Glucosamine-induced Insulin Resistance in 3T3-L1 Adipocytes Is Caused by Depletion of Intracellular ATP

Abstract

Glucosamine, which enters the hexosamine pathway downstream of the rate-limiting step, has been routinely used to mimic the insulin resistance caused by high glucose and insulin. We investigated the effect of glucosamine on insulin-stimulated glucose transport in 3T3-L1 adipocytes. The ⌬-insulin (insulin-stimulated minus basal) value for 2-deoxyglucose uptake was dramatically inhibited with increasing concentrations of glucosamine with an ED 50 of 1.95 mM. Subcellular fractionation experiments demonstrated that reduction in insulin-stimulated 2-deoxyglucose uptake by glucosamine was due to an inhibition of translocation of both Glut 1 and Glut 4 from the low density microsomes (LDM) to the plasma membrane. Analysis of the insulin signaling cascade revealed that glucosamine impaired insulin receptor autophosphorylation, insulin receptor substrate (IRS-1) phosphorylation, IRS-1-associated PI 3-kinase activity in the LDM, and AKT-1 activation by insulin. Measurement of intracellular ATP demonstrated that the effects of glucosamine were highly correlated with its ability to reduce ATP levels. Reduction of intracellular ATP using azide inhibited Glut 1 and Glut 4 translocation from the LDM to the plasma membrane, insulin receptor autophosphorylation, and IRS-1 tyrosine phosphorylation. Additionally, both the reduction in intracellular ATP and the effects on insulin action caused by glucosamine could be prevented by the addition of inosine, which served as an alternative energy source in the medium. We conclude that direct administration of glucosamine can rapidly lower cellular ATP levels and affect insulin action in fat cells by mechanisms independent of increased intracellular UDP-N-acetylhexosamines and that increased metabolism of glucose via the hexosamine pathway may not represent the mechanism of glucose toxicity in fat cells.

Glucosamine activates the plasminogen activator inhibitor 1 gene promoter through Sp1 DNA binding sites in glomerular mesangial cells.

“…Indeed, a reduction of up to 50% of total cellular ATP was found in cultured 3T3-L1 adipocytes exposed to glucosamine in the presence of insulin and 5 mmol/l glucose, but in the absence of insulin, only mild (10-15%) ATP depletion was observed (51). Hresko et al (51) postulated that glucosamine-induced ATP depletion was due to phosphorylation of glucosamine by hexokinase. Glucosamine-induced UTP depletion has been attributed to the consumption of UTP during the formation of UDP-GlcNAc (43,53).…”

PACSIN3 overexpression increases adipocyte glucose transport through GLUT1

“…3T3-L1 adipocyte plasma membranes were isolated as previously described [14]. In brief, 3T3-L1 adipocytes were serum starved in DMEM for several hours.…”

“…Cells were then serum starved for several hours in DMEM prior to initiation of the uptake assay. The assay was performed as previously described [14]. In brief, the cells were incubated for 30 min in DMEM with or without 1 μM insulin for 30 min at 37° C. Cells were then washed in 37° C Krebs Ringers Phosphate (KRP) buffer followed by incubation in 37° C KRP buffer with or without 40 μM cytochalasinB (CB).…”

Nutritional regulation of leptin in humans

“…Infusions of glucose or a lipid emulsion for 3-h increased leptin mRNA and UDP-N-acetylglucosamine concentrations in fat and skeletal muscle, providing additional links between diet and leptin synthesis [60]. The hexosamine hypothesis is an exciting one but requires further studies in light of the finding that glucosamine also depletes cellular ATP concentrations [61]. [39,44,68].…”