The Effect Of Melatonin And Its Receptor Agonist On Insulin Resistance In 3T3-L1 Adipocytes

Objectives: To explore the effects of Melatnoin and its receptor agonist(Tal) on glucose uptake and expression of GLUT-4, IRS-1 and its phosphorylation of serine307(pS307) in 3T3-L1 adipocytes treated with or without free fatty acids.Methods: The experiments were designed as follows: first, 3T3-L1 preadipocytes were maintained in DMEM culture medium supplemented with 10% fetal calf serum for adipogenesis, 3T3-L1 preadipocytes were grown into confluence in a six-well plate, and then were incubated in adipogenic cocktail (5μg/ml insulin, 0.5mM isobutylmethylxanthine, and 1μM dexamethasone) for 2 days. This was followed by incubation in insulin-supplemented medium for additional 4 days. Taking 10～12 days, cells were differentiated into adipocytes as determined by Oil Red O staining. Then, IRS-1 and GLUT-4 protein abundances were monitored in the FFA-treated cells in a time-course study. PS307 and IRS-1 were monitored in the FFA-treated cells in a dose-course study with or without Melatonin or it's receptor agonist Tal. IRS-1, PS307 and GLUT-4 protein expressions were detected by Western blot, and cell glucose uptake was measured by liquid scintillation counter.Results: 3T3-L1 cells were differentiated into adipocytes as determined by Oil Red O staining . 3T3-L1 adipocytes were treated with palmic acid to induce insulin resistance. Insulin-induced glucose uptake was measured to determine insulin sensitivity. The result showed that insulin-induced glucose uptake was inhibited after a 6 h-treatment with palmic acid. IRS-1 and GLUT-4 protein abundances were monitored in the FFA-treated cells in a time-course study. IRS-1 protein decreased gradually. A 50% decrease in GLUT-4 protein expression was detected 6 h after addition of palmic acid. At 24 h, 80% of IRS-1 protein was lost. In a dose-course studey, we found that palmic acid was able to induce the phosphorylation of serine 307 in IRS-1 at 200μM, and the phosphorylation wasn't increased signifficantly at about 300μM. Melatonin at high dose(>1nM)and Tal at 10nM reduced the phosphorylation of serine 307 in IRS-1 (p<0.05 vs FFA alone) and improved adipocytes glucose uptake and GLUT-4 protein expression. Tal alone had no effect on GLUT-4 protein expression and on glucose uptake in normal adipocytes.Conclusion: The results that insulin-induced glucose uptake was inhibited by palmic acid reatment showed FFA was a important pathogenesis of insulin resistance. Phosphorylation of serine 307 in IRS-1 protein was been linked to FFA-associated insulin resistance, this phosphorylation resulted a reduce in GLUT-4 protein expression and glucose uptake. Melatonin at high dose(>1nM)and Tal at 10nM reduced the phosphorylation in IRS-1 and improved adipocytes glucose uptake and GLUT-4 protein expression. And the effect of improving glucose uptake enhanced by insulin. We concluded that melatonin interacts with insulin and it's signaling.