Neu-p11 Ameliorates Insulin Resistance Via Inhibiting The Oxidative Stress In 3T3-L1 Adipocytes

Background Melatonin is a kind of indole neural endocrine hormone secreted by the pineal gland of brain. Melatonin has the function of regulating circadian rhythm and improving sleep. Neu-P11 is a novel melatonin agonist. Our previous studies showed that Neu-P11 can regulate glucolipid metabolism and improve the insulin resistance in high sugar and high fat and cholesterol induced obesity rats. In vitro cell test also proved Neu-P11 can improve insulin resistance by inhibiting the triglyceride accumulation in 3T3-L1 fat cells. Reactive oxygen species mediated oxidative stress and mitochondrial dysfunction are considered as key factors leading to IR. Based on the link among Neu-P11, oxidative stress and insulin resistance, the hypothesis is proposed: whether Neu-P11 can improve the high glucose(25 mmol/L) and high insulin(1 μmol/L)-induced insulin resistance by inhibiting the oxidative stress in 3T3-L1 adipocytes.Methods(1) 3T3-L1 adipocytes culture and induction of differentiation: 3T3-L1 preadipocytes are induced by the classic “cocktail”(0.5 mmol/L IBMX,1 μmol/L DEX,10 μg/ml INS), and differentiation into mature 3T3-L1 adipocytes, and the Oil red O staining were used to observe fat cell morphology and 3T3-L1 pre-adipocytes differentiation.(2) Establish the insulin resistance model: the mature adipocytes were induced by high glucose and high insulin(HGI, 25 mmol/L and 1 μmol/L respectively) coculture for 24 h. To verify whether the model is established successfully, the uptake of glucose was measured by glucose oxidase method.(3) ROS production and MMP was detected by fluorescence probe and rhodamine 123, glucose consumption were evaluated via glucose oxidase-peroxidase respectively at the presence or absence of Neu-P11 or Mel in 3T3-L1 adipocytes.(4) Western Blot was applied to detect JNK/NF-κBp65 protein expression and these changes after Neu-P11 or Mel intervention.Results 3T3-L1 preadipocytes were induced by “cocktail” method for 10 days, oil red O staining found that more than 90% of cells presented a visible “signet ring-like” structure.cells were round, containing a large amount of lipid droplets, and arranged in annular arrangements; It is demonstrated that 3T3-L1 preadipocytes were differentiated into typical mature fatty cells. Our results have demonstrated that HGI incubating led to significant decreases in insulin-stimulated glucose uptake in 3T3-L1 adipocytes significantly, and successfully induced insulin resistance. In addition, the experimental results show that compared withcontrol group, ROS content of the IR group cells increased obviously, mitochondrial membrane potential decreased; the expression of JNK/NF-κBp65 protein increased; the expression of insulin signaling proteins Akt decreased. The Neu-P11 intervention can reverse the situation, it has similar effect as Mel.Conclusions Neu-P11 can improve insulin resistance in 3T3-L1 adipocytes which are induced by high-glucose and insulin, its mechanism may be related to removing ROS, improving mitochondrial function and inhibiting the expression of JNK/NF-κBp65.