in these systems. Cross-inhibition profiles indicated that none of the Na-dependent systems conform to classical A or ACS paradigms. Other notable features of jejunal brush border vesicles include (1) no beta-alanine carrier, and (2) no major proline/glycine interactions.
The amino acids are ions of various charge combinations, and one can argue that historically they were the first ions for which the ongoing problem of membrane transport was presented; also that among transported ions these may undergo a highly detailed molecular recognition. Furthermore, the distribution of charge on the amino acid molecule determines by what route or routes it is conducted across the biological membrane, with what directional and structural specificity, and therefore what regulation is imposed, and where. Cases where a presumably charged chemical group behaves as if it were somehow absent from the amino acid have been observed to fall into several categories: Straightforward cases where the pH has been low enough or high enough to remove the charge by protonation or deprotonation, even in free solution. Cases where that protonation or deprotonation is facilitated at the binding site, and perhaps by the total transport process. The cystine molecule can apparently thus be rendered either a tripolar anion or a tripolar cation for transport. Cases where an otherwise co-transported Na+ is omitted to redress charge, or where a Na+ serves as a surrogate for a missing charged group on the amino acid molecule. A case where the protonation occurs reversibly at the receptor site rather than on the amino acid molecule.
1. Alanine, glutamine and serine were actively accumulated in liver cells isolated from starved rats. 2. This accumulation was inhibited when either Na+ or HCO3- ions were omitted from the incubation medium. In general the degree of dependence on Na+ was quantitatively similar to that on HCO3-. 3. The apparent Km values for the transport of all three amino acids were in the range 3--5mM with Vmax. values in the range 15--25nmol/min per mg of cell protein at 37 degrees C. 4. Alanine and serine transport were mutually competitive; glutamine inhibited the transport of alanine and serine non-competitively. 5. The initial rate of transport of these amino acids was inhibited when the intracellular content of ATP was decreased. 6. Ouabain inhibited the rate of alanine transport without inhibiting the rate of alanine metabolism. 7. It is concluded that a minimum of three transport systems must be postulated to exist in the liver cell plasma membrane to account for the transport of alanine, serine and glutamine. The rate of transport of these amino acids in isolated hepatocytes is unlikely to limit the rate at which they are metabolized.
A carrier for glutamine, identified in rat muscle, has properties in terms of kinetics, ion dependence and hormone sensitivity, and effects of endotoxin and branched-chain aminoacids that point to an important function in the control of whole-body aminoacid metabolism. The existence of a link between the size of the glutamine pool in muscle and the rate of muscle protein synthesis raises possibilities for therapeutic interventions to limit protein loss in injury, sepsis, and chronic disease.
Periportal and perivenous hepatocytes differ in their metabolism of blood glutamate (Glu). Uncertainty about the mechanisms of Glu blood-liver exchange led us to characterise, by paired-tracer dilution, a sodium-dependent dicarboxylate transporter (resembling system X-ag) in sinusoidal membranes of perfused rat liver (Vmax = 0.18 mumol Glu/g per min, Km = 0.29 mM Glu). Tracer Glu transport was depressed 65% after necrosis of perivenous hepatocytes by acute CCl4 treatment, indicating that X-ag transporter activity is located mainly in these cells, the sites of glutamine (Gln) synthesis from glutamate and ammonia. Modulation of Glu transport may influence the extent of hepatic Gln release.
1. We have investigated glutamine transport in the perfused rat hindlimb using the paired-tracer isotope dilution technique. 2. Uptake of L-glutamine was stereospecific, saturable, sodium dependent, insulin sensitive and pH insensitive in the physiological range. The maximum capacity of transport (Vmax) under normal perfusate conditions at 37 degrees C, 145 mM-Na+ and in the absence of insulin was 1156 +/- 193 nmol min-1 g-1 with transport being half-maximal at a perfusate glutamine concentration of 9.25 +/- 1.15 mM. 3. The kinetics of Na+ dependence strongly suggested co-transport of Na+ and glutamine with a stoichiometry of 1:1; furthermore, Na+ activated the carrier without any change in the concentration of glutamine at which transport was half-maximal, i.e. a 'Vmax effect' rather than a 'Km effect'. 4. The characteristics of glutamine transport, especially its substrate specificity and the pattern of competitive and non-competitive inhibition of glutamine transport by other amino acids, suggest that it is mediated by a carrier or carriers for which asparagine and histidine are also suitable substrates. 5. The characteristics of muscle glutamine transport are related but distinct from those of system N identified in hepatocytes; we suggest that they are sufficiently distinct to justify the identification of a new variant of mammalian amino acid transport systems which may be identified by the symbol Nm. 6. The kinetic characteristics of system Nm are such that glutamine is likely to be the most rapidly exchanging amino acid across the muscle membrane at physiological intra- and extracellular glutamine concentrations. Its hormone and ion sensitivities are likely to be important in the physiological modulation of whole-body glutamine metabolism and also during derangements observed in disease and after injury.
Control of urea synthesis was studied in rat hepatocytes incubated with physiological mixtures of amino acids in which arginine was replaced by equimolar amounts of ornithine. The following observations were made.
Intramitochondrial carbamoyl phosphate was always below 0.1 mM. Only when ornithine was absent and when, in addition, the concentration of amino acids was higher than four times their plasma concentration, intramitochondrial carbamoyl phosphate rose up to about 3 mM; under these conditions ammonia accumulated in the medium.
The relationship between ornithine‐cycle flux and the concentration of the cycle intermediates at varying amino acid concentration indicated that under near‐physiological conditions the ornithine‐cycle enzymes are far from being saturated with their substrates.
Moderate concentrations of norvaline had no effect on the rate of urea synthesis unless the cells were severely depleted of ornithine.
Activation of carbamoyl‐phosphate synthetase (ammonia) by addition of N ‐carbamoylglutamate only slightly stimulated urea production at all amino acid concentrations. However, in the presence of the activator the curve relating ornithine‐cycle flux to the steady‐state ammonia concentration was shifted to lower concentrations of ammonia.
The intramitochondrial concentration of carbamoyl phosphate in rat liver in vivo was below 0.1 mM. This value is far below the concentration required for substantial inhibition of carbamoyl‐phosphate synthetase.
It is concluded that in vivo the function of activity changes in carbamoyl‐phosphate synthetase, via the well‐documented alterations in the intramitochondrial concentration of N ‐acetylglutamate, is to buffer the intrahepatic ammonia concentration rather than to affect urea production per se . At constant concentration of ammonia the rate of urea production is entirely controlled by the activity of carbamoyl‐phosphate synthetase.
A study was made of the ability of insulin to stimulate the transport of L [14C] alanine into paired 'intact' rat hemidiaphragms in vitro, and the results were compared with those obtained with α amino [14C] isobutyrate and L [14C] leucine. A variety of agents and conditions were tested for their relative abilities to alter total saturable uptake of the amino acids on the one hand, and the insulin stimulation of uptake on the other. Transport of alanine (0.5 mM) was increased about 20% by the presence of insulin (0.1 unit per ml) under the conditions of the experiment. A 10 to 20 mM level of α aminoisobutyrate eliminated the insulin stimulation of alanine uptake while decreasing its total saturable uptake by only about 10%. Leucine, in contrast, could inhibit at least three fourths of the total saturable alanine uptake without decreasing the increment in uptake produced by insulin. Omitting Na+ from the incubation medium decreased total alanine uptake by about 60%, but abolished insulin stimulation. Decreasing the pH of the incubation medium progressively from 7.4 to 5.4 brought a regular reduction in the insulin stimulation, but at the same time the total saturable alanine uptake was increased. Other agents and conditions gave less sharp distinction between the transport systems used by alanine. α Aminoisobutyrate transport was more than doubled by 0.1 unit of insulin per ml in 1 hr. The pattern of its inhibition by different conditions suggests that this amino acid is transported in diaphragm almost completely by one system, which is Na+ dependent as well as sensitive to insulin. Leucine transport, on the other hand, showed relatively little response to insulin, and was essentially unaltered by changes in buffer [Na+] or pH or by the presence of competitive inhibitors other than leucine and isoleucine. The results suggest that L alanine enters rat diaphragm by at least 3 saturable routes. One is Na+ independent; another is Na+ dependent and sensitive to insulin; while the third is Na+ dependent but not affected by insulin. The insulin sensitive route accounts for no more than about 10 to 20% of the total saturable uptake at the alanine levels used. These facts support the hypothesis that insulin acts on only one neutral amino acid transport system in diaphragm; and that the hormone does not increase alanine transport greatly in this tissue because alanine does not use the insulin sensitive system extensively.
To elucidate the role of blood cells in amino acid metabolism, substrate balance across the forearm was studied in a nitrogen depleted subject fed 200 gm. of meat. After ingestion of the meal, there was the expected outpouring of amino acids from the splanchnic bed into the general circulation. Both cell and plasma levels of most amino acids in arterial blood increased rapidly. Whole blood arterio deep venous amino acid differences frequently differed from that of plasma. In conclusion, it appears that both blood cells and plasma transport amino acids from the splanchnic bed to the periphery and that both participate actively in the deposition of amino acids in the forearm of the subject studied.
1. The rates of entry of twenty of the blood amino acids into skeletal muscle of living rats were measured directly by means of a technique which ensured that a steady concentration of a radioactively labelled amino acid is reached rapidly and is maintained in the bloodstream.
2. The rates of entry were measured in experiments of short duration to avoid possible artifacts caused by amino acids leaving the muscle or by their metabolism.
3. The entry rate of each amino acid increased in direct proportion to its concentration in the blood plasma over the physiological range.
4. The various amino acids had widely different rates of entry. These rates could not be correlated with the physicochemical properties of the amino acids.
5. Two amino acids, L ‐lysine and L ‐threonine, enter muscle against a concentration gradient, while in the case of a third, L ‐arginine, the blood concentration was raised high enough to induce saturation of the entry mechanism.
6. It is concluded that entry takes place in vivo by means of carrier‐mediated transport processes with a high degree of specificity.
7. When the concentration of an amino acid in the bloodstream was increased to about twice normal the proportion of the additional amino acid that was taken up rapidly by the muscle was large enough, especially for the essential amino acids, to suggest that the tissue constitutes a quantitatively important storage system helping to regulate the concentrations of amino acids in the bloodstream.
Twenty one healthy subjects were studied after an overnight fast. Muscle tissue obtained by needle biopsy from m. quadriceps femoris was homogenized and precipitated with 4% sulfosalicylic acid. The supernatant material was analyzed for 23 to 28 free amino acids by a modified Moore-Stein technique. Simultaneously obtained plasma samples were precipitated with 6% sulfosalicylic acid and in all other respects were treated in the same way. Extracellular water in the muscle was estimated using a modified chloride method. The extra cellular (EC) and intracellular (IC) concentration and the IC/EC gradient for each amino acid was calculated. The majority of the amino acids showed much higher concentration in intracellular water than in plasma. The concentration gradient was especially high for taurine, glutamic acid, and glutamine. Valine, leucine, isoleucine, phenylalanine, citrulline, and tyrosine had a concentration gradient below 2.0. The rest of the amino acids showed gradients between 5.0 and 10.0. The free amino acid pool in skeletal muscle tissue was calculated in a normal man weighing 70 kg to be 86.5 g without taurine and 121.5 g with taurine. Of the total pool of muscle free amino acids the 8 essential amino acids represent only 8.4%, whereas glutamine, glutamic acid and alanine constitute about 79%.
The amino acids are ions of various charge combinations, and one can argue that historically they were the first ions for which the ongoing problem of membrane transport was presented; also that among transported ions these may undergo a highly detailed molecular recognition. Furthermore, the distribution of charge on the amino acid molecule determines by what route or routes it is conducted across the biological membrane, with what directional and structural specificity, and therefore what regulation is imposed, and where. Cases where a presumably charged chemical group behaves as if it were somehow absent from the amino acid have been observed to fall into several categories: Straightforward cases where the pH has been low enough or high enough to remove the charge by protonation or deprotonation, even in free solution. Cases where that protonation or deprotonation is facilitated at the binding site, and perhaps by the total transport process. The cystine molecule can apparently thus be rendered either a tripolar anion or a tripolar cation for transport. Cases where an otherwise co-transported Na+ is omitted to redress charge, or where a Na+ serves as a surrogate for a missing charged group on the amino acid molecule. A case where the protonation occurs reversibly at the receptor site rather than on the amino acid molecule.
Dichloroacetate (which activates pyruvate dehydrogenase) decreases the release of alanine, pyruvate and lactate in hemidiaphragm incubations with valine. Dichloroacetate interferes with alanine formation by diverting pyruvate into oxidative pathways, which not only limits pyruvate availability for direct transamination to form alanine but also indirectly affects branched-chain amino acid transamination by limiting 2-oxoglutarate regeneration from glutamate.
This paper reviews studies of cellular uptake of substrates by the gastric mucosa, salivary gland, and pancreas by single-circulation, multiple tracer dilution techniques. The application of this methodology to secretory organs in vivo has permitted the characterization of transport phenomena at the blood-tissue interface of resting and secreting epithelia. To estimate uptake of a test molecule after an intraarterial injection, its venous concentration profile (30 samples in 1 in) was compared with that of 1) an intravascular marker or 2) a diffusible molecule that remains confined to the extracellular space. Among the molecules investigated were 86Rb, 57Co-labeled cyanocobalamin, 125I-labeled insulin, [3H]ouabain, [3H]dopamine, [3H]norepinephrine, and a wide range of labeled amino acids. High tracer uptake (80%) was measured that could be inhibited by specific unlabeled competitors. Unidirectional influx was saturable and Michaelis-Menten kinetic constants could be estimated. The ultimate objective was to identify various transport systems and/or receptors at the basolateral side of these epithelia. However, nerve terminals in the interstitium could be the major site for the uptake of catecholamines.
Studies of leg exchange and urine output of 3-methylhistidine provide the most unequivocal demonstrations to date of the discrepancy between urinary excretion and skeletal muscle protein degradation. We believe that such demonstrations, taken with the evidence previously presented above, leave little room for doubt that the use of 3-methylhistidine production in urine or of the urinary 3-methylhistidine/creatinine ratio as indices of skeletal muscle protein breakdown should be discontinuid. However, we believe that the measurement of arteriovenous differences of 3-methylhistidine across skeletal muscle can be used to provide useful information about the regulation of myofibrillar protein breakdown, and this method deserves wider use.
Glutarnine transport and synthesis: explanation for injury-induced loss of glutamine in isolated rat muscle
- P Babij
- H S Hundal
- M J Rennie
- M R Ward
- P W Watt
BABIJ, P., HUNDAL, H. S., RENNIE, M. J., WARD, M. R. & WATT. P. W. (1986). Glutarnine transport and synthesis: explanation for injury-induced loss of glutamine in isolated rat muscle. Journal of Physiology 380, 52P
Regulation of basolateral amino acid transport activity in the exocrine pancreas by insulin, acetylcholine, cholecystokinin and experimental diabetes
- Mann G E Norman
- P S R J Habara
- M Munoz
- S Peran
MANN. G. E.. NORMAN, P. S. R.. HABARA. J., MUNOZ. M. & PERAN. S. (1985). Regulation of
basolateral amino acid transport activity in the exocrine pancreas by insulin, acetylcholine,
cholecystokinin and experimental diabetes. In Carrier Mediated Transport of Solutes from Blood
to Tissue, ed. YUDILEVICH, D. L. & MANN, G. E., pp. 77-98. London: Longman.
Fundamentals of Enzymnology
- N C Stevens
PRICE, N. C. & STEVENS, L. (1982). Fundamentals of Enzymnology. Oxford: Oxford University
Press.
Organic ion transport during seven decades
CHRISTENSEN, H. N. (1984). Organic ion transport during seven decades. Biochimnica et biophysica
acta 779, 255-269.
Effect of insulin on muscle glutamate uptake
- T T Brennan
- M F Muller
- W A Moore
- F D Cahill
AOKI, T. T., BRENNAN, M. F., MULLER, W. A., MOORE, F. D. & CAHILL, G. F. (1972). Effect of
insulin on muscle glutamate uptake. Journal of Clinical Investigation 51, 2889-2894.
Hindlimb muscle fibre population of five mammals
- M A Armstrong
- R B Edgerton
ARIANO, M. A., ARMSTRONG, R. B. & EDGERTON, V. R. (1973). Hindlimb muscle fibre population
of five mammals. Journal of Histochemistry and Cytochemistry 21, 51-55.