AIB Uptake Research Articles

Regulation of amino acid uptake and deoxyribonucleic acid synthesis in isolated human fetal fibroblasts and myoblasts: effect of human placental lactogen, somatomedin-C, multiplication-stimulating activity, and insulin.

We compared the abilities of human placental lactogen (hPL), somatomedin-C/insulin-like growth factor I (SM-C/IGF-I), multiplication-stimulating activity (MSA), and insulin to induce a rapid anabolic event, the uptake of the nonmetabolizable amino acid [3H]alpha-aminoisobutyric acid ([3H] AIB) or the more long term action of increasing [3H]thymidine incorporation, as a measure of DNA synthesis, in isolated human fetal fibroblasts and myoblasts. Myoblasts were derived from skeletal muscle and fibroblasts from skin explants removed from human fetuses delivered between 12 and 19 weeks gestation after prostaglandin-induced abortion. Each of the four peptides caused a dose-dependent increase in [3H]AIB uptake by both fibroblasts and myoblasts, with mean half-maximal concentrations (ED50) ranging from 0.9-1.9 nM. The concentration of each peptide required to stimulate [3H]thymidine uptake was significantly greater, with the exception of insulin, which was inactive. For myoblast cultures, the mean ED50 values were: hPL, 7.9 nM; SM-C/IGF-I, 2.0 nM; and MSA, 2.2 nM. For fibroblast cultures, the mean ED50 values were: hPL, 2.3 nM; SM-C/IGF-I, 3.3 nM; and MSA, 4.3 nM. Insulin did not stimulate [3H]thymidine incorporation into either cell type at concentrations up to 6.9 nM. Incubation in the presence of monoclonal antibody against SM-C/IGF-I abolished the ability of SM-C/IGF-I to stimulate either [3H]thymidine or [3H]AIB uptake into fetal fibroblasts. The antibody substantially inhibited the incorporation of [3H]thymidine by these cells in response to hPL, but was less effective in blocking hPL-stimulated [3H]AIB uptake. It did not inhibit the uptake of either radioisotope in response to MSA or [3H]AIB uptake in response to insulin. The actions of SM-C/IGF-I and hPL on thymidine incorporation were additive at submaximal concentrations, but not so at maximal individual concentrations. Their actions on AIB uptake were additive at both submaximal and maximal concentrations. The results suggest that hPL as well as the SMs may contribute to the growth stimulus in human fetal connective tissues. Since incubation with SM-C/IGF-I antibody reduced the mitogenic response of fetal cells to hPL, the actions on DNA synthesis may be partially mediated by local release of SM. However, the similar ED50 values with which these peptides stimulated [3H]AIB uptake during a short incubation, and their additive effects at maximal individual concentrations, suggest that hPL may also have direct actions.

Inhibited muscle amino acid uptake in sepsis.

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

Insulin and exercise stimulate muscle alpha-aminoisobutyric acid transport by a Na +K +-ATPase independent pathway

Sodium ions are required for the active transport of amino acids such as alpha-aminoisobutyric acid (AIB) into skeletal muscle. To examine the role of Na +K +-ATPase in this phenomenon, studies were carried out using the isolated perfused rat hindquarter preparation. Perfusion for 30 min with ouabain at a dose sufficient to inhibit the Na +K + pump (10 −4M) inhibited the basal rate of AIB uptake in all muscles studied by up to 80%. However, it failed to inhibit the stimulation of AIB uptake, either by insulin (200 uU/ml) or electrically-induced muscle contractions. The increase in K+ release by the hindquarter in the presence of ouabain was the same under all conditions suggesting comparable inhibition of the Na +K + pump. These studies suggest that the basal, but not insulin or exercise-stimulated AIB transport into muscle is acutely dependent on a functional Na +K + pump. They also suggest that stimulated and basal uptake of AIB involve different mechanisms.

Effect of propranolol on alpha-amino-isobutyric acid and water kinetics in the materno-fetal unit of the rat.

Pregnant Sherman rats were given propranolol (a) per os (50 mg X kg-1, twice a day) on days 17-19 of gestation, (b) by constant rate infusion: osmotic minipumps (6 mg X kg-1 X day-1) from day 13 of gestation. On day 20 (a-treated) or 21 (b-treated), [14C]-AIB + [3H]-H2O were injected intravenously; plasma and tissue samples were collected at different times up to 4 h (a-treated) or at 2 h (b-treated) for radioactivity measurements. Whatever the treatment, no difference was found between control and treated animals for all parameters studied (number of fetuses; weight, protein; DNA; [14C]-AIB or [3H]-H2O tissue levels) except in propranolol-treated mothers where maternal heart [14C]-AIB uptake was decreased and where fetal plasma and tissue [3H]-H2O levels where higher than control at 5 min. Another set of experiments were performed in a-treated rats. On day 20, each mother was anesthetized with ether and injected as above; maternal plasma sample and one feto-placental unit were collected at different times within 15 min after injection. For this period, [14C]-AIB uptake and mainly [3H]-H2O diffusion were increased in placentae and fetuses of propranolol-treated dams. Arterial blood pressure did not change throughout the experiment in propranolol-treated animals, but decreased in controls. Heart rate was slowed down in the former compared to controls. Thus, in our experimental conditions, propranolol treatment: does not modify fetal weight; has no effect on AIB placental transfer and tissue uptake, and could protect towards the stress of anesthesia and surgical injuries, since H2O transfer was impaired in control but not in treated animals.

Stimulative effect of nerve growth factor on alpha-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 alpha-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 degrees C was significantly accelerated by the addition of NGF to the incubation medium in a dose-dependent manner. Although the Km value of the AIB uptake by the SCG did not change with the addition of NGF, Vmax 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 medium 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.

Membrane phosphatase activities in isolated and cultured adult rat hepatocytes

Membrane bound phosphohydrolysing enzymes, such as Na-K-ATPase, Mg-ATPase, ALPase and G-6-Pase were assayed in intact liver, in freshly isolated cells and in cultured hepatocytes to evaluate the effects of the isolation procedure and culture on these enzyme activities. Na-K-ATPase and Mg-ATPase are significantly reduced following cell dispersion while ALPase and G-6-Pase are nearly unaffected. During culture, Na-K-ATPase is restored to the “in vivo” level within the first two days, but rapidly declines in the following days. The Na dependent, energy requiring AIB uptake shows a similar pattern; Mg-ATPase is pratically unmodified. A significant increase in ALPase activity and a net decrease of G-6-Pase activity, as a function of the culture time has been observed.

Effects of antiinsulin receptor antibodies on amino acid uptake by cultured rat hepatocytes.

The effects of two antiinsulin receptor antisera (AIRA) on alpha-amino-2-[14C]isobutyric acid [( 14C]AIB) uptake by and [125I]insulin binding to cultured rat hepatocytes were examined. Diluted 1:100, both antisera inhibited insulin binding by 75-80%, mainly by decreasing available high affinity insulin-binding sites. Diluted 1:500, they decreased insulin binding and high affinity binding sites by 35-40%. Neither antiserum had an effect on basal [14C]AIB uptake, but decreased insulin-stimulated AIB uptake by 83% (dilution, 1:100; 24-h incubation) and 25% (dilution, 1:500; 24-h incubation), respectively. Insulin stimulation of AIB uptake correlated positively (r = 0.96; P less than 0.01) with insulin binding to high affinity receptors on hepatocytes incubated with normal serum or AIRA. We conclude that: 1) insulin stimulation of amino acid uptake by hepatocytes was mediated through insulin receptors, especially high affinity binding sites, and was inhibited by AIRA in parallel with receptor blockade; and 2) AIRA had no insulin-like effect on basal amino acid uptake by hepatocytes.

Muscle alpha-aminoisobutyric acid transport after exercise: enhanced stimulation by insulin.

After exercise the ability of insulin to stimulate glucose transport and glycogen synthesis in rat skeletal muscle is markedly enhanced (25). The present study was designed to determine whether prior exercise augments the stimulation of other processes by insulin and, if so, whether this can be attributed to an increase in insulin binding to its receptor. Toward this end rats were run on a treadmill for 45 min at moderate intensity and the uptake of alpha-aminoisobutyric acid (AIB) by muscle was then assessed using the isolated perfused hindquarter preparation. Approximately 30 min after the cessation of exercise, both the sensitivity and responsiveness of insulin-stimulated AIB uptake were significantly enhanced in the soleus and the red portion of the gastrocnemius. As previously shown for glucose transport and glycogen synthesis, only small effects were observed in the white portion of the gastrocnemius, which unlike the other muscles was not depleted of glycogen during the run. Insulin-stimulated glucose utilization was also enhanced in the incubated soleus muscle of exercised rats; however, insulin binding to the soleus was not altered. These studies indicate that the ability of insulin to stimulate processes other than glucose transport and glycogen synthesis is enhanced in skeletal muscle after exercise and that this is not due to an alteration in insulin binding. The changes in insulin-stimulated AIB uptake and glucose metabolism after exercise are the reverse of those found in denervated and immobilized muscle and in both situations insulin binding is not altered. This suggests that a common factor(s) modulates the increase in insulin effect after exercise and the insulin resistance of disuse.

260 FETAL NUTRITION: CALCIUM REGULATES HUMAN PLACENTAL UPTAKE OF AMINO ACID

Intracellular calcium regulates numerous cell functions, including amino acid uptake by rat diaphragm and hepatocytes (Arch Biochem 23:96; Proc Nat Acad Sci 77:5953). Therefore, we studied the effect of calcium (Ca) on the uptake of α−amino isobutyric acid (AIB) by human placental slices. During 45 minutes preincubation, tissue calcium was maintained (CaM), or was depleted (CaD) by 2.0 mM EGTA in Ca-free medium. Tissues were then incubated with 125 uM 14C-AIB for 60 minutes in medium with (+) or without (−) Ca. AIB uptake, expressed as the ratio of intracellular to extracellular AIB concentrations (Ci/Co), was 3 53 ± 0.13 (mean ± SE) in controls (CaM+), but was markedly reduced in CaD slices incubated without Ca repletion (CaD−): Ci/Co = 1.66 − 0.08 (p<0.001). Ca repletion during incubation (CaD+) resulted in partial restoration of 60 min AIB uptake: Ci/Co = 2.64 ± 0.11 (p<0.01, compared to either CaM+ or CaD−). In time-course experiments, the slope of AIB uptake for CaD+ was parallel to CaM+ by 15 minutes and linear through 60 minutes. However, CaD− showed no AIB uptake after 30 minutes. Finally, 10 uM verapamil, a calcium channel blocker, reduced 60 minute AIB uptake by 17.5% (p<0.05). CONCLUSION: Calcium regulates human placental uptake of the actively transported, neutral amino acid, AIB. SPECULATION: The placenta may serve as a model for studying calcium regulated amino acid transport in human tissue.

Alpha-Aminoisobutyric acid transport in rat soleus muscle during endotoxic shock.

We studied alpha-aminoisobutyric acid (AIB) transport by skeletal muscle and the effect of insulin and Na+ on the transport process during endotoxic shock. Rats (140-160 g) were injected with Salmonella enteritidis endotoxin (20 mg/kg iv) or saline and killed 5 h later. At that time an elevation of blood lactate and hypoglycemia marked the onset of shock in rats given endotoxin. AIB uptake was measured in isolated soleus muscles in vitro. Total cellular AIB uptake in the endotoxic muscles was 25, 28, and 47% lower than control muscles at 1-, 2-, or 3-h incubations, respectively. Insulin stimulated AIB uptake to a lesser extent in endotoxic muscles (from a basal value of 11.62 +/- 0.29 nmol X g dry wt-1 X 3 h-1 to 15.88 +/- 0.64, 19.10 +/- 1.06, and 18.78 +/- 0.52 at 1, 10, and 100 mU/ml insulin, respectively) than in controls (from 17.07 +/- 0.51 to 27.13 +/- 1.16, 27.25 +/- 0.93, and 29.01 +/- 1.09). Na+-dependent AIB uptake, calculated as the difference between AIB uptake in the presence and absence of Na+, was decreased in the endotoxic muscles to 36% of the control value. Na+-independent AIB uptake (measured in Na+-free media) was the same in control and endotoxic muscles. These results suggest that the decrease in both basal and insulin-stimulated AIB transport was due to the decrease in Na+-dependent AIB transport by skeletal muscle during endotoxic shock.

The stimulation of α-aminoisobutyric acid uptake by the nerve growth factor in superior cervical sympathetic ganglia and by the glia maturation factor in nodose ganglia excised from rats

During aerobic incubation at 37°C, active uptake of labeled non-metabolizable α-aminoisobutyric acid (AIB) into isolated nodose ganglia (NG) excised from adult rats was accelerated to nearly twice that of the control, by the addition of glia maturation factor (GMF, 5 μg/ml) in a dose-dependent manner. A similar but moderate stimulative effect on ganglionic AIB uptake was caused by the addition of acetylcholine (ACh, 1 mM) plus eserine (0.1 mM). This effect, however, was not antagonized by nicotinic (hexamethonium, C6, 0.1 mM) or muscarinic (atropine, 0.1 mM) blockers. The GMF-induced amino acid uptake seemed to be inhibited by further addition of ACh. On the other hand, ganglionic Ca2+, Mg2+-ATPase activity was greatly stimulated by either GMF or ACh. These results suggest that the increase in AIB uptake induced by GMF or ACh is possibly linked to Ca2+, Mg2+-ATPase activity in NG cell membranes.

Cellular responses elicited by insulin mimickers in cells lacking detectable plasma membrane insulin receptors.

Madin-Darby canine kidney (MDCK) cells were previously shown to have few or no plasma membrane insulin binding sites (Hofmann et al: J Biol Chem 258:11774, 1983]. Accordingly, neither insulin-stimulated incorporation of [14C]glucose into glycogen, nor insulin-induced uptake of radiolabeled alpha-aminoisobutyrate ([3H]AIB) could be demonstrated. To probe for receptors, MDCK cultures were surface-labeled with Na125I or were labeled with [35S]methionine. When solubilized cells were immunoprecipitated with sera containing antibodies to the insulin receptor, and immunoprecipitates were analyzed on SDS-gel electrophoresis, no evidence for insulin receptor components was found. Also, when intact MDCK cells wee incubated first with serum containing antibodies to the insulin receptor and then with 125I-protein A, no radiolabeling of insulin receptors occurred. Various agents reported to have insulin-like activity were tested on MDCK cells. The insulinomimetic lectins concanavalin A and wheat germ agglutinin as well as hydrogen peroxide enhanced incorporation of [14C]glucose into glycogen and induced stimulated [3H]AIB uptake, whereas trypsin, vanadate, and serum containing antibodies to the insulin receptor were without effects. Altogether, these results showed that MDCK cells had few or no insulin receptors and were correspondingly insulin-insensitive. However, since insulin-associated responses could be elicited by some insulin mimickers, the post-receptor limb of response in MDCK cells was apparently intact.

Effects of ethanol ingestion on amino acid uptake in the dog liver in vivo.

The effect of ethanol ingestion on the uptake of labeled amino acids was studied in the in situ autoperfused dog liver. Ethanol was administered orally, as a 15% water solution, in a dose of 4 g/kg body weight/day as the only source of water for 2 days. Amino acid uptake was measured in anesthetized dogs by means of the single-passage, multiple-tracer dilution technique. In control animals, hepatic uptake of 14C-glycine, 3H-alpha-aminoisobutyric acid (3H-AIB) and 3H-L-leucine were 50, 15 and 66%, respectively. In the ethanol-treated dogs, glycine and AIB uptake was reduced by 70 and 63%, respectively. L-leucine uptake was reduced by only 23%. The plasma concentration of the naturally occurring amino acids was significantly increased after ethanol treatment, probably due to a reduced influx into hepatocytes. Simultaneous measurements of hemodynamic parameters showed a significant increase in the portal vein pressure of ethanol-treated animals, whereas the portal vein blood flow and hepatic extracellular volume were unaffected.

The neuronal differentiation of the Ca 2+ channel by the nerve growth factor

During aerobic incubation at 37°C, active uptake of labeled non-metabolizable α-aminoisobutyric acid (AIB) into isolated nodose ganglia (NG) excised from adult rats was accelerated to nearly twice that of the control, by the addition of glia maturation factor (GMF, 5 μg/ml) in a dose-dependent manner. A similar but moderate stimulative effect on ganglionic AIB uptake was caused by the addition of acetylcholine (ACh, 1 mM) plus eserine (0.1 mM). This effect, however, was not antagonized by nicotinic (hexamethonium, C6, 0.1 mM) or muscarinic (atropine, 0.1 mM) blockers. The GMF-induced amino acid uptake seemed to be inhibited by further addition of ACh. On the other hand, ganglionic Ca2+, Mg2+-ATPase activity was greatly stimulated by either GMF or ACh. These results suggest that the increase in AIB uptake induced by GMF or ACh is possibly linked to Ca2+, Mg2+-ATPase activity in NG cell membranes.

Light-stimulated Electrogenic Uptake of Neutral Amino Acids by Leaf Cells of Egeria densa and Vicia faba

Summary The kinetics and energetics of amino acid uptake into leaf tissues of Egeria and Vicia by means of 14C-amino acids (alanine, aminoisobutyric acid, leucine) and the accompanying electric response at the plasmalemma were compared. Amino acid uptake in the concentration range of 0.1 to 5 mmol/l at pH 6 exhibits biphasic isotherms (Egeria: saturable phase, [S]: 0.1-1.5 mmol/l, linear phase, [S]:, 1.5 mmol/l; Vicia: two Michaelis-Menten terms, without any linear component). Uptake of Ala, AIB and Leu is strongly pH-dependent and is enhanced in the light. Effects of light and various inhibitors on amino acid influx, medium-acidifying activity of the leaf tissues and on the membrane potential prove the existence of a light-stimulated H+ transport system at the plasmalemma, creating a protonmotive force capable to provide transport energy. The transient depolarization of the membrane potential during amino acid influx and the sensitivity of this influx to the membrane potential point to coupling of amino acid transport with electrochemical proton gradient. It is suggested that in Egeria as well as in Vicia neutral amino acids were taken up by proton symport in the range of pH 4 to 8.

Alpha 1-adrenergic inhibition of protein synthesis in rat submandibular cells.

The incorporation of [14C]leucine into protein in dispersed cells from rat submandibular gland was inhibited by epinephrine. The dose required for a half-maximal effect was approximately 10(-6) M. This sensitivity is similar to that of mucin and potassium secretion induced by epinephrine from these cells. The inhibitory effect of epinephrine was mediated by the activation of alpha 1-adrenergic receptors and was partially independent of extracellular calcium. The calcium ionophore A23187 also decreased protein synthesis in the presence and absence of extracellular calcium. Epinephrine diminished alpha-[14C]aminoisobutyric acid (AIB) influx, but the extent of altered AIB uptake was much less than the extent of decrease in protein synthesis. Analysis of newly synthesized protein by gel electrophoresis and autofluorography showed that epinephrine selectively inhibited the synthesis of specific submandibular proteins. In aggregate these results suggest that activation of alpha 1-adrenergic receptors in rat submandibular cells influences the regulation of protein synthesis by a mechanism that uses intracellular calcium.

Induction of amino acid transport by L-triiodothyronine in cultured growth hormone-producing rat pituitary tumor cells (GC cells).

The action of L-triiodothyronine (T3) on amino acid transport in the GC clonal strain of rat pituitary cells was investigated by measurement of the uptake of the nonmetabolizable amino acid, alpha-aminoisobutyric acid (AIB). The uptake of AIB by GC cells appeared to require energy and Na+ and displayed Michaelis-Menten kinetics. In comparison to cultures maintained in the absence of T3, T3 addition resulted in an increase in AIB uptake which seemed due to an increase in the initial rate of AIB transport. T3 addition resulted in increased AIB accumulation at later time points as well. T3 induction of AIB transport did not occur until 3.5 h after addition of T3, and this effect was blocked by cycloheximide. Maximal induction occurred 48 to 72 h later. One-half maximal induction occurred 24 to 48 h after addition of T3. No detectable changes either in AIB uptake or intracellular water space, measured by uptake of the nonmetabolizable sugar, 3-O-methyl-D-glucose, were noted for the first 120 min after addition of T3. Induction of AIB transport occurred at 0.05 nM T3 (total medium concentration) and one-half maximal induction occurred at 0.17 nM T3. The relative potencies of four iodothyronine analogues for AIB transport were in accord with their reported activities in nuclear T3 receptor binding assays. These data suggest that induction of AIB transport by T3 may be mediated by the nuclear T3 receptor and may reflect the pleiotrophic response of GC cells to thyroid hormone.

VIRALLY INDUCED MEMBRANE PERMEABILITY CHANGES AS A POSSIBLE LINK BETWEEN INFLUENZA B AND REYE'S SYNDROME

The mechanism by which viral infection can lead to the multiple metabolic derangements characteristic of Reye's Syndrome is not understood. The possibility that influenza virus could induce changes in membrane permeability to nutrients ordinarily concentrated within the cell was examined. Madin Darbin canine kidney (MDCK) cells were infected by egg grown influenza B virus by two routes: at physiological conditions, pH 7.4, 37°C; and adsorption at 0°C followed by brief exposure to pH 5.0, 37°C. Control cells were mock-infected with allantoic fluid. Transport (uptake and release) of phosphate (Pi) 2-deoxyglucose (dGlc) and α-aminoisobutyric acid (AIB) were measured at various intervals 0 to 10 hours after infection. At physiological pH, Pi uptake by infected cells was inhibited (p< 0.01) at 0 to 2 hours as compared to controls. Uptake of AIB was also inhibited (p< 0.01) at 0 to 2 hours. The uptake of all nutrients was higher at 6 to 10 hours in infected cells as compared to controls (p< 0.01). When release of Pi and dGlc was measured, there was no difference between infected cells and controls at 0-10 hours. At low pH, fusion occurred in infected cells, but not in control cells. Fused infected cells demonstrated both significantly increased release and diminished uptake of nutrients compared to controls. Thus influenza B virus can induce changes in host cell membrane permeability that may have important implications for cell metabolism.

The type I insulin-like growth factor receptor mediates the rapid effects of multiplication-stimulating activity on membrane transport systems in rat soleus muscle.

Multiplication-stimulating activity (MSA) stimulates the uptake of xylose and alpha-aminoisobutyric acid (AIB) by intact rat soleus muscle. It is approximately 50 times less potent than insulin. A native insulin receptor species with apparent Mr = 350,000 in soleus muscle is revealed by affinity cross-linking to 125I-insulin with disuccinimidyl suberate. 125I-labeled insulin-like growth factor (IGF) I with the cross-linker affinity labels two native receptor types with Mr = 360,000 (type I) and Mr = 220,000 (type II). In order to distinguish which of the three insulin and IGF receptor systems identified mediate the rapid insulin-like effects of MSA, the biological actions of the unlabeled ligands at various concentrations were correlated with their ability to inhibit the affinity labeling of these receptor species. The stimulatory action of native insulin is closely related to its ability to inhibit the labeling of the insulin receptor by 125I-insulin such that the uptake of xylose and AIB is maximally stimulated when 80% of insulin receptor 125I-affinity labeling is inhibited. In contrast, MSA only displaced 16% of the 125I-labeling of the insulin receptor when it maximally stimulates the uptake of xylose and AIB, indicating that the insulin receptor is not primarily involved in mediating these effects. The affinity of MSA to the type II IGF receptor is 10 times higher than that to the type I IGF receptor. There is close to a 1:1 relationship between the stimulatory effects of MSA on xylose and AIB uptake and its inhibitory action on the affinity labeling of the type I receptor by 125I-IGF I. In contrast, MSA almost abolishes the labeling of the type II IGF receptor at concentrations which have no detectable effects on the uptake of xylose and AIB by soleus muscle. Thus, a marked dissociation can be observed between the rapid insulin-like action of MSA and its inhibitory effects on the affinity labeling of the type II receptor by 125I-IGF I. We conclude that MSA acts through the type I IGF receptor in soleus muscle to stimulate hexose and amino acid transport. The type II IGF receptor appears to be incapable of modulating these effects in this tissue.

Insulin-induced formation of ruffling membranes of KB cells and its correlation with enhancement of amino acid transport.

Insulin induced the formation of ruffling membranes in cultured KB cells (a cell strain derived from human epidermoid carcinoma) within 1-2 min after its addition. The ruffled regions were stained strongly with antibody to actin but not that to tubulin. Pretreatment of KB cells with agents disrupting microfilaments (cytochalasins), but not with those disrupting microtubules (colcemid, nocodazole, and colchicine) completely inhibited the formation of ruffling membranes. Pretreatment of KB cells with dibutyryl cyclic AMP, but not with dibutyryl cyclic GMP, also inhibited the formation of ruffling membranes. Addition of insulin enhanced Na+-dependent uptake of a system A amino acid (alpha-amino isobutyric acid; AIB) by the cells within 5 min after the addition, and decreased the cyclic AMP content of the cells. Treatments that inhibited insulin-induced formation of ruffling membranes of KB cells also inhibited insulin-induced enhancement of their AIB uptake. From these observations, the mechanism of insulin-induced formation of ruffling membranes and its close correlation with AIB transport are discussed.