DHA And EPA Participate In Myokine-mediated Browning Of White Adipose Tissue By Regulating Ca²⁺ Signal Pathways Under Insulin Resistance

PurposeInsulin resistance(IR)is a key pathological link to chronic non-communicable diseases(NCDs).Moykines produced by skeletal muscle have a prominent effect on improving IR by mediating browning of white adipose tissue(WAT).Exercise is the main inducement for stimulating the production and secretion of myokines.However,there are few studies on the effects and mechanisms of dietary factors on myokines.DHA and EPA have outstanding effects on improving IR and stimulating WAT browning,but whether they mediate the production and secretion of moykines remains unknown.This study aims to investigate the effects of DHA and EPA on Irisin,IL-6,FGF21,and IL-15 in skeletal muscle under IR conditions,and to clarify whether DHA and EPA regulate myogenic Irisin and IL-6 through the Ca2+ signaling pathway.Furthermore,clarify whether DHA and EPA mediate WAT browning and improve IR by regulating the expression and secretion of myokines.This study is expected to provide a theoretical and scientific basis for the rational use of DHA and EPA targeting moykines to prevent and treat the occurrence and development of IR-related NCDs.Method1?C57BL/6J mice were fed with high-fat diet for 16 weeks to establish an IR-mouse-model,then treated with 4%(w/w)composition ratios of DHA and EPA(DHA/EPA=3:1 or 1.5:1 or 1:1 or 1:1.5 or 1:3)to intervene IR-mice for 12 weeks.Measured the weight and fat pad content of mice;detected the levels of blood glucose and insulin,free fatty acids and blood lipids(TG,TC,LDL-c,HDL-c)of IR-mice;assessed glucose tolerance with oral glucose tolerance test(OGTT);calculated HOMA-IR,ISI and Lee's index;detected the key factors of insulin signaling pathway(IRS-1,p-AKT,AKT)in skeletal muscle by western blot;observed the morphological changes of inguinal white fat(iWAT)in mice by hematoxylin-eosin staining;detected UCP1 of iWAT by immunohistochemistry;detected the mRNA expression of browning marker genes and mykines(Irisin,FGF21,IL-6,IL-15)by qPCR;detected myokines content in serum of mice by ELISA.2?C2C12 myotubes were treated with 100-500?M palmitic acid(PA)for 6-24 h to establish IR-cell-model,and 100?M DHA and EPA mixture(DHA/EPA=3:1 or 1:1 or 1:3)was used to intervene IR-myotubes for 24 h.IR-myotubes were intervened by DHA/EPA with or without BAPTA-AM,STO-609 and Compoud C to block Ca2+signaling pathway.Detected the protein levels of IRS-1,p-AKT and AKT in myotubes and adipocytes,NFATcl and PGC-1? in skeletal muscle and myotubes by western blot;detected the mRNA expression of mykines by qPCR;detected myokines content of cell supernatant in myotubes by ELISA;detected the concentration of Ca2+ in myotubes by calcium ion fluorescence probe.3?3T3-L1 adipocytes were treated with 10ng/ml TNF-? for 48 h to establish an IR-cell-model,and IR-adipocytes were treated with cell supernatant from IR-myotubes for 24 h.Detected the mRNA expression of browning marker genes by qPCR.Results1?The weight,visceral fat pad content,Lee's index,TG,TC,HDL-c,free fatty acids,FBG,FINS and HOMA-IR of mice fed with high-fat diet increased,ISI decreased and glucose tolerance impaired,IRS-1 and p-AKT expression in skeletal muscle decreased.The visceral fat pad content(group 3:1 and 1:3)and body fat rate(group 1:3)of mice fed with DHA and EPA decreased,FBG(group 1.5:1),FFAs(group 3:1,1.5:1 and 1:3),HOMA-IR(except group 1:1)of mice decreased;iWAT had been beiged(group 3:1 and 1:3),and the expression of browning marker genes,such as UCP1,were up-regulated(the effect of group 3:1 and 1:3 was more prominent than others);the level of Irisin,IL-6,FGF21 and IL-15 increased in skeletal muscle(except group 1.5:1)and serum secretion of Irisin(group 3:1,group 1.5:1 and group 1:3),IL-6.(group 1,5:1 and group 1:3),FGF21(group 1:1,group 1.5:1 and group 1:3)and IL-15(group 1.5:1 and group 1:3)increased.2?The intervention with 500?M PA for 12 h had the best effect on reducing the sugar uptake capacity and down-regulating the expression of IRS-1 and p-AKT in myotube cells.The expression and secretion of Irisin,IL-6,FGF21 and IL-15 in IR-myotubes treated with DHA/EPA increased(the effect of group 3:1 and 1:3 was more prominent than others),the protein expression of NFATc1 and PGC-1? was up-regulated,and the expression and secretion of Irisin and IL-6 with inhibiting of Ca2+ signal decreased.3?The ability of sugar uptake and IRS-1 expression in 3T3-L1 adipocytes treated with TNF-? reduced.After treated with myotube cell supernatant,the level of browning marker genes in IR-adipocytes increased,of which expression was down-regulated after Ca2+signal transduction was deprived.Conclusion1?Mixed supplementation of DHA and EPA stimulates the expression and secretion of myogenic Irisin,IL-6,FGF21 and IL-15,mediates fat browning and improves IR in mice.2?DHA/EPA stimulates the expression and secretion of Irisin by regulating the Ca2+/CaMK signaling pathway,stimulates the expression and secretion of IL-6 by regulating the Ca2+/CaN signaling pathway in IR myotube cells and stimulates the browning of 3T3-L1 adipocytes.