Neu-p11Ameliorates Insulin Resistance By Enhancing ATGL/HSL And Underlying Mechanisms

Background and purposeNeu-p11, which was developed by Neurim Pharmaceuticals Ltd, not only was amelatonic agonist, but also had the same structure and biological function as melatonin.Both of them could inhibit the triglyceride accumulation. Increased triglyceride intent inadipose tissue was associated with the relative diseases, such as obesity and insulinresistance. HSL was discovered more than40years ago, and recently ATGL was identifiedto be predominantly responsible for the TG breakdown. ATGL and HSL, the majorenzymes, contributed to hydrolyze more than95%TG in murine WAT. Therefore, theprimary aim of this present study is to examine the protein level of ATGL and HSL in3T3-L1adipocytes induced by high glucose and high insulin, the changes of these proteinsafter Neu-p11intervention, and to elucidate the role of ATGL and HSL in the mechanismof insulin resistance.Methods(1) Cell culture and induction of differentiation: the classic "cocktail" induced thedifferentiation of3T3-L1preadipocytes into mature3T3-L1adipocytes, and then theywere stained by Oil red O in order to observe fat cell morphology and3T3-L1preadipocytes differentiation.(2) Insulin resistance model: incubating mature adipocyteswith high glucose and insulin in combination for24hours resulted in insulin resistance.Glucose consumption was detected via glucose oxidase to verify whether the model isestablished successfully.(3) Intracellular triglyceride levels and glucose consumption wereevaluated via triglycerides assay kit and glucose oxidase-peroxidase (GOD-POD)respectively at the presence or absence of Neu-p11/Luzindole in3T3-L1adipocytes.(4)Western Blot was applied to detect ATGL and HSL protein expression and these changes after Neu-p11intervention at the presence of Luzindole or not.ResultsMouse3T3-L1preadipocytes were induced by "cocktail" method for10days, morethan90%cells presented a visible “signet ring-like” appearance by Oil red O staining.Cells were round, containing a large number of lipid droplets which were arranged inannular arrangements; all of these demonstrated that3T3-L1preadipocytes weredifferentiated into typical mature fatty cells. Our results have demonstrated that thecombination with high glucose and insulin in3T3-L1adipocytes significantly decreasedcellular glucose uptake, and successfully induced insulin resistance. Our experimentalresults also showed that intracellular TG content in HGI group was significantly higherthan the Con group, while compared with the Con group, concomitant with ATGL and HSLprotein down regulation. The Neu-p11intervention can significantly reduce TG content inthe insulin-resistant3T3-L1adipocytes, promotes cell uptake of glucose and upregulatesthe levels of ATGL and HSL protein expression higher in insulin resistant3T3-L1adipocytes, it has similar effect as Mel. Luzindole (LZD), the MT2competitive antagonist,significantly inhibited the above-mentioned effects of Neu-p11, and the differences werestatistically significant.ConclusionsNeu-p11ameliorates insulin resistance by inhibiting triglyceride accumulation viaMT2receptor-dependent mechanism in3T3-L1adipocytes which are induced byhigh-glucose and insulin. ATGL and HSL may participate in this process.