Regulation of glycogen synthesis by amino acids in cultured human muscle cells

Insulin and a number of metabolic factors stimulate glycogen synthesis and the enzyme glycogen synthase. Using human muscle cells we find that glycogen synthesis is stimulated by treatment of the cells with lithium ions, which inhibit glycogen synthase kinase 3. Insulin further stimulates glycogen synthesis in the presence of lithium ions, an effect abolished by wortmannin and rapamycin. We report also that amino acids stimulate glycogen synthesis and glycogen synthase, these effects also being blocked by rapamycin and wortmannin. Amino acids stimulate p70(s6k) and transiently inhibit glycogen synthase kinase 3 without effects on the activity of protein kinase B or the mitogen-activated protein kinase pathway. Thus, the work reported here demonstrates that amino acid availability can regulate glycogen synthesis. Furthermore, it demonstrates that glycogen synthase kinase 3 can be inactivated within cells independent of activation of protein kinase B and p90(rsk).     

Albumin synthesis in cultured hempatoma cells. Regulation by essential amino acids.

The effects of essential amino acids on albumin synthesis by a mouse hepatoma cell line have been investigated. The amino acids tested were tryptophan, phenylalanine, histidine, isoleucine and leucine. Cellular rates of synthesis (molecules albumin/cell per min) were determined from rates of [3H]leucine incorporation into immunoprecipitable albumin in the culture medium. The effects of amino acids on albumin synthesis fall into three distinct groups. The concentration of tryptophan producing half-maximal synthesis is 4 micronM. The corresponding concentration for leucine is 100 micronM. Histidine, phenylalanine and isoleucine were very similar, the half-maximal concentrations being approximately 15 micronM. The concentrations of amino acids producing half-maximal synthesis correlate directly with the amino acid composition of albumin. The levels of these essential amino acids necessary to saturate albumin synthesis have been compared with amino acid levels in normal plasma.     

Characterization of sulfate transport in cultured tobacco cells

Sulfate transport by tobacco cells (Nicotiana tabacum L. var. Xanthi) cultured in liquid medium was investigated.     

Translation kinetics in cultured mouse hepatoma cells. Regulation of albumin synthesis by amino acids

The synthesis of albumin in the liver has been shown to correlate with the availability of essential amino acids in the diet. We have investigated this phenomenon in the highly differentiated mouse hepatoma cell line, Hepa. Cells were grown for three days in complete medium with daily changes. The cells were then incubated for 22 h in media containing varying concentrations of individual essential amino acids. The deficient media were then changed; 1.5 h later the cells were labeled for 0.5 h with [3H]leucine. Albumin was immunoprecipitated and total protein was acid-precipitated from postribosomal supernatants of detergents-solubilized cells. With the exception of isoleucine, the relative rates of albumin synthesis decreased as a function of amino acid concentration from 4.3% in complete medium to 2.5% in totally deficient media. This specific reduction in albumin synthesis was confirmed by analysis of labeled Hepa proteins displayed on sodium dodecyl sulfate/polyacrylamide gels. Essential amino acid limitation reduced total protein synthesis by 50%. This is the result of a decrease in the translation efficiency of total mRNA from 5 to 3 polypeptides/message min-1 and is consistent with a reduction in the initiation rate. In contrast, the 70% decrease in albumin synthesis was a result of a reduced number of functional albumin messages/cell. The translation efficiency of these albumin messages remained unchanged at 1.     

Regulation of phosphatase synthesis by orthophosphate in cultured tobacco cells

Phosphatase activity in cultured tobacco cells XD-6 increasedremarkably under phosphate-deficient culture conditions. Suchan increase did not occur in the activities of -amylase, ß-galactosidase,succinate dehydrogenase and catalase under the same cultureconditions. By replenishment with Pi, the increase in totalphosphatase activity was suppressed and the specific activitywas reduced to a low level. The suppression of increase in theactivity resulted form a repression of de novo synthesis ofthe enzyme. Added Pi also suppressed the release of phosphataseinto the culture medium. The effect of Pi was found to be greaterthan the effect on the increase in the intracellular activity. At least three phosphatases were extracted from XD-6 cells.One of the enzymes increased when the phosphatase synthesiswas increased by phosphate deficiency. ¹ Present address: Laboratory of Applied Microbiology, Facultyof Agriculture, Yamagata University, Tsuruoka, Yamagata 997,Japan. (Received May 18, 1977; )     

Boron uptake by sunflower, squash and cultured tobacco cells

Boron uptake was studied in sunflower ( Helianthus annuus cv. Ha301), squash ( Cucurbita pepo cv. Early prolific straight neck) and cultured tobacco ( Nicotiana tobacum L. cv. TXD Monsanto cell line) cells with the use of stable B isotopes and inductively coupled plasma mass spectrometry. Boron uptake increased linearly with increasing B concentrations in the uptake medium, did not exhibit multiphasic kinetics and was not saturable over a wide concentration range. The addition of respiratory inhibitors to the uptake solution or exposure to low (2°C) or high (42°C) temperatures did not inhibit B uptake. The majority of the B within the plants, including recently absorbed B, was present in a nonexchangeable form and could not be removed by repeated rinsing with deionized water or exchange with B isotope. These results demonstrate that in these species B uptake is a passive, nonmetabolic process and that the formation of nonexchangeable B-complexes within the cytoplasm and cell wall is a key factor in determining the uptake of B by plants.     

Interaction of sulfate and glutathione transport in cultured tobacco cells

Photoheterotrophic and heterotrophic suspension cultures of tobacco (Nicotiana tabacum L.) were grown with 1 mM glutathione (reduced; GSH) as sole source of sulfur. Addition of sulfate to both cultures did not alter the rate of exponential growth, but affected the removal of GSH and sulfate in different ways. In photoheterotrophic suspensions, addition of sulfate caused a decline in the net uptake of GSH, whereas sulfate was taken up by the green cells immediately. In heterotrophic suspensions, however, addition of sulfate did not affect the net uptake of GSH and sulfate was only taken up by the cells after the GSH supply in the medium had been exhausted. Apparently, GSH uptake in photoheterotrophic cells is inhibited by sulfate, whereas sulfate uptake is inhibited by GSH in heterotrophic cells. The differences in the effect of GSH on sulfate uptake in photoheterotrophic and heterotrophic tobacco suspensions cannot be attributed to differences in the kinetic properties of sulfate carriers. In short-time transport experiments, both cultures took up sulfate almost entirely by an active-transport system as shown by experiments with metabolic inhibitors; sulfate transport of both cultures obeyed monophasic Michaelis-Menten kinetics with similar app. K_m (photoheterotrophic cells: 16.0±2.0 M; heterotrophic cells: 11.8±1.8 M) and V_max (photoheterotrophic cells: 323±50 nmol·min^-1·g^-1 dry weight; heterotrophic cells: 233±3 nmol·min^-1·g^-1 dry weight). Temperature- and pH-dependence of sulfate transport showed almost identical patterns. However, the cultures exhibited remarkable differences in the inhibition of sulfur influx by GSH in short-time transport experiments. Whereas 1 mM GSH inhibited sulfate transport into heterotrophic tobacco cells completely, sulfate transport into photoheterotrophic cells proceeded at more than two-thirds of its maximum velocity at this GSH concentration. The mode of action of GSH on sulfate transport in chloroplast-free tobacco cell does not appear to be direct: a 14-h exposure to 1 mM GSH was found to be necessary to completely block sulfate transport; a 4-h time of exposure did not affect this process. Consequently, glutathione does not seem to be a product of sulfur metabolism acting on sulfate-carrier entities by negative feedback control. When transferred to the whole plant, the observed differences in sulfate and glutathione influx into green and chloroplast-free cells may be interpreted as a regulatory device to prevent the uptake of excess sulfate by plants.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - DNP dinitrophenol - DW dry weight - FW fresh weight - GSH reduced glutathione     

Regulation of ornithine decarboxylase activity by amino acids, cyclic AMP and luteinizing hormone in cultured mammalian cells

Any one of five amino acis (alanine, asparagine, glutamine, glycine, and serine) is an essential requirement for the induction of ornithine decarboxylase (EC 4.1.1.17) in cultured chinese hamster ovary (CHO) cells maintained with a salts/glucose, medium. Each of these amino acids induced a striking activation of ornithine decarboxylase in the presence of dibutyryl cyclic AMP and luteinizing hormone. The effect of the other amino acids was considerably less or negligible. The active amino acids at optimal concentrations (10 mM) induced only a 10-20 fold enhancement of enzyme activity alone, while in the presence of dibutyryl cyclic AMP, ornithine decarboxylase activity was increased 40-50 fold within 7-8 h. Of the hormones and drugs tested, luteinizing hormone resulted in the highest (300-500 fold) induction of ornithine decarboxylase with optimal concentrations of dibutyryl cyclic AMP and asparagnine. Omission of dibutyryl cyclic AMP reduced this maximal activation to one half while optimal levels of luteinizing hormone alone caused no enhancement of ornithine decarboxylase activity. The induction of ornithine decarboxylase elicited by dibutyryl cyclic AMP, amino acid and luteinizing hormone was diminished about 50% with inhibitors of RNA and protein synthesis. The specific amino acid requirements for ornithine decarboxylase induction in chinese hamster ovary cells was similar to the requirements for induction in two other transformed cell lines. Understanding the mechanism of enzyme induction requires an identification of the essential components of the regulatory system. The essential requirement for enzyme induction is one of five amino acids. The induction of ornithine decarboxylase by dibutyryl cyclic AMP and luteinizing hormone was additive in the presence of an active amino acid.     

Regulation of uptake of purines, pyrimidines and amino acids by Candida utilis

Uptake of uracil by Candida utilis is increased by addition of leucine to a minimal medium in which organisms are growing. This response requires protein synthesis and has kinetics consistent with the induction of additional uracil transport by the amino acid or a derivative. Consequently, the contribution of exogenous radioactive uracil to the pyrimidine nucleotide pools increases so that RNA made after the amino acid is added is of greater specific radioactivity. Some other amino acids are as effective as leucine in increasing the incorporation of uracil into RNA. Growth with leucine present also increases to different extents the initial rates of uptake of adenine, cytosine, uridine, lysine, histidine, threonine, phenylalanine, aspartic acid and leucine itself. The action of leucine on lysine transport appears to involve induction. These effects are not restricted to leucine; growth with aspartic acid or phenylalanine in the medium gives similar results. Lysine, on the other hand, is without action on the uptake of leucine, aspartic acid, phenylalanine, threonine or uracil but decreases the initial rates of uptake of both histidine and lysine. We suggest that lysine represses its own transport. Similarly, there is a specific decrease in uracil uptake caused by growth with this pyrimidine. Thus in C. utilis there are complex interrelationships in the uptake of nitrogen-containing compounds.     

Regulation of uptake of inositol by glucose in cultured retinal pigment epithelial cells

Long term and acute effects of glucose on myo-inositol (MI) uptake were studied in primary cultures of bovine retinal pigment epithelial (RPE) cells. RPE cells were grown under low (5 mM) or high (20, 40, or 50 mM) glucose levels in the growth medium for up to 18 days. The concentrative capacity of confluent RPE cells to accululate [3H]MI (10 microM) was reduced up to 41% as the glucose concentration in the growth medium increased. When the growth medium glucose was switched from 5 to 40 mM, or vice versa, the capacity of cells to accumulate MI was reversed. Treatment of cells grown in 40 or 50 mM glucose with 0.1 mM Sorbinil (an aldose reductase inhibitor) minimally reversed the ability of cells to accumulate MI. RPE cells, grown in 5 mM glucose, were incubated with 10-60 mM D-glucose or its nonmetabolizable analogues (acute effect). Kinetics of MI uptake inhibition by alpha-methyl glucose according to Dixon plots were characteristic of competitive inhibition (Ki = 28 mM). MI uptake was strongly inhibited by phlorizin. The ability of RPE cells to bind 5 microM [3H]phlorizin also was reduced by increased glucose levels in the growth medium. These studies indicated that MI and glucose shared the same transporter system. Glucose in the incubation medium directly interfered with MI binding to the transporter. High glucose feeding of the cells also down-regulated the transporter density. The uptake and function of solutes such as MI in tissues that operate on the glucose carrier system may be severely impaired in diabetes.     

Regulation of nitrate uptake by amino acids in maize cell suspension culture and intact roots

The effect of amino acids on nitrate transport was studied in Zea mays cell suspension cultures and in Zea mays excised roots. The inclusion of aspartic acid, arginine, glutamine and glycine (15mM total amino acids) in a complete cell-culture media containing 1.0 mM NO₃ ^- strongly inhibited nitrate uptake and the induction of accelerated uptake rates. The nitrate uptake rate increased sharply once solution amino acid levels fell below detection limits. Glutamine alone inhibited induction in the cell suspension culture. Maize seedlings germinated and grown for 7 days in a 15 mM mixture of amino acids also had lower nitrate uptake rates than seedlings grown in 0.5 mM Ca(NO₃)₂ or 1 mM CaCl₂. As amino acids are the end product of nitrate assimilation, the results suggest an end-product feed-back mechanism for the regulation of nitrate uptake.     

Effects of amino acids on calcium uptake by glial and neuroblastoma cells

The uptake of [⁴⁵Ca] has been studied in clonal glial and neuronal cells. It was somewhat more efficient in the neuroblastoma clone M₁ compared to glial clones. In all cases [⁴⁵Ca] uptake was shown to depend on the phosphate concentration in the incubation medium. It was decreased by the ionophore A 23187 at 200 μM concentration in both neuronal and glial clones. The influence of amino acids some of which are putative neurotransmitters was investigated; the interactions between [⁴⁵Ca] uptake and these amino acids were related to their concentration and the type of cells used (neuronal or glial). L-aspartate and taurine for example had two opposite effects on [⁴⁵Ca] uptake by the glial clone NN at two different concentrations; they could therefore play a role in the control of calcium level in the synaptic cleft.     

Characterization of neutral amino acid uptake by cultured epithelial cells from pig kidney

Two transport systems for neutral amino acids have been characterised in LLC-PK₁ cells. The first, which transport alanine in a sodium-dependent manner, also mediates alanine exchange and is preferentially inhibited by serine, cysteine, and α-amino-n-butyric acid. This system resembles the ASC system in Ehrlich ascites and some other cell types. There is only a small contribution of other systems to alanine uptake. The second, which transports leucine with no requirement for sodium and mediates leucine exchange, is blocked by 2-aminonorbornane-2-carboxylic acid and hydrophobic amino acids. This system is similar to the L system described in other cell types. LLC-PK₁ cells retain several other features implying renal proximal tubule origin; our results thus suggest that these transport systems may be involved in the reabsorption of neutral amino acids by the nephron in vivo.     

Methylated amino acids and lysosomal function in cultured heart cells

Methylated amino acids inhibit lysosomal function in cultured rat heart myocytes more effectively than the classically employed lysosomotropic weak bases. Moreover, L-leucine methyl ester (L-Leu-OMe) or L-methionine methyl ester (L-Meth-OMe) do not alter lysosomal pH or inactivate lysosomal cysteine proteinases, but do inhibit protein degradation more efficiently than either chloroquine or NH4Cl. These observations suggest that amino acid methyl esters are more effective probes to investigate lysosomal function in cultured myocytes than chloroquine or NH4Cl.