Defective Akt activation is associated with glucose- but not glucosamine-induced insulin resistance

3T3-L1 adipocytes develop insulin-resistant glucose transport upon preincubation with high glucose or glucosamine, provided insulin (0.6 nM) is present during preincubation. Insulin receptor substrate-1 (IRS-1)-associated phosphatidylinositol (PI) 3-kinase activity is unaffected (30). Total cellular IRS-1, PI 3-kinase, or Akt concentrations were unchanged. Akt activation in subcellular fractions was assessed by immunoblotting with two phospho-Akt-specific antibodies. Upon acute 100 nM insulin stimulation, plasma membrane (PM)-associated phospho-Akt was highest in cells preincubated in low glucose with no insulin, less in high glucose with no insulin, even less in low glucose+insulin, and lowest in high glucose+insulin. Only high glucose+insulin caused insulin-resistant glucose transport. Acute insulin stimulation increased total PM-Akt about twofold after preincubation without insulin in low or high glucose. Preincubation with 0.6 nM insulin decreased Akt PM translocation by approximately 25% in low and approximately 50% in high glucose. Preincubation with glucosamine did not affect Akt phosphorylation or translocation. Conclusions: chronic exposure to high glucose or insulin downregulates acute insulin-stimulated Akt activation, acting synergistically distal to PI 3-kinase. Maximal insulin activates more Akt than required for maximal glucose transport stimulation. Insulin resistance may ensue when PM-associated phospho-Akt decreases below a threshold. High glucose and glucosamine cause insulin resistance by different mechanisms in 3T3-L1 adipocytes.