title: Discovery of a metabolic pathway mediating glucose-induced desensitization of the glucose transport system. Role of hexosamine biosynthesis in the induction of insulin resistance.
abstract: Based on our previous finding that desensitization of the insulin-responsive glucose transport system (GTS) requires three components, glucose, insulin, and glutamine, we postulated that the routing of incoming glucose through the hexosamine biosynthesis pathway plays a key role in the development of insulin resistance in primary cultured adipocytes. Two approaches were used to test this hypothesis. First, we assessed whether glucose-induced desensitization of the GTS could be prevented by glutamine analogs that irreversibly inactivate glutamine-requiring enzymes, such as glutamine:fructose-6-phosphate amidotransferase (GFAT) the first and the rate-limiting enzyme in hexosamine biosynthesis. Both O-diazoacetyl-L-serine (azaserine) and 6-diazo-5-oxonorleucine inhibited desensitization in 18-h treated cells without affecting maximal insulin responsiveness in control cells. Moreover, close agreement was seen between the ability of azaserine to prevent desensitization of the GTS in intact adipocytes (70% inhibition, ED50 = 1.1 microM), its ability to inactivate GFAT in intact adipocytes (64% inhibition, ED50 = 1.0 microM) and its ability to inactivate GFAT activity in a cytosolic adipocyte preparation (ED50 = 1.3 microM). From these results we concluded that a glutamine amidotransferase is involved in the induction of insulin resistance. As a second approach, we determined whether glucosamine, an agent known to preferentially enter the hexosamine pathway at a point distal to enzymatic amidation by GFAT, could induce cellular insulin resistance. When adipocytes were exposed to various concentrations of glucosamine for 5 h, progressive desensitization of the GTS was observed (ED50 = 0.36 mM) that culminated in a 40-50% loss of insulin responsiveness. Moreover, we estimated that glucosamine is at least 40 times more potent than glucose in mediating desensitization, since glucosamine entered adipocytes at only one-quarter of the glucose uptake rate, yet induced desensitization at an extra-cellular dose 10 times lower than glucose. In addition, we found that glucosamine-induced desensitization did not require glutamine and was unaffected by azaserine treatment. Thus, we conclude that glucosamine enters the hexosamine-desensitization pathway at a point distal to GFAT amidation. Overall, these studies indicate that a unique metabolic pathway exists in adipocytes that mediates desensitization of the insulin-responsive GTS, and reveal that an early step in this pathway involves the conversion of fructose 6-phosphate to glucosamine 6-phosphate by the first and rate-limiting enzyme of the hexosamine pathway, glutamine:fructose-6-phosphate amidotransferase.
authors.affiliation: Department of Biochemistry, University of Tennessee, Memphis 38163.
authors.forename: S
authors.initials: S
authors.lastname: Marshall
authors.forename: V
authors.initials: V
authors.lastname: Bacote
authors.forename: R R
authors.initials: RR
authors.lastname: Traxinger
mesh_terms: D000818:Animals; D001377:Azaserine / Q000494:pharmacology; D001692:Biological Transport; D005944:Glucosamine / Q000378:metabolism; D005947:Glucose / Q000378:metabolism*; D005973:Glutamine / Q000378:metabolism; D006595:Hexosamines / Q000096:biosynthesis*; D007333:Insulin Resistance; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains
chemical_list: D006595:Hexosamines; D005973:Glutamine; D001377:Azaserine; D005947:Glucose; D005944:Glucosamine