Regulation of System A amino acid transport in L6 rat skeletal muscle cells by insulin, chemical and hyperthermic stress

Amino acid starvation markedly stimulates the activity of system A, a widely distributed transport route for neutral amino acids. The involvement of MAPK (mitogen-activated protein kinase) pathways in this adaptive increase of transport activity was studied in cultured human fibroblasts. In these cells, a 3-fold stimulation of system A transport activity required a 6-h amino acidfree incubation. However, a rapid tyrosine phosphorylation of ERK (extracellular regulated kinase) 1 and 2, and JNK (Jun N-terminal kinase) 1, but not of p38, was observed after the substitution of complete medium with amino acid-free saline solution. ERK1/2 activity was 4-fold enhanced after a 15-min amino acid-free incubation and maintained at stimulated values thereafter. A transient, less evident stimulation of JNK1 activity was also detected, while the activity of p38 was not affected by amino acid deprivation. PD98059, an inhibitor of ERK1/2 activation, completely suppressed the adaptive increase of system A transport activity that, conversely, was unaffected by inhibitors of other transduction pathways, such as rapamycin and wortmannin, as well as by chronic treatment with phorbol esters. In the presence of either L-proline or 2-(methylaminoisobutyric) acid, two substrates of system A, the transport increase was prevented and no sustained stimulation of ERK1/2 was observed. To identify the stimulus that maintains MAPK activation, cell volume was monitored during amino acid-free incubation. It was found that amino acid deprivation caused a progressive cell shrinkage (30% after a 6-h starvation). If proline was added to amino acid-starved, shrunken cells, normal values of cell volume were rapidly restored. However, proline-dependent volume rescue was hampered if cells were pretreated with PD98059. It is concluded that (a) the triggering of adaptive increase of system A activity requires a prolonged activation of ERK1 and 2 and that (b) cell volume changes, caused by the depletion of intracellular amino acid pool, may underlie the activation of MAPKs.

show abstract

“…5). The stimulatory changes in system A activity induced by chemical and hyperthermic stress also appear to be ERK-independent (36).…”

Section: Discussionmentioning

confidence: 93%

Adaptive Increase of Amino Acid Transport System A Requires ERK1/2 Activation

Franchi-Gazzola

Visigalli

Bussolati

et al. 1999

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…System A is extensively regulated by changes in substrate availability with expression and stability of the SNAT2 protein being enhanced in cells subjected to amino acid withdrawal (42,43). In addition to this adaptive increase in SNAT2 protein, System A activity has long been known to be stimulated acutely by insulin (32,44,45), growth factors (31), and cell stresses (31,32). These stimuli induce a rapid increase in the V max of system A transport, which involves molecules participating in proximal insulin and growth factor signaling.…”

Section: Discussionmentioning

confidence: 99%

Use of Akt Inhibitor and a Drug-resistant Mutant Validates a Critical Role for Protein Kinase B/Akt in the Insulin-dependent Regulation of Glucose and System A Amino Acid Uptake

Green

Göransson

Kular

et al. 2008

Journal of Biological Chemistry

102

View full text Add to dashboard Cite

Protein kinase B (PKB)/Akt has been strongly implicated in the insulin-dependent stimulation of GLUT4 translocation and glucose transport in skeletal muscle and fat cells. Recently an allosteric inhibitor of PKB (Akti) that selectively targets PKB␣ and -␤ was reported, but as yet its precise mechanism of action or ability to suppress key insulin-regulated events such as glucose and amino acid uptake and glycogen synthesis in muscle cells has not been reported. We show here that Akti ablates the insulin-dependent regulation of these processes in L6 myotubes at submicromolar concentrations and that inhibition correlates tightly with loss of PKB activation/phosphorylation. Similar findings were obtained using 3T3-L1 adipocytes. Akti did not inhibit IRS1 tyrosine phosphorylation, phosphatidylinositol 3-kinase signaling, or activation of Erks, ribosomal S6 kinase, or atypical protein kinases C but significantly impaired regulation of downstream PKB targets glycogen synthase kinase-3 and AS160. Akti-mediated inhibition of PKB requires an intact kinase pleckstrin homology domain but does not involve suppression of 3-phosphoinositide binding to this domain. Importantly, we have discovered that Akti inhibition is critically dependent upon a solvent-exposed tryptophan residue (Trp-80) that is present within the pleckstrin homology domain of all three PKB isoforms and whose mutation to an alanine (PKB W80A ) yields an Akti-resistant kinase. Cellular expression of PKB W80A antagonized the Akti-mediated inhibition of glucose and amino acid uptake. Our findings support a critical role for PKB in the hormonal regulation of glucose and system A amino acid uptake and indicate that use of Akti and expression of the drug-resistant kinase will be valuable tools in delineating cellular PKB functions.It is now widely accepted that the serine/threonine kinase PKB 3 (also known as Akt) acts as a major node in the cellular signaling network downstream of phosphatidylinositol 3-kinase (PI3K) regulating diverse pathways that impact upon cellular metabolism, growth, proliferation, and apoptosis (1). Activation of PKB by insulin and growth factors relies upon its recruitment to the plasma membrane. Precisely how this occurs is unclear, but the process is facilitated by the binding of 3-phosphoinositides (such as phosphatidylinositol 3,4,5-triphosphate (PIP 3 )) to the N-terminal pleckstrin homology (PH) domain of the kinase (2). PIP 3 binding induces conformational changes in PKB (3) that expose two regulatory sites, Thr 308 and Ser 473 (in PKB␣), whose phosphorylation is required for full activation of the kinase. Phosphorylation of Thr 308 is mediated by PDK1, whereas that of Ser 473 relies upon the TORC2 complex (4, 5). Activated PKB is then able to target a number of important downstream molecules including, for example, members of the FOXO family of forkhead transcription factors (6), the pro-apoptotic protein BAD as well as TSC2 and PRAS40, which have been implicated in regulation of the mTOR pathway (7). PKB also targets a protein ca...

show abstract

“…Recent identification of the proto-oncogene Rheb (Ras homologue enriched in brain), an effector of TSC tumor suppressor function (Garami et al, 2003;Saucedo et al, 2003;Stocker et al, 2003;Zhang et al, 2003) should aid in delineating these interactions ( Figure 1 and see below). Growth factors such as insulin also positively regulate nutrient uptake, including glucose and amino acids (Munoz et al, 1995;McDowell et al, 1998;Jefferson and Kimball, 2001). In particular, branched-chain amino acids, most notably leucine, exert a potent stimulatory effect on protein synthesis, an effect that is in part mediated by mTOR and its downstream effectors, the S6Ks and 4E-BPs Schmelzle and Hall, 2000;Gingras et al, 2001b) (Figure 1).…”

Section: Mtormentioning

confidence: 99%