Amphibolic role of the Krebs cycle in the insulin-stimulated protein synthesis.

It has been a generally held view that insulin does not significantly affect the incorporation of amino acids into liver protein. This interpretation was based on data obtained from studies using the branched chain amino acids, which are poorly metabolized by the hepatic tissue. The effect of insulin on 14CO2 formation and protein incorporation of several 1-14C-labeled or U-14C-labeled amino acids was studied in isolated rat hepatocytes and diaphragm pieces. It was shown that insulin enhanced 14CO2 formation and protein incorporation primarily of those carbons of amino acids which are metabolized through the mitochondrial Krebs cycle. Using aminooxyacetic acid (0.5 mM), a potent inhibitor of the transamination reaction, it was shown that there exists an "insulin-sensitive" pool of glutamate which is preferentially utilized for protein synthesis in the presence of insulin. The insulin effect on protein incorporation of 14C-labeled glutamate generated in the Krebs cycle was abolished in the presence of aminooxyacetic acid. We interpret these results to signify that mitochondrial transamination of alpha-ketoglutarate to glutamate is essential for insulin stimulation of 14C incorporation into hepatocyte protein.     

Some factors affecting the response of muscle to insulin

1. The effect of various changes in the composition of the supporting medium on the capacity of isolated rat diaphragm to incorporate amino acids into its protein has been studied. 2. Replacement of most of the normal ionic constituents by sucrose is inhibitory towards protein synthesis, as is also substitution of choline or K(+) for Na(+). 3. The capacity of the tissue to respond to a stimulatory effect of insulin is impaired in the sucrose media and under certain conditions in the absence of Na(+), particularly when Na(+) is replaced by K(+) and the (14)C-labelled amino acid is presented at a relatively high concentration. 4. Cutting of the tissue before incubation also decreases incorporating capacity and markedly decreases responsiveness to insulin. 5. In abnormal media the cellular content of ATP falls sharply. 6. The ATP content of the tissue also declines in the presence of 2-deoxyglucose. This change is prevented by the addition of glucose but not of pyruvate and succinate. 7. Although affecting the rate of amino acid incorporation the ATP content is not thought generally to limit sensitivity to insulin.     

The influence of electrical stimulation in vitro on protein synthesis and other metabolic parameters of rat extensor digitorum longus muscle

1. Apparatus is described in which rat extensor digitorum longus muscle can be incubated in buffer under conditions of light tension and be subject to contractures induced by electrical stimulation in vitro. Under these conditions the tissue retains its weight, its content of potassium and size of the extracellular space at values similar to those in vivo. 2. Though uptake of glucose was enhanced on addition of insulin, there was little increase in glucose consumption on stimulation. Breakdown of glycogen and enhancement of lactate output were found on stimulation. 3. Incorporation into protein of several labelled amino acids was diminished during stimulation. Accumulation of [(14)C]leucine was enhanced whereas that of glycine was decreased. 4. There were no very consistent changes in the content of free unlabelled amino acids during incubation with or without stimulation. Comparison of actual amino acid concentrations in tissue and incubation mixture with accumulation of (14)C-labelled amino acid indicated that full equilibration of the cell pool of amino amino acids with the medium is slow. 5. Substantial oxidation of several (14)C-labelled acids was observed. 6. The ATP content of the tissue declined a little during incubation and somewhat faster after a period of stimulation. 7. The results are discussed in relation to the way in which exercise can induce muscle hypertrophy.     

Incorporation of amino acids into protein of foetal rabbit diaphragm: effect of insulin.

The incorporation of six L-amino acids into the protein fraction of the diaphragm of foetal and neonatal rabbits has been measured. Insulin stimulates the incorporation of lysine from the 29th day and of glycine from the 31st day of gestation onwards. The incorporation of histidine, serine and threonine is only enhanced in the diaphragm of 16-20 hour-old rabbits; no effect of insulin on the incorporation of leucine has been shown for the period under study.     

Amino acid uptake in the developing chick embryo heart. The effect of insulin on glycine and leucine accumulation

1. The accumulation of [1-(14)C]glycine and the uptake, accumulation, incorporation (into protein, lipid, glycogen) and oxidation of l-[1-(14)C]leucine in 5-day-old chick embryo hearts were investigated in vitro, and the effects of insulin, puromycin and 4-methyl-2-oxopentanoic acid on these processes were studied. 2. With glycine, the ratio of concentration of the labelled amino acid in the cell water to that in medium markedly exceeded unity. Insulin significantly increased this ratio. Puromycin did not prevent the insulin effect. 3. With leucine, the concentration ratio of the labelled amino acid between intracellular and extracellular water approached unity in the absence of puromycin and was doubled by its presence. In neither case did insulin substantially alter this ratio. The addition of 4-methyl-2-oxopentanoic acid had no effect in the absence of insulin, but produced a significant increase of the concentration ratio in the presence of the hormone. 4. Leucine uptake was increased slightly by insulin in all experimental conditions except in the presence of puromycin, where a more pronounced stimulation was observed. The hormone had no effect on the incorporation of the labelled amino acid into protein, but accelerated its oxidation to carbon dioxide; the latter effect was particularly evident in the presence of puromycin and disappeared after the addition of 4-methyl-2-oxopentanoic acid.     

Hippurate synthesis and ammonia metabolism in isolated hepatocytes

The incorporation of benzoate into hippurate by isolated hepatocytes was limited by the availability of glycine. This limitation was unaffected by the addition of ammonia. When isolated hepatocytes were incubated in a solution containing urease, benzoate was completely ineffective in combating ammonia accumulation, while a mixture of α-keto analogues of several amino acids reduced ammonia accumulation by 50%. These findings do not support the use of benzoate to combat hyperammonemia, but they do support the current use of α-keto analogues of amino acids for that purpose.     

Action of insulin and growth hormone on protein synthesis in muscle from non-hypophysectomized rabbits

The separate effects of insulin and growth hormone on the uptake and incorporation of five amino acids into diaphragm muscle from non-hypophysectomized rabbits has been examined. Both growth hormone and insulin, when present in the medium separately, stimulated the incorporation into protein of the amino acids, leucine, arginine, valine, lysine and histidine. Insulin also stimulated amino acid uptake, but growth hormone did not. When insulin and growth hormone were present in the incubation medium together, the uptake and incorporation of valine, the only amino acid studied under these conditions, tended to be greater than the sum of the separate effects of the two hormones.     

The specific radioactivity of the tissue free amino acid pool as a basis for measuring the rate of protein synthesis in the rat in vivo

1. Rats were infused in vivo with [U-(14)C]glycine for periods of 2-6h, during which time the specific radioactivity of the free glycine in plasma and tissue approached a constant value. 2. Free serine also became labelled. The ratio of specific radioactivity of serine to that of glycine in the protein of liver, kidney, brain, jejunum, heart, diaphragm and gastrocnemius muscle was closer to the ratio in the free amino acid pool of the tissue than that of the plasma. 3. The kinetics of incorporation of [(14)C]glycine and [(14)C]serine into the protein of gastrocnemius muscle further suggested that the plasma free amino acids were not the immediate precursors of protein. 4. Infusion of rats with [U-(14)C]serine resulted in labelling of free glycine. The ratio of specific radioactivity of glycine to serine in the protein of liver, kidney, brain, jejunum and heart again suggested incorporation from a pool similar to the free amino acid pool of the tissue. 5. Rates of tissue protein synthesis calculated from the incorporation into protein of both radioactive glycine and serine, either infused or derived, were very similar when the precursor specific radioactivity was taken to be that in the total free amino acids of the tissue. Except for gastrocnemius muscle and diaphragm during the infusion of radioactive serine, the rates of tissue protein synthesis calculated from the specific radioactivity of the free glycine and serine in plasma differed markedly.     

The effect of insulin on free amino acid pools and protein synthesis in rat skeletal muscle in vitro

1. The effects of insulin in vitro on tissue pools and incorporation into protein of glycine and leucine in the extensor digitorum longus muscle of the rat are reported. 2. It was found that insulin decreased the lag period before the establishment of a linear rate of incorporation of radioactive glycine into protein. 3. The hormone increased the size of the free intracellular glycine pool. No such effect was found for leucine. The accumulation of radioactive glycine in the intracellular fluid compartment was increased. The content of radioactive leucine in the intracellular compartment was decreased. 4. Insulin decreased the specific radioactivity of both glycine and leucine in the extracellular fluid. 5. The hormone also decreased protein catabolism. 6. The effect on protein synthesis was not caused by an increase in the specific radioactivity of the extracellular pool but was possibly related to increased amino acid concentrations in this pool, which could in turn have affected the aggregation of ribosomes.     

Kinetic analysis of insulin action on amino acid uptake by isolated chick embryo heart cells

1. Isolated chick embryo heart cells were used to investigate the mode of action of insulin on the transport of three naturally occurring amino acids: l-proline, l-serine and glycine. Initial velocities of uptake were measured over a period of 5min with an 80-fold range of amino acid concentration. Corrections for amino acid diffusion, incorporation into protein and conversion into carbon dioxide were introduced. 2. The uptake processes approximated Michaelis-Menten kinetics within definite ranges of amino acid concentrations. A single transport system for proline and at least two transport systems for serine and glycine were detected. 3. The kinetic effects of insulin on transport systems for the amino acids tested were consistent with an acceleration of the maximal velocity of the process, without substantial changes in substrate concentration for half-maximal transport velocity. 4. These hormonal effects were not essentially altered by the corrections for amino acid incorporation into protein and conversion into carbon dioxide.     

Amino acids augment muscle protein synthesis in neonatal pigs during acute endotoxemia by stimulating mTOR-dependent translation initiation

In skeletal muscle of adults, sepsis reduces protein synthesis by depressing translation initiation and induces resistance to branched-chain amino acid stimulation. Normal neonates maintain a high basal muscle protein synthesis rate that is sensitive to amino acid stimulation. In the present study, we determined the effect of amino acids on protein synthesis in skeletal muscle and other tissues in septic neonates. Overnight-fasted neonatal pigs were infused with endotoxin (LPS, 0 and 10 microg.kg(-1).h(-1)), whereas glucose and insulin were maintained at fasting levels; amino acids were clamped at fasting or fed levels. In the presence of fasting insulin and amino acids, LPS reduced protein synthesis in longissimus dorsi (LD) and gastrocnemius muscles and increased protein synthesis in the diaphragm, but had no effect in masseter and heart muscles. Increasing amino acids to fed levels accelerated muscle protein synthesis in LD, gastrocnemius, masseter, and diaphragm. LPS stimulated protein synthesis in liver, lung, spleen, pancreas, and kidney in fasted animals. Raising amino acids to fed levels increased protein synthesis in liver of controls, but not LPS-treated animals. The increase in muscle protein synthesis in response to amino acids was associated with increased mTOR, 4E-BP1, and S6K1 phosphorylation and eIF4G-eIF4E association in control and LPS-infused animals. These findings suggest that amino acids stimulate skeletal muscle protein synthesis during acute endotoxemia via mTOR-dependent ribosomal assembly despite reduced basal protein synthesis rates in neonatal pigs. However, provision of amino acids does not further enhance the LPS-induced increase in liver protein synthesis.     

Effects of hormones on protein and amino acid metabolism in mammary-gland explants of mice.

The effects of insulin, cortisol and prolactin on amino acid uptake and protein biosynthesis were determined in mammary-gland explants from mid-pregnant mice. Insulin stimulated [3H]leucine incorporation into protein within 15 min of adding insulin to the incubation medium. Insulin also had a rapid stimulatory effect on the rate of aminoiso[14C]butyric acid uptake, but it had no effect on the intracellular accumulation of [3H]leucine. Cortisol inhibited the rate of [3H]leucine incorporation into protein during the initial 4h of incubation, but it had no effect at subsequent times. [3H]Leucine uptake was unaffected by cortisol, but amino[14C]isobutyric acid uptake was inhibited after a 4h exposure period to this hormone. Prolactin stimulated the rate of [3H]leucine incorporation into protein when tissues were exposed to this hormone for 4h or more; up to 4h, however, no effect of prolactin was detected. At all times tested, prolactin had no effect on the uptake of either amino[14C]isobutyric acid or [3H]leucine. Incubation with actinomycin D abolished the prolactin stimulation of protein biosynthesis, but this antibiotic did not affect the insulin response. A distinct difference in the mechanism of action of these hormones on protein biosynthesis in the mammary gland is thus apparent.     

Properties of yeast grown anaerobically in media limiting in potassium

1. Saccharomyces cerevisiae was grown anaerobically in media with different concentrations of K(+) down to less than 1mm. Below 3.2mm the K(+) concentration limited the growth rate and yield. 2. Yeast extract was essential for maximum growth. The yield of cells when the medium contained 0.83mm-K(+) was only 30% of the yield with 90mm-K(+). 3. At the end of anaerobic growth the cells grown in 0.83mm-K(+) had a higher concentration of oxidative enzymes than cells grown in 90mm-K(+). 4. The cells grown anaerobically in 0.83mm-K(+) could adapt to aerobic conditions if K(+) was present in the adaptation medium, but not otherwise. 5. The enzyme pattern of the yeast grown aerobically in 0.83mm-K(+) was very similar to the anaerobically grown cells and did not change markedly after the glucose was consumed.     

Relationship between intracellular amino acids and protein synthesis in the extensor digitorum longus muscle of rats

1. The incorporation into protein, and the accumulation into the free amino acid pools, of radioactive l-leucine and glycine was studied in rat extensor digitorum longus muscle. 2. The tissue was incubated first with (14)C-labelled and then with (3)H-labelled amino acid. 3. The experimental results were consistent with a model based on the premise that the amino acids in protein were incorporated directly from the extracellular pool.     

Amino acid and putative neurotransmitter transport in human Y79 retinoblastoma cells. Effect of insulin and insulin-like growth factor

The binding of insulin and insulin-like growth factor I (IGF-I) and their effect on amino acid and neurotransmitter transport was studied in cultured human Y79 retinoblastoma cells. Y79 cells possess specific receptors for both insulin and IGF-I. Insulin binding to Y79 cells is characterized by a curvilinear Scatchard plot suggesting a two-site or two-affinity binding system. In contrast, IGF-I binding has a linear plot indicative of a one-site, one-affinity binding system. The uptake of glycine, a putative neurotransmitter in the retina occurs by a specific transport system in Y79 cells, independent of the uptake of other neutral amino acids. The uptake of glycine was increased 25-50% by either insulin or IGF-I. The response to insulin or IGF-I on glycine uptake is gradual and concentration dependent. The accumulation of other amino acids and putative retinal neurotransmitters by Y79 cells was not significantly affected by insulin of IGF-I. In addition, the activity of Na+/K+-ATPase was not influenced. The analysis of high affinity glycine uptake indicates that insulin and IGF-I are stimulating glycine transport by increasing the V'max without significantly affecting the K'm. Further analysis suggests that insulin and IGF-I are causing a recruitment of additional glycine transporters at the cell surface or activating otherwise nonfunctional transporters by an unexplained mechanism. Because of the implication that glycine responds as a neuroactive amino acid in Y79 cells these studies suggest that insulin and IGF-I may influence neuroactivity in the human retina by regulating the transport of glycine.     

Further observations on the polynucleotide-induced stimulation of protein synthesis by cell-free preparations from rabbit reticulocytes.

1. The effect of high-molecular-weight RNA from reticulocyte polyribosomes (messenger RNA) on protein synthesis by subcellular fractions derived from reticulocytes, reported by Arnstein, Cox & Hunt (1964), has now been studied in detail. Optimum response of the cell-free system requires 30-50mm-K(+) and approx. 5mm-Mg(2+) in the pH range 7.4-7.6. 2. RNA stimulates the incorporation into protein of both free amino acids and of aminoacyl residues from s-RNA. Stimulation by either RNA or polyuridylic acid is dependent on a labile factor or enzyme, which is present in the ;pH5 fraction' and may be concerned with the formation of new polysomes. Quantitatively the response of the cell-free system to RNA is similar to that of polyuridylic acid, and there appears to be competition between messenger RNA and polyuridylic acid or polyadenylic acid.     

Studies on metabolism of vitamin A. The effect of hormones on gestation in retinoate-fed female rats

1. The preparation of cell suspensions by treatment of chick embryo hearts with collagenase at various stages of development is described. 2. Measurements of oxygen consumption, incorporation of labelled leucine into protein and accumulation of labelled alpha-aminoisobutyric acid against a concentration gradient indicated a long-lasting viability of the isolated heart cells in vitro; a satisfactory preservation of subcellular structures, including plasma membrane, was assessed by electron-microscopic examination. 3. The rate of alpha-aminoisobutyric acid accumulation by cardiac cells isolated from hearts at different stages of embryological development decreased with aging; insulin stimulated the intracellular accumulation of this amino acid analogue. 4. Insulin increased the uptake by isolated heart cells of several (14)C-labelled naturally occurring amino acids; however, the fraction of amino acid taken up by the cells that was recovered free intracellularly, and therefore the concentration ratio (between intracellular water and medium), was enhanced by the hormone only with glycine, proline, serine, threonine, histidine and methionine. When isolated heart cells were incubated in the presence of a mixture of labelled amino acids, the addition of insulin increased the disappearance of radioactivity from the medium. 5. The general pattern of amino acid transport (in the absence and in the presence of insulin) in isolated cardiac cells was similar to that found in intact hearts, suggesting that the biological preparation described in this paper might be useful for studies of cell permeability and insulin action.     

Stoicheiometrical proton and potassium ion movements accompanying the absorption of amino acids by the yeast Saccharomyces carlsbergensis

1. Proton uptake into the yeast Saccharomyces carlsbergensis, was studied at pH4.5-5.5 in the presence of both antimycin and 2-deoxyglucose to inhibit energy metabolism. Previous work had shown that the cells then absorbed about 20nmol of glycine or l-phenylalanine against a considerable amino acid concentration gradient. The addition of the amino acid immediately stimulated the rate of uptake of protons two- to three-fold. About 2 extra equivalents of H(+) accompanied a given amount of the amino acids into the yeast preparations exposed to the metabolic inhibitors for 2-4min and about 1.2 equivalents after 20min exposure. 2. Analogous observations were made during serial additions of glycine, l-phenylalanine, l-leucine and l-lysine to preparations lacking the metabolic inhibitors and deficient in substrates needed for energy metabolism. In fresh cellular preparations the influx of glycine was then closely coupled to a stimulated flow of 2.1 equiv. of H(+) into the yeast. A similar number of K(+) ions left the cells. About 30% of the extra protons was subsequently ejected from the yeast. Deoxyglucose and antimycin together inhibited the ejection of protons. When the yeast had been fed with glucose energy metabolism was stimulated and almost as many protons as were absorbed with the amino acid were apparently ejected again. 3. Yeast preparations containing Na(+), instead of K(+), as the principal cation absorbed about 1 extra equivalent of H(+) after the addition of phenylalanine, glycine or leucine. This response was not observed in the presence of both deoxyglucose and antimycin. 4. The observations show that H(+) and, in certain circumstances, K(+) are co-substrates in the transport of the amino acids into the yeast. An analogy is drawn with the roles of Na(+) and K(+) as co-substrates in certain mammalian systems. The results lead to various models relating the physical flow of the co-substrate ions on the amino acid carrier to the transduction of chemical energy in an associated ion pump forming part of the mechanism for transporting amino acids into the yeast.     

Compartmental analysis of sulphate transport in Lemna minor L., taking plant growth and sulphate metabolization into consideration

Rat hepatoma cells accumulate considerably less 2-aminoisobutyrate after cultivating in the absence of serum the change in rate of aminoisobutyrate uptake takes place within 1 h of serum starvation. Starvation of amino acids by contrast raises aminoisobutyrate uptake in the presence or absence of serum, but the cells are much less responsive to amino acid supply than to availability of serum. Phosphate (10 mM) reduced aminoisobutyrate uptake by cells grown in serum to that exhibited by serum-starved cells. Aminoisobutyrate uptake by cells grown in serum was reduced by glycine, proline, alanine, serine, glutamine, methylaminoisobutyrate and 2-aminonorbornane-2-carboxylate, the effects of methylaminoisobutyrate and 2-aminonorbornane-2-carboxylate being additive. However, similar inhibition phenomena were not seen for cells deprived of serum where aminoisobutyrate uptake tended to a relatively constant level insensitive to inhibitory influences, yet substantially greater than that arising by simple diffusion. The comparative insensitivity of our hepatoma line when starved of serum to competition and repression phenomena is in contrast to findings of others. Our results also suggest a lack of clear delineation of specificities for the A and L transport systems as usually defined.