Glucose-induced insulin resistance of phosphatidylinositol 3 '-OH kinase and AKT/PKB is mediated by the hexosamine biosynthesis pathway

Hyperglycemia is responsible for many of the vascular complications and met abolic derangements seen in diabetes. One potential regulator of the effect s of glucose is the hexosamine biosynthesis pathway (HBP). Glutamine: fruct ose -6-phosphate amidotransferase (GFA), the first and rate-limiting enzyme in this pathway, catalyzes the transfer of an amino group from glutamine t o fructose-6-phosphate to form glucosamine-6-phosphate. Overexpression of C FA in rat-1 fibroblasts results in insulin resistance fur glycogen synthase (GS activity, and renders these cells more sensitive to the effects of glu cose. Using rat-1 cells, we examine further the mechanisms whereby hexosami nes lead to insulin resistance, Insulin stimulated GS activity was found to occur via a PI-3 kinase (PI-3k)-dependent pathway as wortmannin, an inhibi tor of PI-3K, blocked insulin's ability to stimulate GS activity. Subsequen tly, we examined the effect of hexosamines on PI-3K and Akt/PKB activity. C ells were cultured in 1 mM glucose (low glucose, LG, 20 mM glucose thigh gl ucose, l-IG), or I mM glucose plus 3 mM glucosamine (GlcN) for 16-20 h. Aft er treatment with insulin (100 nM) for 5 min, cell extracts were assayed fo r IRS-1 associated and total PI-3K activity. At LG, insulin increased PI-3K activity by 43%. There was no insulin stimulation of PI-3k activity in cel ls cultured in Hg or GlcN. There was a trend for IRS-1 protein levels to de crease in HG but not GlcN. PI-3K protein levels were not altered by HG or G lcN. Finally PKB activity was assayed. At LG, insulin stimulated PKB activi ty. Again, both HG and GlcN significantly reduced insulin's ability to stim ulate PKB activity. We conclude that the hexosamine-mediated insulin resist ance of GS activity seen in rat-1 cells is mediated by hexosamine regulatio n of PI-3K and PKB. (C) 2001 Elsevier Science Inc. All rights reserved.