Different effects of two proteinase inhibitors on insulin-induced cellular responses in rat adipocytes.
Among various proteinase inhibitors, N-acetyl-L-tyrosine ethyl ester (ATEE), a chymotrypsin substrate analog, and N alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), a trypsin inhibitor, showed significant inhibitory effects on insulin stimulated glucose transport in rat adipocytes. ATEE did not affect insulin binding, but inhibited insulin internalization. In intact adipocytes, ATEE inhibited tyrosine phosphorylation of the beta-subunit of the insulin receptor, a 170 kDa protein and a 60 kDa protein at almost the same concentration (ID50 = 0.24 +/- 0.05 mM, n = 4, mean +/- S.E.), but in a plasma membrane fraction, ATEE did not appreciably inhibit the tyrosine phosphorylation of the beta-subunit of the insulin receptor, TLCK did not inhibit insulin binding. At 0.25 mM, TLCK did not inhibit insulin internalization, but inhibited 70% of the insulin-stimulated glucose transport (ID50 = 0.19 +/- 0.02 mM, n = 7). TLCK inhibited insulin internalization at more than 0.25 mM. TLCK did not inhibit the tyrosine phosphorylation of the beta-subunit of the insulin receptor in intact cells or in the plasma membrane fraction. In intact cells, TLCK inhibited the phosphorylation of the 60 kDa protein and simultaneously it stimulated the phosphorylation of the 170 kDa protein more than 3-fold. These results indicate that there are at least two sites in the insulin-induced signal transduction pathway where proteinase inhibitors act to suppress the insulin signal transduction. A major ATEE site is very close to phosphorylation of the beta-subunit of the insulin receptor. On the other hand, TLCK inhibits a step(s) in the signal transduction pathway after the insulin receptor but before the glucose transporter.
Kasahara
T
T
Laboratory of Biophysics, School of Medicine, Teikyo University, Hachioji, Japan.
Ezaki
O
O
Kasahara
M
M
D019325:3-O-Methylglucose; D000273:Adipose Tissue / Q000187:drug effects* / Q000201:enzymology; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology*; D000818:Animals; D002462:Cell Membrane / Q000187:drug effects; D002478:Cells, Cultured / Q000187:drug effects; D007328:Insulin / Q000378:metabolism*; D008297:Male; D008757:Methylglucosides / Q000378:metabolism; D010766:Phosphorylation; D011480:Protease Inhibitors / Q000494:pharmacology*; D051381:Rats; D011919:Rats, Inbred Strains; D011972:Receptor, Insulin / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D014107:Tosyllysine Chloromethyl Ketone / Q000494:pharmacology*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology
D000590:Amino Acid Chloromethyl Ketones; D007328:Insulin; D008757:Methylglucosides; D011480:Protease Inhibitors; D019325:3-O-Methylglucose; D014107:Tosyllysine Chloromethyl Ketone; D014443:Tyrosine; C007651:ethyl N-alpha-acetyl-tyrosinate; D011972:Receptor, Insulin