Adipocyte-derived Exosomal MiR-27a Induces Insulin Resistance In Skeletal Muscle Through Repressing Of PPAR?

Obesity is an important pathological basis leading to insulin resistance in type 2 diabetes.Studies have shown that miR-27 a is highly expressed in sera of obese individuals with prediabetes and T2 DM.In patients with metabolic syndrome,sera mi R-27 a levels were positively correlated with fasting blood glucose and obesity.Adipose tissue is a major source of circulating exosomal microRNAs in the obese state,and a significant fraction of miRNAs existing in circulation is in the form of exosomes.MiR-27 a is highly expressed in adipose tissue,and was initially proposed as a negative regulator of adipogenic and lipogenic pathways by targeting PPAR?.Moreover,miR-27 a has been confirmed to be secreted in the form of exosome by MCF-7 cell.Skeletal muscle is the main effector organ of peripheral glucose uptake,and activated PPAR? heterodimerizes with RXR to maintain glucose homeostasis through direct regulation of genes harboring PPAR response elements,including the glucose transporter GLUT4 and the insulin receptor substrate IRS-1.Therefore,we hypothesize that increasingly exosomal serum miR-27 a derived from adipocytes could be taken up by skeletal muscle tissue,and induce insulin resistance in skeletal muscle in obese state.In this study,the association between miR-27 a and insulin resistance in skeletal muscle was determined in obese children,high-fat diet-induced miR-27 a knockdown obese mice,db/db mice and C2C12 cells overexpressing miR-27 a.The crosstalk mediated by exosomal miR-27 a between adipose tissue and skeletal muscle was determined in C2C12 cells incubated with conditioned medium prepared from palmitate-treated 3T3-L1 adipocytes with or without miR-27 a knockdown.The dissertation consists of three parts.The first part: to identify the key role of miR-27 a in obesity-induced insulin resistance.In this part,clinical sera of obese children were collected,and high-fat diet-induced miR-27 a knockdown obese mice,db/db mice model were established,and the levels of sera miR-27 a,blood glucose,insulin,glucose tolerance and insulin tolerance were detected.The expressions of IRS-1/Akt/GLUT4 in glucose uptake signaling pathway of skeletal muscle were determined.The results showed that sera mi R-27 a were positively correlated with BMI,fasting blood glucose and various pro-inflammatory cytokines including Visfatin,TNF-? and IL-6 in obese children sera;the levels of miR-27 a in sera and skeletal muscle high-fat diet-fed C57BL/6J mice increased progressively with the course of high-fat diet-fed,and were consistent with the abnormal levels of systemic insulin resistance,glucose tolerance and skeletal muscle insulin resistance.After knockdown miR-27 a in high-fat diet-fed C57BL/6J mice,impaired insulin resistance,glucose intolerance and insulin intolerance of skeletal muscle were partly restored.In high-fat diet-fed C57BL/6J mice,the expressions of IRS-1 and GLUT4 in glucose uptake signal pathway of skeletal muscle were significantly decreased,while the expressions of IRS-1 and GLUT4 were restored after mi R-27 a knockdown.The results cleared that mi R-27 a play a key role in obesity induced insulin resistance.The second part: to investigate the mechanism of miR-27 a in regulating skeletal muscle insulin resistance via targeting PPAR?.In this part,the expressions of PPAR? in high-fat diet-induced miR-27 a knockdown obese mice were examined,and C2C12 cells overexpressing miR-27 a in the absence or presence with rosiglitazone(PPAR? activator)were used to test glucose consumption,glucose uptake,and glucose uptake signaling pathways of skeletal muscle.The results showed that the expression of PPAR? in skeletal muscle of high-fat diet-fed C57BL/6J mice was significantly decreased,while was restored by mi R-27 a knockdown.In C2C12 cells overexpressing miR-27 a,the expression of PPAR? was significantly decreased,consisting with the decreased glucose consumption and glucose uptake,decreased mRNA expressions of IRS-1 and GLUT4,and the decreased activations of p-IRS-1 and p-Akt under insulin stimulation.After overexpressing miR-27 a with rosiglitazone in C2C12,PPAR? expression along with glucose consumption and glucose uptake,mRNA expressions of,IRS-1 and GLUT4,and the activations of p-IRS-1 and p-Akt stimulated by insulin were all significantly up-regulated compared with the miR-27 a overexpressing group.The above results indicate that miR-27 a level in skeletal muscle is elevated in obese state,and miR-27 a could induce insulin resistance in skeletal muscle cells,maybe mainly via targeting PPAR?.The third part: to investigate whether adipocyte-derived exosomal miR-27 a could induce insulin resistance in skeletal muscle in obesity.In this part,we first validated whether adipocytes could secrete exosomal miR-27 a.The expressions of miR-27 a in adipose tissues and palmitate-treated 3T3-L1 adipocytes were examined.The contents of FABP4,an adipocyte derived marker,were detected in sera of obese mice and supernatant of palmitate-treated adipocytes.Then exosomes from sera of obese mice and supernatant of palmitate-treated adipocytes were extracted and identified by TEM and CD63.At last,fluorescence co-localization experiments among FABP4,exosomes and miR-27 a from exosomes abovementioned were conducted.The results showed that the expression of miR-27 a in visceral adipose of high-fat diet-fed C57BL/6J mice was significantly decreased after 4w feeding.The levels of FABP4 in sera of high-fat diet-fed C57BL/6J mice and conditioned medium prepared from palmitate-treated 3T3-L1 adipocytes were significantly increased.The exosomes in the sera of high-fat diet-fed C57BL/6J mice were positive for FABP4,and exosomes from conditioned medium prepared from palmitate-treated 3T3-L1 adipocytes contained miR-27 a.The results suggested adipocytes could secrete exosomal miR-27 a.In this part,next,the levels of FABP4 and miR-27 a in skeletal muscle tissues of high-fat diet-fed C57BL/6J mice and C2C12 cells incubated with conditioned medium prepared from palmitate-treated 3T3-L1 adipocytes were detected.Then we used conditioned medium prepared from palmitate-treated 3T3-L1 adipocytes with or without miR-27 a knockdown to treat with C2C12 cells,and detected the miR-27 a expression,glucose consumption and glucose uptake,along with the expressions of PPAR?,IRS-1 and GLUT4 to investigate whether adipocyte-derived miR-27 a could induce insulin resistance in skeletal muscle.The results showed the contents of FABP4 in skeletal muscle were significantly increased.After incubation with conditioned medium prepared from palmitate-treated 3T3-L1 adipocytes,the contents of FABP4 and miR-27 a were increased,while glucose consumption and glucose uptake,along with the expressions of PPAR?,IRS-1 and GLUT4 in C2C12 cells were significantly decreased,and the activations of p-IRS-1 and p-Akt were significantly reduced by insulin stimulation,which all could be restored by miR-27 a knockdown in 3T3-L1.However,glucose consumption,glucose uptake and insulin-stimulated glucose uptake were unaltered in C2C12 cells incubated with conditioned medium prepared from palmitate-treated or untreated 3T3-L1 cells in which miR-27 a was knocked down.The results showed that adipocyte-derived exosomal miR-27 a induces insulin resistance in skeletal muscle.In the current study,we demonstrated that adipose-derived exosomal miR-27 a is taken up by skeletal muscle,and this results in impairment of insulin-dependent glucose uptake into skeletal muscle in the obesity-triggered insulin resistance state.The principal findings of our study are 1.MiR-27 a plays a key role in obesity-induced insulin resistance in skeletal muscle;2.Increasingly exosomal sera miR-27 a is secreted by lipid-loaded adipocytes in obese state,and is taken up by skeletal muscle cells;and 3.Increasing miR-27 a in skeletal muscle in obese state may be mainly uptaken from circulatory system,and adipose-derived mi R-27 a induces insulin resistance in skeletal muscle,maybe with the underlying mechanism via PPAR? repression.This experiment provides a new mechanism for the cross-talk between adipose tissue and skeletal muscle in obese state,and provides a new target for obesity treatment and drug intervention.