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

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

Amino acid metabolism in several tissues of the obese Zucker rat as indicated by the tissue accumulation of α-amino[1-14C]isobutyrate

Measurements of the tissue accumulation of α-amino[1-¹⁴C]isobutyrate [1-¹⁴C]AIB) in lean (+/?) and obese (fa/fa) Zucker rats showed an augmented tissue/plasma ratio in the liver of the obese animals. In contrast, brown adipose tissue AIB accumulation was lower in the fa/fa animals. In response to a 24h starvation period AIB accumulation was significantly elevated in the liver and plasma of the lean animals and was unchanged in the liver of the fa/fa animals. The circulating concentration of alanine and branched-chain amino acids was elevated in the fa/fa animals as compared to their lean counterparts. These observations suggest that amino acid uptake is not involved in the impaired muscle development observed in the obese Zucker rat and that the ability of brown adipose tissue for amino acid utilization is decreased in the obese animals suggesting that this may partially explain the impaired thermoregulatory capacity observed in brown adipose tissue of obese Zucker rats.     

A transient increase in amino acid transport modulated by insulin in differentiating muscle cells.

During synchronous differentiation of embryonic chick muscle cells in cultures, the Na-dependent uptake of an amino acid analog, alpha-amino isobutyric acid (AIB) undergoes in abrupt, transient increase. The increase in AIB uptake is concomitant with the rapid fusion of mononucleated myoblasts, and precedes the accumulation of muscle-specific proteins. Subsequently, Na-dependent AIB transport diminishes markedly during postfusional differentiation of myotubes. The rate of AIB uptake is increased by insulin both before and after myoblast fusion. This stimulation by insulin is restricted to the Na-dependent component of total AIB uptake but is apparently not the result of insulin-mediated increase in the trans-membrane Na gradient.     

Uptake of 14C-α-aminoisobutyric acid by Phaseolus vulgaris

Specific uptake (S.U.) of α-aminoisobutyric acid ([1-¹⁴C]AIB), a non-metabolizable neutral amino acid analog, by dwarf bush bean plants (Phaseolus vulgaris cv Top Crop) demonstrated wide differences in active transport between various plant organs. The kinetic and timed uptake data reported were expressed as S.U. because this corrects for the diffusion of AIB which is part of the total AIB uptake process. Roots accumulated AIB to concentrations up to 18 times and leaf disks to twice those of the incubation medium. Stem tissue showed very little uptake, if any, that could not be accounted for by simple diffusion or water free space. Although initial rate kinetic studies demonstrated the presence of a normal transport system, timed uptake studies revealed greatly decreased transport by etiolated plants, suggesting a relationship between active transport and the lack of photosynthate. The reproducibility of the AIB uptake pattern by mature roots strongly supports the concept that the transport of neutral amino acids is biphasic and suggested one or more carrier systems are inducible by either low intracellular concentrations or repressed by high intracellular concentrations of the amino acid.     

Insulin resistance in soleus muscle from obese Zucker rats. Involvement of several defective sites

1. The effect of insulin upon glucose transport and metabolism in soleus muscles of genetically obese (fa/fa) and heterozygote lean Zucker rats was investigated at 5–6 weeks and 10–11 weeks of age. Weight-standardized strips of soleus muscles were used rather than the intact muscle in order to circumvent problems of diffusion of substrates. 2. In younger obese rats (5–6 weeks), plasma concentrations of immunoreactive insulin were twice those of controls, whereas their circulating triacylglycerol concentrations were normal. Insulin effects upon 2-deoxyglucose uptake and glucose metabolism by soleus muscles of these rats were characterized by both a decreased sensitivity and a decrease in the maximal response of this tissue to the hormone. 3. In older obese rats (10–11 weeks), circulating concentrations of insulin and triacylglycerols were both abnormally elevated. A decrease of 25–35% in insulin-binding capacity to muscles of obese rats was observed. The soleus muscles from the older obese animals also displayed decreased sensitivity and maximal response to insulin. However, at a low insulin concentration (0.1m-i.u./ml), 2-deoxyglucose uptake by muscles of older obese rats was stimulated, but such a concentration was ineffective in stimulating glucose incorporation into glycogen, and glucose metabolism by glycolysis. 4. Endogenous lipid utilization by muscle was calculated from the measurements of O₂ consumption, and glucose oxidation to CO₂. The rate of utilization of fatty acids was normal in muscles of younger obese animals, but increased in those of the older obese rats. Increased basal concentrations of citrate, glucose 6-phosphate and glycogen were found in muscles of older obese rats and may reflect intracellular inhibition of glucose metabolism as a result of increased lipid utilization. 5. Thus several abnormalities are responsible for insulin resistance of muscles from obese Zucker rats among which we have observed decreased insulin binding, decreased glucose transport and increased utilization of endogenous fatty acid which could inhibit glucose utilization.     

A mathematical model for membrane transport of amino acid and Na+ in vesicles

Summary A model with a carrier having sites for both amino acid and Na⁺ can account for AIB (-aminoisobutyric acid) transport kinetics observed in membrane vesicles from SV3T3 (simian virus 40-tranformed Balb/c3T3 cells) and 3T3 (the parent cell line). The main feature of this cotransport model is that Na⁺ binding to carrier decreases the effectiveK _m for AIB transport, Na⁺ transport kinetics observed in both vesicle systems can be described by passive (possibly facilitated) diffusion. The lag of Na⁺ transport across the membrane compared to that for AIB, coupled to the Na⁺-dependent decrease in theK _m for AIB, accounts for the overshoot in intravesicular AIB observed for SV3T3 in the presence of an initial Na⁺ gradient. Extra-vesicular Na⁺ maintains a derease in theK _m for AIB influx before intra-vesicular Na⁺ has accumulated to balance it with a comparable decrease in theK _m for AIB efflux. 3T3 vesicles display little overshoot, and this finding can be explained mostly by a lower carrier affinity for Na⁺.     

Energetic basis of development of salt-tolerant transport in a moderately halophilic bacterium,Vibrio costicola

Active transport of -aminoisobutyric acid (AIB) in Vibrio costicola utilizes a system with affinity for glycine, alanine and, to some extent, methionine. AIB transport was more tolerant of high salt concentrations (3–4 M NaCl) in cells grown in the presence of 1.0 M NaCl than in those grown in the presence of 0.5 M NaCl. The former cells could also maintain much higher ATP contents than the latter in high salt concentrations.Transport kinetic studies performed with bacteria grown in 1.0 M NaCl revealed three effects of the Na⁺ ion: the first effect is to increase the apparent affinity (K _t) of the transport system for AIB at Na⁺ concentrations <0.2 M, the second to increase the maximum velocity (V _max) of transport (Na⁺ concentrations between 0.2 and 1.0 M), and the third to decrease the V _max without affectig K _t (Na⁺ concentrations >1.0 M). Cells grown in the presence of 0.5 M or 1.0 M NaCl had similar affinity for AIV. Thus, the differences in salt response of transport in these cells do not seem due to differences in AIB binding. Large, transport-inhibitory concentrations of NaCl resulted in efflux of AIB from cells preloaded in 0.5 M or 1.0 M NaCl, with most dramatic efflux occurring from the cells whose AIB transport was more salt-sensitive. Our results suggest that the degree to which high salt concentrations affect the transmembrane electrochemical energy source used for transport and ATP synthesis is an important determinant of salt tolerance.Abbreviations AIB -aminoisobutyric acid - pmf proton motive force     

Amino acid and22Na+ uptake in membrane vesicles from confluent simian virus 40 transformed Balb/c3T3 and Balb/c3T3

Summary The studies reported here were carried out to characterize further previously described changes in membrane localized amino acid transport associated with simian virus 40 transformation of the mammalian cell line, Balb/c3T3. Membrane vesicles were prepared from confluent cultures of both simian virus 40 transformed Balb/c3T3 (SV3T3) and the untransformed parent line, Balb/c3T3 (3T3). An initial, externally imposed out>in, 100mm Na⁺ gradient produces acceleration of early ingress of -aminoisobutyric acid (AIB) in vesicles from both cell lines, but transient, concentrative uptake (overshooting) only in SV3T3 vesicles. Early ingress ofl-leucine is also accelerated in SV3T3 vesicles by a Na⁺ gradient, and overshooting is also demonstrable.Na⁺-gradient independent AIB permeability of SV3T3 and 3T3 membranes was estimated using uptake data, a first order rate equation and measurements of vesicle size derived from quasi-elastic light-scattering studies. AIB permeability of SV3T3 membranes is greater than that of 3T3 membranes (113 Å/min and 43 Å/min, respectively), suggesting that overshooting in 3T3 vesicles is not attenuated by a Na⁺-independent AIB leak. Na⁺ permeability of the two membranes is similar, ruling out the possibility that a slower rate of Na⁺ equilibration across the SV3T3 membrane allows development of the overshoot.In SV3T3 vesicles the height of a Na⁺-gradient dependent overshoot varies with the initial [Na⁺] _o /[Na⁺] _i ratio, and [Na⁺] _o /[Na⁺] _i is linearly related to ln AIB uptake at overshoot peak/AIB uptake at equilibrium, consistent with the possibility that for [Na⁺] _o /[Na⁺] _i ratios in the range studied, AIB overshoot is energized by a constant proportion of the energy available from the initial electrochemical gradient for Na⁺.These results are consistent with the possibility that Na⁺-gradient dependent overshooting in SV3T3 vesicles is produced by Na⁺-amino acid carrier interactions resulting in either an increase in maximum transport velocity or an incrase in carrier affinity for AIB.Abbreviations used 3T3 Balb/c3T3 - SV3T3 simian virus 40 transformed Balb/c3T3 - AIB -aminoisobutyric acid     

Derepression of amino Acid-h cotransport in developing soybean embryos

The uptake of the unnatural amino acid α-aminoisobutyric acid (AIB) and glutamine by developing soybean (Glycine max Merr. cv Chippewa 64) embryos was investigated. In freshly excised embryos, the accumulation ratio (cytoplasmic concentration/external concentration) of AIB did not exceed 1.0. After an 18-hour preincubation in nitrogen-free medium the accumulation ratio of AIB exceeded 4.5 at an external AIB concentration of 10 micromolar. This indicates the derepression of an active amino acid uptake mechanism operative at low external amino acid concentration. The presence of sucrose, NH₄NO₃, or glutamine during a 21-hour preincubation prior to measuring glutamine uptake inhibited the enhancement of uptake by 43%, 51%, and 96%, respectively. The time course of the decline in free amino acids and the time course of enhancement of amino acid uptake was not consistent with enhanced uptake resulting from relief of transinhibition, but suggested instead the derepression of synthesis of new carriers. The time course of enhancement of amino acid uptake was paralleled by an increase in glutamine-induced depolarization of the membrane potential. The kinetics of glutamine uptake indicated the presence of a saturable and a nonsaturable component of uptake. The saturable component of uptake is attributed to a mechanism of amino acid-H⁺ cotransport which is derepressed by nitrogen and/or carbon starvation. At physiological concentrations of amino acids, uptake through the saturable system in freshly excised embryos is negligible. Thus, uptake through the nonsaturable system is of primary importance in the nitrogen nutrition of developing soybean embryos.     

Regulation of amino acid transport in L6 muscle cells: I. Stimulation of transport system a by amino acid deprivation

The regulation of amino acid transport in L6 muscle cells by amino acid deprivation was investigated. Proline uptake was Na⁺-dependent, saturable and concentrative, and was predominantly through system A. Proline uptake was inhibited by alanine, α-amino isobutyric acid (AIB), and by α-methylamino isobutyric acid, but not by lysine or valine. At 25°C, Km of proline uptake was 0.5 mM. Amino acid-deprivation resulted in a progressive increase in the rate of proline uptake, reaching up to 6-fold stimulation after 6 hours. The basal and stimulated transport were equally Na⁺-dependent, and both were inhibited by competition with the same amino acids. Kinetic analysis showed that Km decreased by a factor of 2.4 and Vmax increased 1.9-fold in deprived cells. Amino acid-deprivation did not stimulate amino acid uptake through systems other than system A. This suggests that the higher Km in proline-supplemented cells is not due to release of intracellular amino acids into unstirred layers surrounding the cells. The presence of amino acids which are substrates of system A (including AIB) during proline-deprivation, prevented stimulation of proline uptake, whereas those transported by systems Ly⁺ or L exclusively were ineffective. The stimulation of the transport-rate in deprived cells could be reversed by subsequent exposure to proline or other substrates of system A. L6 cells, deprived of proline for 6 hours, retained the stimulation of transport after detachment from the monolayers with trypsin. Uptake rates were comparable in suspended and attached cells in monolayer culture. Thus, amino acid-depreivation of L6 cells results in an adaptive increase in proline uptake, which is not due to unstirred layers but appears to be mediated by other mechanisms of selective transport regulation.     

Hormonal regulation of hepatic amino acid transport

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

Effect of the pH of culture medium on the alkalophilicity of a species of Bacillus

The amino acid incorporation and -aminoisobutyric acid (AIB) uptake of an alkalophilic Bacillus grown at pH 8.2 (the pH 8-bacteria) were much less pH dependent (less alkalophilic) than those of the organisms grown at pH 10.0 (the pH 10-bacteria), respectively. The rate of AIB uptake of the pH 10-bacteria was almost the same as that of the pH 8-bacteria, while the rate of amino acid incorporation of the pH 10-bacteria was higher than that of the pH 8-bacteria in alkaline environments. The colloidal titration with clupein showed that the amount of negative charge on the pH 10-bacteria was greater than that of the pH 8-bacteria in alkaline environments. Considerable difference in protein composition was observed between the membranes of the pH 8-and 10-bacteria, while no difference was observed in phospholipid composition.Abbreviations AIB Amino-isobutyric acid     

Endogenous respiration reflects the energy load imposed by transport of nonmetabolizable substrates and by induced de novo protein synthesis in Rhodotorula glutinis

Uptake of the nonmetabolizable sugars 6-deoxy-d-glucose, l-rhamnose and l-xylose, which are taken up by a common carrier, stimulated significantly cell respiration in Rhodotorula glutinis. The extra oxygen consumption for uptake (0.5–0.7 equivalents O₂/mol transported sugar) was proportional to the uptake rate and was independent of the K _tvalue of the transport system. Sugars that become metabolized after induction, d-arabinose and methyl--d-glucoside, caused a higher stimulation, 1.4 and 3.6 equivalents O₂/mol respectively, which was reduced to 0.6 equivalents O₂/mol when de novo protein synthesis was blocked by cycloheximide. The stimulation of respiration thus includes a fraction related purely to the energy demand for uptake and another one related to the induced de novo protein synthesis. The net uptake-induced respiration boost was similar with all sugars under study irrespective of their transport systems. The estimated energy demand was equivalent to about 2 ATP/sugar molecule. For comparison, the amino acid analogue -aminoisobutyric acid (AIB) was also investigated; the overall energy demand for its uptake corresponded to the equivalent of about 4 ATP/molecule.Abbreviation AIB -aminoisobutyric acid     

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

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

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

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

Absorption of different amino acids by an intestinal preparation from normal rats and from rats exposed to supralethal X-Irradiation

Summary Absorption of different amino acids [lysine,-amino isobutyrate (AIB), methionine, alanine, glycine] was studied in an intestinal in vivo preparation from normal and X-irradiated rats (1 and 3 days after 2000 R). Absorption of amino acids under loading and in presence of other amino acids was also followed. Lysine and AIB absorption follow a saturation kinetics with a maximal velocity of 35 and 85.6 µmoles/min and aK _t of 10 and 67 µmoles for lysine and AIB respectively. Interference in absorption exists not only between amino acids of the same class, but also by lysine on neutral amino acids. One day after irradiation, absorption is normal or even enhanced, although active transport appears impaired as indicated by the increase inK _t. Three days after exposure, absorption, particularly the active one, is severely depressed. This is indicated by the low Vm and highK _t, as well as by the fact that loading has little influence on absorption.Supported by the Schutzkommission am Innenministerium der BRD. Publication No. 1389 of the Euratom Biology Division     

Effect of radiotherapy and chemoradiotherapy on circulating antioxidant system of human uterine cervical carcinoma

Rats bearing the Yoshida AH-130 ascites hepatoma showed important changes in lipid metabolism. The presence of this rapidly growing tumour induced a significant reduction in the intestinal absorption of an oral [^l4C]triolein load but without changes in whole body oxidation of the tracer to CO₃. Both white (WAT) and brown (BAT) adipose tissue lipoprotein lipase (LPL) activities were increased at day 4 of tumour growth, changes that seem to be related with those observed in [¹⁴C] lipid accumulation; however, heart LPL activity was increased at day 7 but there was no change at day 4. In addition, there was a marked hyperlipemia in the tumour-bearing animals, whereas the blood ketone body concentrations were lower in these animals in comparison with the corresponding pair-fed group. The in vivo lipogenic rate was increased in liver of the tumour-bearing animals (day 4); conversely, it was decreased in WAT and skeletal muscle (day 4) and IBAT (day 7) of the AH-130-bearing rats. It may be suggested that the increased liver lipogenic rate associated with tumour burden is the main factor contributing to the hyperlipidaemia present in the Yoshida AH-130 bearing rats.     

Stimulative effect of nerve growth factor on α-aminoisobutyric acid uptake and Na,K-ATPase activity in superior cervical sympathetic ganglia excised from adult rats

Effects of nerve growth factor (NGF) on the uptake of non-metabolizable -aminoisobutyric acid (AIB) and on Na,K-ATPase activity in superior cervical sympathetic ganglia (SCG) excised from adult rats were examined during aerobic incubation in vitro. Active uptake of labelled AIB into isolated SCG during 1 to 5 hours incubation at 37°C was significantly accelerated by the addition of NGF to the incubation medium in a dose-dependent manner. Although theK _m value of the AIB uptake by the SCG did not change with the addition of NGF,V _max was nearly doubled. The NGF-evoked increase in AIB uptake was antagonized by the further addition of its specific antiserum in a dose-dependent fashion, and was largely suppressed in a mdium containing ouabain. In SCG, axotomized one week prior to the examination, from which most of the neurons had disappeared and reactive proliferation of satellite glial components was in progress, the NGF-induced acceleration of AIB uptake was completely absent. The ganglionic Na,K-ATPase activity was greatly stimulated in the presence of NGF, and the effect was completely eliminated in the axotomized SCG. These results strongly suggest that the NGF-induced acceleration of active AIB uptake by the isolated SCG occurs not in glial cells but exclusively in the neuronal components with the apparent coupling of an Na ion extrusion process.Dedicated to Professor Yasuzo Tsukada.     

Regulation of amino acid transport and protein metabolism in myotubes derived from chicken muscle satellite cells by insulin-like growth factor-I

The effects of insulin and insulin-like growth factor-I (IGF-I) on amino acid transport and protein metabolism were compared in myotubes derived from chicken breast muscle satellite cells. Protein synthesis was assessed by continuous labelling with [³H]-tyrosine. Protein degradation was estimated by the release of trichloroacetic acid (TCA) soluble radioactivity by cells which had been previously labelled with [³H]-tyrosine for 3 days. Amino acid transport was measured in myotubes incubated in Dulbecco's modified Eagle's medium (DMEM) 0.5% bovine serum albumin (BSA) with or without insulin or IGF-I. Subsequent [³H]-aminoisobutyric acid (AIB) uptake was then measured in amino acid-free medium. IGF-I was more efficient than insulin at equimolar concentration (3.2 nmol/l) in stimulating protein synthesis (127 and 113% of basal, respectively) and inhibiting protein degradation (32% and 13% inhibition of protein degradation following 4 h incubation). Half maximal effective concentrations for stimulation of AIB uptake were 0.27 ± 0.03 nmol/l and 34.8 ± 3.1 nmol/l for IGF-I and insulin respectively, with maximal stimulation of about 340% of basal. Cycloheximide (3.6 μmol/l) diminished IGF-I-stimulated AIB uptake by 55%. Chicken growth hormone had no effect on basal AIB uptake in these cells and neither glucagon nor dexamethasone had an effect on basal or IGF-I-stimulated AIB uptake. This study demonstrates an anabolic effect for IGF-I in myotubes derived from primary chicken satellite cells which is mediated by the type I IGF receptor, since the cation-independent mannose 6-phosphate receptor does not bind IGF-II in chicken cells. © 1993 Wiley-Liss, Inc.     

Reabsorption and accumulation of α-amino-iso-butyric acid in the nephridia of Sabella pavonina Sa vigny (Annelida polychaeta)

• 1.l. High amino acid concentrations were found in the anterior coelomic fluid of a Polychaeta (Sabella pavonina Savigny).
• 2.2. The concentrations being much higher in the fluid which penetrates the nephrostomia into the nephridia lumen than in the final urine indicates that the nephridia reabsorbs large amounts of amino acids.
• 3.3. Nephridial perfusion experiments showed that an amino acid analogue (α-amino-iso-butyric acid, AIB) is transported by the nephidia.
• 4.4. The transport took place across the nephridial wall owing to the presence of a carrier-mediated transport system and a diffusion system.
• 5.5. For the carrier-mediated transport, the V_max was 0.234 ± 0.025 nmol·min⁻ and the K_m 3.715 ± 0.315mmol·l⁻.
• 6.6. AIB accumulated in the nephridial cells up to a maximum rate of 01.17 nmol·min⁻.
• 7.7. Intracellular accumulation stopped increasing when the V_max for reabsorption was reached.
• 8.8. These results indicate that the carrier-mediated transport of AIB is located at the apical membrane of the nephridial cell, and that AIB transport by simple diffusion takes place through the paracellular pathway.