Glucosamine infusion in rats rapidly impairs insulin stimulation of phosphoinositide 3-kinase but does not alter activation of Akt/Protein kinase B in skeletal muscle

Glucosamine, a metabolite of glucose via the hexosamine biosynthetic pathwa y, potently induces insulin resistance in skeletal muscle by impairing insu lin-induced GLUT4 translocation to the plasma membrane. Activation of phosp hoinositide (PI) 3-kinase is necessary for insulin-stimulated GLUT4 translo cation, and the serine/threonine kinase Akt/protein kinase B (PKB) is a dow nstream mediator of some actions of PI 3-kinase. To determine whether gluco samine-induced insulin resistance could be due to impaired signaling, me me asured insulin receptor substrate (IRS)-1 and insulin receptor tyrosine pho sphorylation; PI 3-kinase activity associated with IRS-1, IRS-2, and phosph otyrosine; and Akt activity and phosphorylation in skeletal muscle of rats infused for 2 h with glucosamine (6.0 mg.kg(-1).min(-1)) or saline. Euglyce mic-hyperinsulinemic clamp studies (12 mU.kg(-1).min(-1) insulin) in awake rats showed that; glucosamine infusion resulted in rapid induction of insul in resistance, with a 33% decrease in glucose infusion rate (P < 0.01). Tis sues were harvested after saline alone (basal), 1 min after an insulin bolu s (10 U/kg), or after 2 h of insulin clamp in saline- and glucosamine-infus ed rats, After 1 min of insulin stimulation, phosphorylation of IRS-1 and i nsulin receptor increased 6- to 8-fold in saline-infused rats and 7- to 10- fold in glucosamine-infused rats. In saline-infused rats, 1 min of insulin stimulation increased PI 3-kinase activity associated with IRS-1, IRS-2, or phosphotyrosine 7.6-, 6.4-, and 10-fold, respectively. In glucosamine-infu sed rats treated for 1 min with insulin, PI 3-kinase activity associated wi th IRS-1 was reduced 28% (P < 0.01) and that associated with phosphotyrosin e was reduced 43% (P < 0.01). Insulin for 1 min stimulated Akt/PKB activity similar to 5-fold in both saline- and glucosamine-infused rats; insulin-in duced hyperphosphorylation of Akt/PKB was not different between groups. Glu cosamine infusion alone had no effect on tyrosine phosphorylation of the in sulin receptor or IRS-I or on stimulation of PI 3-kinase or Akt/PKB activit y. However, 2 h of insulin clamp reduced PI 3-kinase activity associated wi th IRS-1, IRS-2, or phosphotyrosine to <30% of that seen with 1 min of insu lin. No effect of glucosamine was seen on these signaling events when compa red with 2 h of insulin clamp without glucosamine. Our data show that 1) gl ucosamine infusion in rats is associated with an impairment in the early ac tivation of PI 3-kinase by insulin in skeletal muscle, 2) this insulin-resi stant state does not involve alterations in the activation of Akt/PKB, and 3) prolonged insulin infusion under clamp conditions results in a blunting of the PI 3-kinase response to insulin.