Amino acids and insulin control autophagic proteolysis through different signaling pathways in relation to mTOR in isolated rat hepatocytes.
Autophagy, a major bulk proteolytic pathway, contributes to intracellular protein turnover, together with protein synthesis. Both are subject to dynamic control by amino acids and insulin. The mechanisms of signaling and cross-talk of their physiological anabolic effects remain elusive. Recent studies established that amino acids and insulin induce p70 S6 kinase (p70(S6k)) phosphorylation by mTOR, involved in translational control of protein synthesis. Here, the signaling mechanisms of amino acids and insulin in macroautophagy in relation to mTOR were investigated. In isolated rat hepatocytes, both regulatory amino acids (RegAA) and insulin coordinately activated p70(S6k) phosphorylation, which was completely blocked by rapamycin, an mTOR inhibitor. However, rapamycin blocked proteolytic suppression by insulin, but did not block inhibition by RegAA. These contrasting results suggest that insulin controls autophagy through the mTOR pathway, but amino acids do not. Furthermore, micropermeabilization with Saccharomyces aureus alpha-toxin completely deprived hepatocytes of proteolytic responsiveness to RegAA and insulin, but still maintained p70(S6k) phosphorylation by RegAA. In contrast, Leu(8)-MAP, a non-transportable leucine analogue, did not mimic the effect of leucine on p70(S6k) phosphorylation, but maintained the activity on proteolysis. Finally, BCH, a System L-specific amino acid, did not affect proteolytic suppression or mTOR activation by leucine. All the results indicate that mTOR is not common to the signaling mechanisms of amino acids and insulin in autophagy, and that the amino acid signaling starts extracellularly with their "receptor(s)," probably other than transporters, and is mediated through a novel route distinct from the mTOR pathway employed by insulin.
Kanazawa
Takumi
T
Department of Applied Biological Chemistry, Faculty of Agriculture, Niigata University, Ikarashi, Niigata 950-2181, Japan.
Taneike
Ikue
I
Akaishi
Ryuichiro
R
Yoshizawa
Fumiaki
F
Furuya
Norihiko
N
Fujimura
Shinobu
S
Kadowaki
Motoni
M
D000596:Amino Acids / Q000494:pharmacology*; D000598:Amino Acids, Cyclic / Q000494:pharmacology; D000818:Animals; D001427:Bacterial Toxins / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006460:Hemolysin Proteins / Q000494:pharmacology; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism*; D007328:Insulin / Q000494:pharmacology*; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D008297:Male; D010447:Peptide Hydrolases / Q000378:metabolism*; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000378:metabolism; D015398:Signal Transduction; D020123:Sirolimus / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases
D000596:Amino Acids; D000598:Amino Acids, Cyclic; D001427:Bacterial Toxins; D004791:Enzyme Inhibitors; D006460:Hemolysin Proteins; D007328:Insulin; D047428:Protein Kinase Inhibitors; C021091:staphylococcal alpha-toxin; C006504:2-aminobicyclo(2,2,1)heptane-2-carboxylic acid; D011494:Protein Kinases; C546845:mTOR protein, rat; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D058570:TOR Serine-Threonine Kinases; D010447:Peptide Hydrolases; D007930:Leucine; D020123:Sirolimus