Research On The Mechanism Of MiR-15b Reducing Insulin Sensitivity Of Adipocytes

Objective: Adipose insulin sensitivity has an important effect on systemic insulin sensitivity.Previous studies have shown that the level of miR-15 b in serum of obese patients and patients with fatty liver is significantly increased,and adipocytes are the main source of circulating miRNA.However,the role of miR-15 b in adipocyte insulin sensitivity has not been studied.In this study,we will explore whether miR-15 b is involved in adipose insulin resistance and explore its potential molecular mechanism.Methods: The obese mouse model was constructed by feeding high-fat diet(HFD).The insulin-resistant 3T3-L1 adipocyte model was constructed by hyperinsulin treatment.The expression of miR-15 b was detected by q RT-PCR.Target Scan was used to predict the potential target genes of miR-15 b.Western Blot was used to determine the protein levels of INSR,IRS-1 and AKT as well as their phosphorylation levels.miR-15 b minics/inhibitors and p CMV3-INSR-Flag plasmids were transiently transfected with transfection reagent.The glucose uptake capacity of cells was examined by the 2-NBDG uptake assay.Results: 1.The expression level of miR-15 b was elevated and the protein expression of INSR was decreased both in adipose tissue of obese mice and in insulin-resistant adipocytes.And the INSR tyrosine phosphorylation and AKT serine phosphorylation levels also decreased significantly after insulin stimulation.2.Bioinformatics analysis suggested that INSR,the key gene for insulin signaling,was a potential target gene of miR-15 b.3.Overexpression of miR-15 b in adipocytes resulted in reduced INSR protein expression,decreased phosphorylation levels of INSR,IRS-1 and AKT,as well as inhibited glucose uptake stimulated by insulin.4.Compared with controls,inhibition of miR-15 b expression in normal or insulin-resistant adipocytes significantly increased the protein expression level of INSR,improved the tyrosine phosphorylation of INSR and IRS-1 and serine phosphorylation of AKT under insulin stimulation,and promoted glucose uptake.5.Overexpression of INSR partially rescued the impaired insulin signaling and glucose uptake caused by overexpression of miR-15 b.Conclusion: Collectively,under the pathological conditions of obesity and insulin resistance,the expression of miR-15 b in adipocytes was elevated,while the expression of INSR protein was declined.Besides,INSR was the downstream target gene of miR-15 b and was negatively regulated by miR-15 b.miR-15 b may mediate obesity-related insulin resistance by suppressing INSR to inhibit insulin signaling in adipocytes.