Coordinated regulation of glutamine:fructose-6-phosphate amidotransferase activity by insulin, glucose, and glutamine. Role of hexosamine biosynthesis in enzyme regulation

R.R Traxinger,S Marshall

doi:10.1016/s0021-9258(18)99202-1

R.R Traxinger, S Marshall

Open Access

https://doi.org/10.1016/s0021-9258(18)99202-1

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Abstract

We reported previously that glutamine:F-6-P amidotransferase (GFAT) plays an integral role in the development of insulin resistance by directing the flow of incoming glucose into the hexosamine biosynthesis pathway. To determine whether the enzymatic activity of GFAT is altered during desensitization of the glucose transport system, we treated isolated rat adipocytes with various combinations of insulin, glucose, and glutamine. Treatment with insulin or glucose alone (or in combination) failed to reduce cytosolic GFAT activity after 4 h, whereas combined treatment with all three components elicited a progressive loss of GFAT activity that was rapid (t1/2 of 2 h) and extensive (70% loss). A pronounced loss of GFAT activity was also seen in cells exposed to glucosamine, an agent known to directly enter the hexosamine pathway (55% loss at 4 h, ED50 of 360 microM). Moreover, a close correlation was observed between the induction of desensitization and the loss of GFAT activity as a function of glucose, insulin, glutamine, and glucosamine concentrations. When total intracellular hexosamine products were measured, we found that hexosamine formation was unaltered by insulin or glucose (or a combination) but was elevated by greater than 4-fold in the presence of insulin, glucose, and glutamine (t1/2 of 22 min), a condition known to cause both desensitization and loss of GFAT activity. Additional studies indicated that the loss of GFAT activity under desensitizing conditions is not due to allosteric regulation since removal of potential allosteric factors from the cytosol of desensitized cells by G-25 column chromatography failed to restore enzyme activity. Overall, these studies indicate that 1) GFAT is an insulin-regulated enzyme; however, control of enzyme activity is not due to a direct action of insulin, but rather is mediated by insulin-induced enhancement of glucose uptake; 2) the routing of incoming glucose through the hexosamine pathway and the formation of hexosamine products appears to regulate GFAT activity; and 3) the progressive loss of GFAT activity over several hours is probably not due to allosteric regulation.

Highlights

Desensitization of the insulin-responsiveglucose transport dotransferase (GFAT) playsanintegralroleinthe system (GTS)’ in primary culturedadipocytes requires three development of insulin resistanceby directing theflow components, insulin,glucose, and glutamine[1,2,3,4,5]
Since we demonstrated previously that desensitization requires simultaneous treatmentwith insulin, glucose, and glutamine [6], we assessed whether loss of GFAT activity requiresall three componentsA. s can beclearly seen in Fig. 2, when cells were incubated for 4 h in HBSS containing various combinations of glucose (20 mM), insulin
We demonstrated that desensitization of the insulin-responsive GTS requires insulin, glucose, and glutamine [1, 2,4, 5]

Summary

RESULTS

Individual and Combined Effects ofGlucose, Insulin, and Glutamine on GFATActiuity-Shown in Fig. 1B is an experiment in which isolated adipocytes were suspended in HBSS containing 20 mM glucose and 1 X amino acids and treated with 25 ng/ml insulin t o induce desensitization of the GTS. The dose-response curves shown in Fig. 3 expands this basic finding by revealing that the ED,, values for glucose, insulin, and glutamine on loss of GFAT activity (A-C) are remarkably similar to the EDsovalues for desensitization of the GTS (D-F). The loss 1-3), we of GFAT and the induction postulated that productsof and concentrationof amino acids found inDulbecco's modified Eagles the hexosamine biosynthesis pathway coordinately regulate medium and contains 584 mg of glutamine/liter) At the indicated both desensitization and thloess of GFAT activity. Cells were washed and glutamine:fructose-6-P amidotransferase activity was measured in a crude cytosol preparation asdescribed inthemethods section. Each point inA represents the mean S.E. of six experiments, and inB, of 10 experiments This idea by examining theeffects of glucosamine and insulin treatment on GFAT activity. Control; D-Glc, D-glucose; Man, Dmannose; 3MG, 3-O-methylglucose; Fru, D-fructose; Gal, D-galactose

GlcN Insulin Insulin

Glucosamine Mannosamine Galactosamine

DISCUSSION