FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1970989980> ?p ?o ?g. }

• W1970989980 endingPage "3179" @default.
• W1970989980 startingPage "3173" @default.
• W1970989980 abstract "The insulin-stimulated uptake of 2-(methylamino)isobutyric acid (MeAIB), a nonmetabolizable substrate for system A, in 3T3-L1 adipocytes was investigated. As cells took on a more adipogenic phenotype, the insulin-stimulatedversus the saturable basal MeAIB uptake increased by 5-fold. The induced transport activity showed properties characteristic of system A, with a K m value of 190 μm. The half-life of the induced system A activity was independent of de novo mRNA and protein synthesis and was not accelerated by ambient amino acids, therefore, it was mechanistically distinct from the previously described adaptive and hormonal regulation of system A. Inhibition of mitogen-activated protein kinase kinase by PD98059, Ras farnesylation by PD152440 and B581, p70S6K by rapamycin, and phosphatidylinositol 3-kinase (PI 3′-K) by wortmannin and LY294002 revealed that only wortmannin and LY294002 inhibited the insulin-induced MeAIB uptake with IC50 values close to that previously reported for inhibition of PI 3′-K. These results suggest that the Ras/mitogen-activated protein kinase and pp70S6K insulin signaling pathways are neither required nor sufficient for insulin stimulation of MeAIB uptake, and activation of PI 3′-K or a wortmannin/LY294002-sensitive pathway may play an important role in regulation of system A transport by insulin in 3T3-L1 cells. The insulin-stimulated uptake of 2-(methylamino)isobutyric acid (MeAIB), a nonmetabolizable substrate for system A, in 3T3-L1 adipocytes was investigated. As cells took on a more adipogenic phenotype, the insulin-stimulatedversus the saturable basal MeAIB uptake increased by 5-fold. The induced transport activity showed properties characteristic of system A, with a K m value of 190 μm. The half-life of the induced system A activity was independent of de novo mRNA and protein synthesis and was not accelerated by ambient amino acids, therefore, it was mechanistically distinct from the previously described adaptive and hormonal regulation of system A. Inhibition of mitogen-activated protein kinase kinase by PD98059, Ras farnesylation by PD152440 and B581, p70S6K by rapamycin, and phosphatidylinositol 3-kinase (PI 3′-K) by wortmannin and LY294002 revealed that only wortmannin and LY294002 inhibited the insulin-induced MeAIB uptake with IC50 values close to that previously reported for inhibition of PI 3′-K. These results suggest that the Ras/mitogen-activated protein kinase and pp70S6K insulin signaling pathways are neither required nor sufficient for insulin stimulation of MeAIB uptake, and activation of PI 3′-K or a wortmannin/LY294002-sensitive pathway may play an important role in regulation of system A transport by insulin in 3T3-L1 cells. Mammalian cells contain multiple systems for uptake of neutral amino acids. System A is an ubiquitous amino acid transport system that mediates the Na+-dependent uptake of a wide range of neutral amino acids (1Collarini E.J. Oxender D.L. Annu. Rev. Nutr. 1987; 7: 75-90Crossref PubMed Scopus (100) Google Scholar, 2Christensen H.N. Biochim. Biophys. Acta. 1984; 779: 255-269Crossref PubMed Scopus (210) Google Scholar, 3Oxender D.L. Christensen H.N. J. Biol. Chem. 1963; 238: 3686-3699Abstract Full Text PDF PubMed Google Scholar), many of which are gluconeogenic (4Kilberg M.S. Barber E.F. Handlogten M.E. Curr. Top. Cell. Regul. 1985; 25: 133-163Crossref PubMed Scopus (69) Google Scholar). In a number of cell types system A is regulated by a variety of external stimuli and conditions, such as hormones, amino acid starvation (adaptive regulation), cell growth, differentiation, hyperosmotic stress, and trans-inhibition produced by high levels of endogenous amino acid substrates (1Collarini E.J. Oxender D.L. Annu. Rev. Nutr. 1987; 7: 75-90Crossref PubMed Scopus (100) Google Scholar, 2Christensen H.N. Biochim. Biophys. Acta. 1984; 779: 255-269Crossref PubMed Scopus (210) Google Scholar, 4Kilberg M.S. Barber E.F. Handlogten M.E. Curr. Top. Cell. Regul. 1985; 25: 133-163Crossref PubMed Scopus (69) Google Scholar, 5McGivan J.D. Pastor-Anglada M. Biochem. J. 1994; 299: 321-334Crossref PubMed Scopus (339) Google Scholar). It is generally believed that hormonal and adaptive regulation of system A occurs at the level of transcription (4Kilberg M.S. Barber E.F. Handlogten M.E. Curr. Top. Cell. Regul. 1985; 25: 133-163Crossref PubMed Scopus (69) Google Scholar, 5McGivan J.D. Pastor-Anglada M. Biochem. J. 1994; 299: 321-334Crossref PubMed Scopus (339) Google Scholar). Although the molecular mechanisms of the signal transduction in insulin action have been extensively investigated (6Saltiel A.R. FASEB J. 1994; 8: 1034-1040Crossref PubMed Scopus (45) Google Scholar), the mechanism responsible for hormonal regulation of system A transport remains largely unknown. An insulin-insensitive Chinese hamster ovary cell line has been isolated (7Moffett J. Perier F. Jones M. Englesberg E. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8040-8043Crossref PubMed Scopus (8) Google Scholar). These authors proposed that, when insulin binds to its receptor, it regulates system A activity directly or indirectly by inactivation of a regulatory protein designated r2, but the molecular events leading to neutralizing this protein by insulin have not been resolved (5McGivan J.D. Pastor-Anglada M. Biochem. J. 1994; 299: 321-334Crossref PubMed Scopus (339) Google Scholar). In 3T3-L1 adipocytes, insulin induces the dose- and time-dependent uptake of MeAIB, 1The abbreviations used are: MeAIB, 2-(methylamino)isobutyric acid; PDGF, platelet-derived growth factor; AIB, α-aminoisobutyric acid; GH, growth hormone; IGF, insulin-like growth factor; IL-11, interleukin 11; IRS, insulin receptor substrate; MEK, mitogen-activated protein kinase kinase; MAP, mitogen-activated protein; PI 3′-K, phosphatidylinositol 3′-kinase; p70S6K, M r 70,000 S6 kinase; TPA, 12-O-tetradecanoylphorbol-13-acetate; DMEM, Dulbecco's modified Eagle's medium; PMSF, phenylmethylsulfonyl fluoride; PBS, phosphate-buffered saline. a nonmetabolizable substrate of system A (8Grunfeld C. Jones D.S. Endocrinology. 1983; 113: 1763-1769Crossref PubMed Scopus (11) Google Scholar). However, the specificity and kinetics of the insulin-induced amino acid uptake in 3T3-L1 adipocytes and the signaling events potentially involved in insulin stimulation of system A transport in mammalian cells have not previously been explored in great detail. In this report, we investigate the regulation of system A transport by insulin in 3T3-L1 adipocytes. Cell culture reagents, epidermal growth factor, PDGF, IGF-I, and IGF-II were purchased from Life Technologies, Inc. [14C]MeAIB was purchased from American Radiolabeled Chemicals, Inc., and [γ-32P]ATP from Amersham Corp. Wortmannin, LY294002, B518, and rapamycin were purchased from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA). Anti-p42/p44 MAP kinase antiserum used for Western blotting was prepared from rabbits immunized with a C-terminal peptide (amino acids 425–445) of p44 MAP kinase (p44MAPK) expressed as a glutathioneS-transferase fusion protein. PD98059 and PD152440 were synthesized by Parke-Davis. Phosphatidylinositol was purchased from Avanti (Birmingham, AL). The S6 kinase kit and the mouse anti-phosphotyrosine monoclonal antibody were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). The GH and insulin were obtained from Eli Lilly; IL-11 from R&D Systems (Minneapolis, MN), and all other chemicals from Sigma. 3T3-L1 fibroblasts were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% calf serum in an atmosphere of 5% CO2, air. Differentiation to adipocytes was induced by incubating confluent monolayers (day 0) for 2 days in DMEM containing 10% fetal bovine serum, 0.5 mm 3-isobutyl-1-methylxanthine, and 0.4 μg/ml dexamethasone, followed by incubation for 2 more days in DMEM containing 10% fetal bovine serum and 1 μg/ml insulin. Two days after transfer to the same medium without insulin, greater than 90% of the cells expressed the adipocyte phenotype. Unless otherwise stated, experiments were performed on adipocytes 2–4 days after withdrawal from the differentiation medium (days 6–8). This assay was carried out as described previously (9Lazar D.F. Wiese R.J. Brady M.J. Mastick C.C. Waters S.B. Yamauchi K. Pessin J.E. Cuatrecasas P. Saltiel A.R. J. Biol. Chem. 1995; 270: 20801-20807Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar, 10Wiese R.J. Mastick C.C. Lazar D.F. Saltiel A.R. J. Biol. Chem. 1995; 270: 3442-3446Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). In brief, 3T3-L1 adipocytes were serum-starved for about 16 h. After hormone stimulation for 10 min, the cells (10-cm culture dishes) were harvested in cold lysis buffer (1% Nonidet P-40, 150 mm NaCl, 5 mm EDTA, 20 mm Tris, pH 7.5, 1 mm Na3VO4, 100 μm phenylmethylsulfonyl fluoride (PMSF), 20 μm leupeptin, 20 μm pepstatin A) and kept on ice for 30 min. Insoluble materials were removed by centrifugation and the supernatants were incubated with anti-phosphotyrosine antibody under agitation at 4 °C for 1 h. Protein A-agarose beads were added and incubated for another hour. The immunoprecipitates were sequentially washed with lysis buffer, washing buffer (100 mm Tris, pH 7.5, 0.5 m LiCl, 1 mmNa3VO4, 100 μm PMSF, 20 μm leupeptin, 20 μm pepstatin A), and PI 3′-kinase buffer (20 mm MgCl2, 10 mm Tris, pH 7.5, 0.2 mm EDTA, 100 mm NaCl, 1 mm Na3VO4, 100 μm PMSF, 20 μm leupeptin, 20 μm pepstatin A). The precipitates were suspended in 50 μl of PI 3′-kinase buffer. The reaction was carried out at 25 °C for 20 min in the presence of 0.4 mg/ml phosphatidylinositol and 10 μm ATP (containing 0.2 mCi/ml [γ-32P]ATP). The reaction was terminated by adding to each tube 100 μl of CHCl3:MeOH:HCl (100:200:2). To the mixture 100 μl of CHCl3 and H2O each were then added, and the mixture was vortexed. After centrifugation, the lower phase (organic phase) was spotted on a thin layer chromatography plate. The plate was developed in CHCl3:MeOH:H2O:NH4OH (43:38:7:5). The dried plate was visualized by autoradiography on x-ray film. For the assay of p70 S6 kinase (p70S6K), 3T3-L1 adipocytes were serum-starved for 16 h. The cells were treated with or without rapamycin (100 ng/ml) for 1 h followed by treatment with insulin for 10 min. The cells were harvested and the S6 kinase activity of cytosolic extracts was detected according to the procedure provided by Upstate Biotechnology, Inc. using a peptide substrate. The S6 kinase activity was obtained by subtracting the values for unstimulated cells from the values for the insulin-induced cells. MAP kinase was assayed as described previously (9Lazar D.F. Wiese R.J. Brady M.J. Mastick C.C. Waters S.B. Yamauchi K. Pessin J.E. Cuatrecasas P. Saltiel A.R. J. Biol. Chem. 1995; 270: 20801-20807Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar). In brief, 3T3-L1 adipocytes were serum-starved overnight and then pretreated or untreated with 50 μm PD98059 for 1 h before stimulation by 100 nm insulin for 15 min. Cell lysates were collected in cold buffer (50 mm HEPES, pH 7.5, 150 mm NaCl, 5 mm EDTA, 0.5% deoxycholate, 1% Nonidet P-40, 50 mm NaF, 10 mmNa-pyrophosphate, 1 mm p-nitrophenylphosphate, 25 mm β-glycerophosphate, 1 mmNa3VO4, 10 μg/ml aprotinin, 1 μg/ml leupeptin, 1 mm PMSF, and 1 mm benzamidine). The precleared lysates (10,000 × g, 10 min) containing 10 μg of protein were subjected to 12% SDS-polyacrylamide gel electrophoresis with 75:1 of acrylamide:bisacrylamide (w/w) and 0.5m Tris-HCl. After electrophoresis, the gel was transferred to nitrocellulose and probed with anti-p42/p44 MAP kinase antiserum. The immunoreactive proteins were visualized by enhanced chemiluminescence detection. The sodium-containing buffer for transport assay was phosphate-buffered saline (PBS) consisting of 137 mm NaCl, 2.7 mm KCl, 10.6 mmNa2HPO4, and 1.5 mmKH2PO4. Prior to use, PBS buffer (pH 7.4) was supplemented with 20 mmd-glucose, 0.49 mm MgCl2, 0.9 mm CaCl2, and 0.2% bovine serum albumin. Before stimulation, the cells were incubated in serum-free DMEM for 3–4 h and then switched to the same medium containing stimuli and incubated for 5–6 h. The cluster tray transport assay was used as described previously (11Su T.Z. Campbell G.W. Oxender D.L. Brain Res. 1997; 757: 69-78Crossref PubMed Scopus (55) Google Scholar). To eliminate trans-inhibition, the intracellular pool of amino acids was depleted by incubation in PBS for 40 min, with a change to fresh PBS at 20 min in the presence or absence of stimuli. An appropriate amount of choline chloride was added to each reaction mixture to keep all solutions at equal osmolarity. Since uptake of MeAIB was linear at 37 °C for at least 15 min, 10-min uptake was used for determining initial uptake rates. Unless otherwise noted, the MeAIB concentration for initial rate of transport measurements was 50 μm, the concentration of insulin was 100 nm, and all transport rates were referred to as saturable uptake rates. The saturable uptake rates were calculated by subtracting the MeAIB uptake rates in the presence of 10 mm excess unlabeled MeAIB from the total uptake rates. The differentiation of 3T3-L1 fibroblasts to adipocytes is accompanied by a dramatic increase in insulin sensitivity (12Rubin C.S. Lai E. Rosen O.M. J. Biol. Chem. 1977; 252: 3554-3557Abstract Full Text PDF PubMed Google Scholar). To evaluate the regulation of amino acid transport in these cells, the uptake of 50 μm MeAIB was determined for a period of 14 days after induction of differentiation. In preadipocytes insulin produced only about a 2-fold increase over the saturable basal uptake of MeAIB (Fig. 1). The maximal insulin response occurred 3 days after induction of differentiation. The maximal insulin-stimulated transport decreased from days 4 to 5, reaching levels about 2-fold higher than that of preadipocytes. Basal MeAIB uptake was increased 3–4 days after initiation of differentiation. However, the basal uptake rates at these time points were determined 8 h after switching from insulin-containing to insulin-free medium, suggesting that the increased basal uptake may represent residual activity resulting from previous stimulation (see below). As cells took on a more adipogenic phenotype, basal activity was reduced to about 20% of that seen in fibroblasts. Thus the effects of insulin to stimulate amino acid transport increased to 10-fold. This pattern of reduced basal and increased insulin-dependent activity in fully differentiated adipocytes is similar to that observed for glycogen synthesis in these cells (13Brady M.J. Nairn A.C. Saltiel A.R. J. Biol. Chem. 1997; 272: 29698-29703Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Protein synthesis in 3T3-L1 cells was also significantly enhanced during the incubation with insulin for 2 days (days 3 to 4) and reached a steady state level after switching to insulin-free medium (Fig. 1). An increase in adipocyte protein content (about 20%) was consistently observed during 5–6 h of stimulation of amino acid transport by insulin. Thus the transport velocity when expressed as the amount of substrate incorporated per unit surface area of cell monolayer (8Grunfeld C. Jones D.S. Endocrinology. 1983; 113: 1763-1769Crossref PubMed Scopus (11) Google Scholar) may not be representative of the insulin action specific for system A transport (14Marshall S. Monzon R. J. Biol. Chem. 1989; 264: 2037-2042Abstract Full Text PDF PubMed Google Scholar). To evaluate the kinetics of stimulation of amino acid transport, MeAIB uptake was assayed after exposure to insulin for different times (Fig. 2 A). The effect of insulin was maximal by 5–6 h. Evaluation of the dose response to insulin revealed an EC50 of 5.2 nm (Fig. 2 B). To determine the initial uptake rate kinetics in differentiated adipocytes, the concentration dependence of the process was examined. The basal MeAIB uptake increased in direct proportion to concentration (Fig. 2 C), suggesting that the unstimulated or basal entry of MeAIB in adipocytes was predominantly mediated by a nonsaturable process (60–70%). In contrast, the insulin-stimulated MeAIB uptake was saturable, exhibiting typical Michaelis-Menten kinetics with aK m value of 190 μm and V max of 390 pmol/min/mg of protein. Saturable uptake of MeAIB has been regarded as the simplest indication for mediation by system A (2Christensen H.N. Biochim. Biophys. Acta. 1984; 779: 255-269Crossref PubMed Scopus (210) Google Scholar). To determine that system A mediates the insulin-stimulated amino acid transport in 3T3-L1 adipocytes, the MeAIB transport was characterized. Inhibition of MeAIB uptake by various amino acids produced a typical system A transport pattern (Fig. 3, left). Alanine, serine, cysteine, proline, and histidine inhibited MeAIB uptake by more than 90%, whereas the cationic amino acids, arginine and lysine, had no effect. It is noteworthy that the anionic amino acids, glutamate and aspartate, showed moderate inhibition of MeAIB uptake (30–40%). Inhibition by glutamate was inversely proportional to pH (data not shown), indicating that the anionic amino acids could be interacting with system A as uncharged zwitterions (2Christensen H.N. Biochim. Biophys. Acta. 1984; 779: 255-269Crossref PubMed Scopus (210) Google Scholar). Consistent with the major properties of system A transport, uptake of MeAIB in 3T3-L1 adipocytes was strictly Na+-dependent. More than 90% of the MeAIB entry was eliminated when sodium was replaced with choline. In addition, system A activity decreased by 35% in cells preloaded with 50 μm MeAIB for 40 min (trans-inhibition), and by 65% at pH 5.0 as compared with pH 7.4 (Fig. 3, right). The substrate specificity, hormonal stimulation, Na+ dependence, decreased transport activity at lower pH, and trans-inhibition indicate that the insulin-stimulated MeAIB uptake in 3T3-L1 adipocytes is mediated by a typical A-type transport system (1Collarini E.J. Oxender D.L. Annu. Rev. Nutr. 1987; 7: 75-90Crossref PubMed Scopus (100) Google Scholar). In contrast to the insulin-stimulated MeAIB uptake, the basal or unstimulated transport was resistant to inhibition by histidine, leaving open the possibility that the constitutively expressed low transport activity is mediated by a different subtype of system A. Instability is a mechanistically important feature of the regulation of system A transport (15Handlogten M.E. Kilberg M.S. J. Biol. Chem. 1984; 259: 3519-3525Abstract Full Text PDF PubMed Google Scholar, 16Bracy D.S. Handlogten M.E. Barber E.F. Han H.P. Kilberg M.S. J. Biol. Chem. 1986; 261: 1514-1520Abstract Full Text PDF PubMed Google Scholar, 17Kilberg M.S. Trends Biochem. Sci. 1986; 11: 183-186Abstract Full Text PDF Scopus (38) Google Scholar). To determine the half-life of the stimulated MeAIB transport in 3T3-L1 adipocytes, the time course of its activity was followed after removal of insulin from the adipocyte growth medium. At the onset of adipocyte differentiation (day 2), the insulin-stimulated uptake decayed rapidly with an estimated half-life of 1.5 h (Fig. 4, PBS), similar to what is observed in hepatocytes (15Handlogten M.E. Kilberg M.S. J. Biol. Chem. 1984; 259: 3519-3525Abstract Full Text PDF PubMed Google Scholar). In fully differentiated adipocytes, the insulin-induced transport activity decayed at much slower rates, with at½ of 4.3 h. To determine if the decay of system A activity was associated with a newly synthesized repressor-type protein or repression by ambient amino acids (15Handlogten M.E. Kilberg M.S. J. Biol. Chem. 1984; 259: 3519-3525Abstract Full Text PDF PubMed Google Scholar, 16Bracy D.S. Handlogten M.E. Barber E.F. Han H.P. Kilberg M.S. J. Biol. Chem. 1986; 261: 1514-1520Abstract Full Text PDF PubMed Google Scholar), cycloheximide and actinomycin D (1 μg/ml) were added 1 h before removal of insulin, or the cells were incubated in insulin-free medium in the presence or absence of ambient amino acids. As shown in Fig. 4, these agents had no effect on the half-life. Moreover, the similarity in half-life between amino acid-free and amino acid-containing media (PBS versus DMEM) suggests that the decay is not caused by ambient amino acids. Following decay of the insulin-stimulated uptake, the MeAIB transport activity could be re-established by switching to insulin-containing medium. As with the stimulated transport activity before the decay, most of the re-established MeAIB uptake was also substantially inhibited by cycloheximide (81 ± 2%) and actinomycin D (95 ± 2%) (Fig. 3, right), suggesting that the insulin-induced or the decayed MeAIB uptake is associated, respectively, with de novo synthesis or degradation of protein(s) essential for system A function. The regulation of protein phosphorylation is believed to play a central role in insulin action (6Saltiel A.R. FASEB J. 1994; 8: 1034-1040Crossref PubMed Scopus (45) Google Scholar). The two best characterized pathways leading to insulin-dependent serine phosphorylation involve either MAP kinase or p70S6K (18Cheatham B. Vlahos C.J. Cheatham L. Wang L. Blenis J. Kahn C.R. Mol. Cell. Biol. 1994; 14: 4902-4911Crossref PubMed Scopus (1001) Google Scholar). To determine if Ras/MAP kinase pathway is required for the stimulation of system A transport by insulin, PD98059, a specific inhibitor of MEK (19Dudley D.T. Pang L. Decker S.J. Bridges A.J. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7686-7689Crossref PubMed Scopus (2593) Google Scholar), was examined. At 10 μm concentration, PD98059 is sufficient to block the activation of MEK, MAP kinase, and pp90rsk, and the induction of c-fos transcription (9Lazar D.F. Wiese R.J. Brady M.J. Mastick C.C. Waters S.B. Yamauchi K. Pessin J.E. Cuatrecasas P. Saltiel A.R. J. Biol. Chem. 1995; 270: 20801-20807Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar, 10Wiese R.J. Mastick C.C. Lazar D.F. Saltiel A.R. J. Biol. Chem. 1995; 270: 3442-3446Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 19Dudley D.T. Pang L. Decker S.J. Bridges A.J. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7686-7689Crossref PubMed Scopus (2593) Google Scholar, 20Gabbay R.A. Sutherland C. Gnudi L. Kahn B.B. O'Brien R.M. Granner D.K. Flier J.S. J. Biol. Chem. 1996; 271: 1890-1897Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). However, this agent did not significantly affect the insulin-stimulated MeAIB uptake even at concentrations up to 50 μm, while the same concentration of PD98059 caused nearly complete inhibition of the insulin-induced activation of MAP kinases (Fig. 5). To further exclude involvement of this signaling pathway, inhibition of the farnesylation of Ras, the upstream mediator for activation of the MAP kinase pathway, by specific inhibitors PD152440 (20Gabbay R.A. Sutherland C. Gnudi L. Kahn B.B. O'Brien R.M. Granner D.K. Flier J.S. J. Biol. Chem. 1996; 271: 1890-1897Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar) and B581 (21Garcia A.M. Rowell C. Ackermann K. Kowalczyk J.J. Lewis M.D. J. Biol. Chem. 1993; 268: 18415-18418Abstract Full Text PDF PubMed Google Scholar) was tested. At concentrations of 10 and 50 μm, respectively, these two compounds have been shown to effectively block activation of MAP kinase (20Gabbay R.A. Sutherland C. Gnudi L. Kahn B.B. O'Brien R.M. Granner D.K. Flier J.S. J. Biol. Chem. 1996; 271: 1890-1897Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar), but at the same concentrations these two reagents had no effect on the insulin-stimulated transport activity, suggesting further that activation of MAP kinase is not responsible for this action of insulin. Rapamycin, a macrolide immunosuppressant, has been widely used to block activation of p70S6K (18Cheatham B. Vlahos C.J. Cheatham L. Wang L. Blenis J. Kahn C.R. Mol. Cell. Biol. 1994; 14: 4902-4911Crossref PubMed Scopus (1001) Google Scholar, 22Price D.J. Grove J.R. Calvo V. Avruch J. Bierer B.E. Science. 1992; 257: 973-977Crossref PubMed Scopus (589) Google Scholar). This drug completely eliminates activation of p70S6K at 10–20 ng/ml (22Price D.J. Grove J.R. Calvo V. Avruch J. Bierer B.E. Science. 1992; 257: 973-977Crossref PubMed Scopus (589) Google Scholar, 23Fingar D.C. Hausdorff S.F. Blenis J. Birnbaum M.J. J. Biol. Chem. 1993; 268: 3005-3008Abstract Full Text PDF PubMed Google Scholar). Fig. 5 shows that rapamycin at concentrations up to 200 ng/ml did not produce significant inhibition of the insulin-stimulated MeAIB uptake, while 100 ng/ml of rapamycin eliminated activation of p70S6K by 90%. The same results were obtained when rapamycin and PD98059 were added together, suggesting that the pp70S6K signaling pathway is not likely involved in the insulin stimulation of system A transport in 3T3-L1 adipocytes. To explore the role of PI 3′-K in the regulation of system A transport, the two structurally distinct inhibitors of PI 3′-K, wortmannin and LY294002, were employed (24Ui M. Okada T. Hazeki K. Hazeki O. Trends Biochem. Sci. 1995; 20: 303-307Abstract Full Text PDF PubMed Scopus (521) Google Scholar, 25Vlahos C.J. Matter W.F. Hui K.Y. Brown R.F. J. Biol. Chem. 1994; 269: 5241-5248Abstract Full Text PDF PubMed Google Scholar). As seen in Fig. 5, both compounds were potent inhibitors of the insulin-stimulated system A activity with IC50 values of 17 nm and 9.8 μm, respectively. These IC50 values are similar to those reported for inhibition of insulin-activation of PI 3′-K (20Gabbay R.A. Sutherland C. Gnudi L. Kahn B.B. O'Brien R.M. Granner D.K. Flier J.S. J. Biol. Chem. 1996; 271: 1890-1897Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 26Tsakiridis T. McDowell H.E. Walker T. Downes C.P. Hundal H.S. Vranic M. Klip A. Endocrinology. 1995; 136: 4315-4322Crossref PubMed Google Scholar, 27Sanchez-Margalet V. Goldfine I.D. Vlahos C.J. Sung C.K. Biochem. Biophys. Res. Commun. 1994; 204: 446-452Crossref PubMed Scopus (122) Google Scholar) and phospholipase A2 (28Cross M.J. Stewart A. Hodgkin M.N. Kerr D.J. Wakelam M.J. J. Biol. Chem. 1995; 270: 25352-25355Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar), but are significantly lower than that for inhibition of phospholipase D and phospholipase C (29Bonser R.W. Thompson N.T. Randall R.W. Tateson J.E. Spacey G.D. Hodson H.F. Garland L.G. Br. J. Pharmacol. 1991; 103: 1237-1241Crossref PubMed Scopus (108) Google Scholar), myosin light chain kinase (30Nakanishi S. Kakita S. Takahashi I. Kawahara K. Tsukuda E. Sano T. Yamada K. Yoshida M. Kase H. Matsuda Y. Hashimoto Y. Nonomura Y. J. Biol. Chem. 1992; 267: 2157-2163Abstract Full Text PDF PubMed Google Scholar), mammalian target of rapamycin (31Brunn G.J. Williams J. Sabers C. Wiederrecht G. Lawrence Jr, J.C. Abraham R.T. EMBO J. 1996; 15: 5256-5267Crossref PubMed Scopus (626) Google Scholar), and phosphatidylinositol 4′-kinase (32Meyers R. Cantley L.C. J. Biol. Chem. 1997; 272: 4384-4390Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). To explore the link between activation of PI 3′-K and stimulation of system A transport by insulin, we examined the effects of a series of hormones and growth factors that differentially regulate these processes. As seen in Fig. 6, epidermal growth factor, glucagon, and TPA were ineffective in activation of PI 3′-K, however, GH, PDGF, and IL-11 produced a marked elevation of PI 3′-K as detected in anti-phosphotyrosine immunoprecipitates. However, capability for activation of PI 3′-K did not correlate well with stimulation of MeAIB uptake. GH, PDGF, and IL-11 did not produce substantial stimulation of system A activity. Several models have been proposed to interpret the kinetic and genetic bases for the regulation of system A amino acid transport (5McGivan J.D. Pastor-Anglada M. Biochem. J. 1994; 299: 321-334Crossref PubMed Scopus (339) Google Scholar,15Handlogten M.E. Kilberg M.S. J. Biol. Chem. 1984; 259: 3519-3525Abstract Full Text PDF PubMed Google Scholar, 16Bracy D.S. Handlogten M.E. Barber E.F. Han H.P. Kilberg M.S. J. Biol. Chem. 1986; 261: 1514-1520Abstract Full Text PDF PubMed Google Scholar, 17Kilberg M.S. Trends Biochem. Sci. 1986; 11: 183-186Abstract Full Text PDF Scopus (38) Google Scholar). According to these models, a “transport inactivation protein” or an active form of “repressor/inactivator” is thought to be responsible for the decay or adaptive regulation of system A activity. Evidence suggests that the histone-like nuclear proteins (16Bracy D.S. Handlogten M.E. Barber E.F. Han H.P. Kilberg M.S. J. Biol. Chem. 1986; 261: 1514-1520Abstract Full Text PDF PubMed Google Scholar,17Kilberg M.S. Trends Biochem. Sci. 1986; 11: 183-186Abstract Full Text PDF Scopus (38) Google Scholar) or heat shock protein P1-related proteins (33Jones M. Gupta R.S. Englesberg E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 858-862Crossref PubMed Scopus (45) Google Scholar) may serve to inactivate the system A transporter. However, data presented here indicate that the decay of system A transport in adipocytes is not caused by synthesis of a repressor-type protein. Moreover, it also appears to be independent of ambient amino acids. Taken together, these data suggest that the insulin-stimulated system A transport in adipocytes is mediated by a mechanism different from that reported in hepatocytes (15Handlogten M.E. Kilberg M.S. J. Biol. Chem. 1984; 259: 3519-3525Abstract Full Text PDF PubMed Google Scholar, 16Bracy D.S. Handlogten M.E. Barber E.F. Han H.P. Kilberg M.S. J. Biol. Chem. 1986; 261: 1514-1520Abstract Full Text PDF PubMed Google Scholar, 17Kilberg M.S. Trends Biochem. Sci. 1986; 11: 183-186Abstract Full Text PDF Scopus (38) Google Scholar). The lack of repressor-type control has been reported for regulation of system A transport by other factors such as osmotic stress (5McGivan J.D. Pastor-Anglada M. Bioc" @default.
• W1970989980 created "2016-06-24" @default.
• W1970989980 creator A5018176504 @default.
• W1970989980 creator A5030999547 @default.
• W1970989980 creator A5033151808 @default.
• W1970989980 creator A5069635995 @default.
• W1970989980 creator A5078858200 @default.
• W1970989980 date "1998-02-01" @default.
• W1970989980 modified "2023-09-26" @default.
• W1970989980 title "Regulation of System A Amino Acid Transport in 3T3-L1 Adipocytes by Insulin" @default.
• W1970989980 cites W1490166974 @default.
• W1970989980 cites W1505315584 @default.
• W1970989980 cites W1507117319 @default.
• W1970989980 cites W1529396499 @default.
• W1970989980 cites W1537820722 @default.
• W1970989980 cites W1544277307 @default.
• W1970989980 cites W1551443142 @default.
• W1970989980 cites W1560473424 @default.
• W1970989980 cites W1566749908 @default.
• W1970989980 cites W1577885145 @default.
• W1970989980 cites W1581635008 @default.
• W1970989980 cites W1583256888 @default.
• W1970989980 cites W1633537473 @default.
• W1970989980 cites W1797700808 @default.
• W1970989980 cites W1838741822 @default.
• W1970989980 cites W1947575656 @default.
• W1970989980 cites W195923807 @default.
• W1970989980 cites W1969130431 @default.
• W1970989980 cites W1969161473 @default.
• W1970989980 cites W1969393623 @default.
• W1970989980 cites W1971915141 @default.
• W1970989980 cites W1974589864 @default.
• W1970989980 cites W1976040992 @default.
• W1970989980 cites W1977654932 @default.
• W1970989980 cites W1991016557 @default.
• W1970989980 cites W1992975639 @default.
• W1970989980 cites W1995672565 @default.
• W1970989980 cites W1997460544 @default.
• W1970989980 cites W2024918809 @default.
• W1970989980 cites W2024942502 @default.
• W1970989980 cites W2025998518 @default.
• W1970989980 cites W2029383686 @default.
• W1970989980 cites W2032194743 @default.
• W1970989980 cites W203563334 @default.
• W1970989980 cites W2044301435 @default.
• W1970989980 cites W2051781413 @default.
• W1970989980 cites W2053907246 @default.
• W1970989980 cites W2058899879 @default.
• W1970989980 cites W2063117678 @default.
• W1970989980 cites W2071083249 @default.
• W1970989980 cites W2075326322 @default.
• W1970989980 cites W2078411393 @default.
• W1970989980 cites W2081698610 @default.
• W1970989980 cites W2089127518 @default.
• W1970989980 cites W2092207721 @default.
• W1970989980 cites W2107207654 @default.
• W1970989980 cites W2109300167 @default.
• W1970989980 cites W2115020513 @default.
• W1970989980 cites W2135185526 @default.
• W1970989980 cites W2156475226 @default.
• W1970989980 cites W2160746910 @default.
• W1970989980 cites W2163976499 @default.
• W1970989980 cites W2171424906 @default.
• W1970989980 cites W2176732357 @default.
• W1970989980 cites W2344973376 @default.
• W1970989980 doi "https://doi.org/10.1074/jbc.273.6.3173" @default.
• W1970989980 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9452428" @default.
• W1970989980 hasPublicationYear "1998" @default.
• W1970989980 type Work @default.
• W1970989980 sameAs 1970989980 @default.
• W1970989980 citedByCount "30" @default.
• W1970989980 crossrefType "journal-article" @default.
• W1970989980 hasAuthorship W1970989980A5018176504 @default.
• W1970989980 hasAuthorship W1970989980A5030999547 @default.
• W1970989980 hasAuthorship W1970989980A5033151808 @default.
• W1970989980 hasAuthorship W1970989980A5069635995 @default.
• W1970989980 hasAuthorship W1970989980A5078858200 @default.
• W1970989980 hasBestOaLocation W19709899801 @default.
• W1970989980 hasConcept C126322002 @default.
• W1970989980 hasConcept C134018914 @default.
• W1970989980 hasConcept C142536801 @default.
• W1970989980 hasConcept C171089720 @default.
• W1970989980 hasConcept C185592680 @default.
• W1970989980 hasConcept C2776175234 @default.
• W1970989980 hasConcept C2779306644 @default.
• W1970989980 hasConcept C55493867 @default.
• W1970989980 hasConcept C71924100 @default.
• W1970989980 hasConcept C86803240 @default.
• W1970989980 hasConcept C95444343 @default.
• W1970989980 hasConceptScore W1970989980C126322002 @default.
• W1970989980 hasConceptScore W1970989980C134018914 @default.
• W1970989980 hasConceptScore W1970989980C142536801 @default.
• W1970989980 hasConceptScore W1970989980C171089720 @default.
• W1970989980 hasConceptScore W1970989980C185592680 @default.
• W1970989980 hasConceptScore W1970989980C2776175234 @default.
• W1970989980 hasConceptScore W1970989980C2779306644 @default.
• W1970989980 hasConceptScore W1970989980C55493867 @default.
• W1970989980 hasConceptScore W1970989980C71924100 @default.

• next