Complete inhibition of glucose-induced desensitization of the glucose transport system by inhibitors of mRNA synthesis. Evidence for rapid turnover of glutamine:fructose-6-phosphate amidotransferase.
Glutamine:fructose-6-phosphate amidotransferase (GFAT) plays a key role in desensitizing the insulin-responsive glucose transport system (GTS), and recent studies have revealed that loss of GFAT activity accompanies desensitization. To gain insights into the mechanisms underlying loss of enzyme activity, we have used primary cultured adipocytes and two well established inhibitors of mRNA synthesis to estimate GFAT turnover. Both actinomycin D and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) caused a rapid and extensive loss in GFAT activity (greater than 70% loss, t1/2 of 45 min) indicating that GFAT has a relatively short half-life. Since induction of insulin resistance requires GFAT, we next examined the ability of mRNA inhibitors to block glucose-induced desensitization. When adipocytes were cultured for 18 h with 20 mM glucose, amino acids, and 25 ng/ml insulin, maximal insulin responsiveness of the GTS was reduced by greater than 70%. Both actinomycin D and DRB rapidly and completely prevented desensitization in a dose-dependent manner (ED50 of 16 nM and 15 microM, respectively). These findings are the predicted functional consequence of diminished GFAT activity. Evidence that actinomycin D acts selectively on GFAT without influencing other steps within the desensitization pathway was obtained using glucosamine, an agent that enters the hexosamine biosynthesis pathway at a point distal to the action of GFAT. Actinomycin D inhibited glucose-induced desensitization but failed to block glucosamine-induced desensitization. From these studies we conclude that 1) glucose-induced desensitization of the GTS can be completely prevented by actinomycin D and DRB, two potent and diverse inhibitors of mRNA synthesis; 2) the functional integrity of the desensitization pathway is maintained by a short-lived protein; and 3) the identity of this short-lived protein is most likely GFAT, the first and rate-limiting enzyme of the hexosamine biosynthesis pathway.
Marshall
S
S
Department of Biochemistry, University of Tennessee, Memphis 38163.
Bacote
V
V
Traxinger
R R
RR
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D003609:Dactinomycin / Q000494:pharmacology*; D004004:Dichlororibofuranosylbenzimidazole / Q000494:pharmacology*; D005944:Glucosamine / Q000378:metabolism; D005947:Glucose / Q000378:metabolism*; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / Q000378:metabolism*; D008297:Male; D012333:RNA, Messenger / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D051381:Rats; D011919:Rats, Inbred Strains
D012333:RNA, Messenger; D003609:Dactinomycin; D004004:Dichlororibofuranosylbenzimidazole; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing); D005947:Glucose; D005944:Glucosamine