Hyperglycemia is responsible for many of the vascular complications and metabolic derangements seen in diabetes. One potential regulator of the effects of
glucose is the
hexosamine biosynthesis pathway (HBP).
Glutamine:
fructose-6-phosphate amidotransferase (GFA), the first and rate-limiting
enzyme in this pathway, catalyzes the transfer of an amino group from
glutamine to
fructose-6-phosphate to form glucosamine-6-phosphate. Overexpression of GFA in rat-1 fibroblasts results in
insulin resistance for
glycogen synthase (GS) activity, and renders these cells more sensitive to the effects of
glucose. Using rat-1 cells, we examine further the mechanisms whereby
hexosamines lead to
insulin resistance.
Insulin stimulated GS activity was found to occur via a
PI-3 kinase (PI-3K)-dependent pathway as
wortmannin, an inhibitor of
PI-3K, blocked
insulin's ability to stimulate GS activity. Subsequently, we examined the effects of
hexosamines on
PI-3K and Akt/PKB activity. Cells were cultured in 1 mM
glucose (low
glucose, LG), 20 mM
glucose (high
glucose, HG), or 1 mM
glucose plus 3 mM
glucosamine (GlcN) for 16--20 h.
After treatment with
insulin (100 nM) for 5 min,
cell extracts were assayed for 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 cells cultured in HG or GlcN. There was a trend for IRS-1
protein levels to decrease in HG but not GlcN.
PI-3K protein levels were not altered by HG or GlcN. Finally PKB activity was assayed. At LG,
insulin stimulated PKB activity. Again, both HG and GlcN significantly reduced
insulin's ability to stimulate PKB activity. We conclude that the
hexosamine-mediated
insulin resistance of GS activity seen in rat-1 cells is mediated by
hexosamine regulation of
PI-3K and PKB.