| As a common chronic disease in the world,type 2 diabetes has seriously affected the quality of life of patients.The level of free fatty acids in plasma of patients with type 2 diabetes is higher than that of normal people.And the accumulation of free fatty acids has an adverse effect on the function of islet β cells.Free saturated fatty acids,in particular,not only cause a variety of stress responses in the body,but also induce the secretion of cytokines,thus playing an important role in insulin resistance.Skeletal muscle is an important organ involved in energy metabolism,responsible for 70-80% of the body’s insulin-stimulated glucose uptake.In addition,it acts as an endocrine organ to produce and secrete a variety of cytokines including myogenic factors.Musclin and irisin are two important myogenic factors which play an important role in glycolipid metabolism.Mu sclin can block glucose uptake and glycogen synthesis by muscle cells,while irisin can act on white adipocytes and activate uncoupling protein-1(UCP-1)to achieve the "Browning" of white fat.At present,there are few studies on the regulation mechanism of saturated fatty acids on myosin and irisin.In this study,we analyzed the expression changes of musclin and irisin in response to high-fat diet and palmitate.Moreover,the regulatory pathways and factors involved in their expression were predicted by RNA sequencing and bioinformatics and were tested by a series of experiments.Firstly,high-fat diet rich in saturated fatty acids or palmitate was used to treat male ICR mice aged eight-week-old to construct diabetic mouse model.And the establishment was recorded through tracking diet,weight and blood glucose.Blood glucose test results showed that high-fat diet and palmitate could cause the increase of blood glucose in mice after treatment for about 6 weeks.High-fat diet and palmitate could both induce obvious insulin resistance in mice and cause shallow staining of the lymph follicle center in the spleen and slight atrophy of the glomerular in the kidney.High-fat diet and palmitate could also cause the significant difference of musclin and irisin expression.Secondly,this study analyzed the pathways involved in the expression regulation of musclin and irisin in response to high-fat and palmitate through RNA sequencing.The analysis results showed that saturated fatty acids could cause the activation of ER stress,TNF,PI3K-Akt and TGFβ signaling pathways.Meanwhile,through ENCODE/Caltech,Gene Transcription Regulation Database(GTRD)and Find Individual Motif Occurences(FIMO),this study identified the regulatory factors related to musclin and irisin expression.The results showed that C/EBPβ and Smad3 bound to the promoter region of musclin and irisin,respectively.Finally,this study performed cell and molecular biology experimental analysis on the pathways and regulatory factors related to musclin and irisin.By blocking the signaling pathway,interference or overexpression of related regulatory factors,it was found that C/EBPβ played an important regulatory role in the high expression of palmitate-induced musclin gene.Ch IP results showed that C/EBPβ could directly bind to the promoter of musclin gene for positive regulation,which was directed by the PERK/ATF4 pathway in response to palmitate.In addition,Smad3 played a negative role in the process of palmitate inhibiting irisin expression.Palmitate could weaken the insulin signaling pathway through the down-regulation of irisin mediated by Smad3.Taken together,palmitate regulated transcription factor C/EBPβ through activating PERK/ATF4 signaling pathway,thereby further affecting the expressi on of musclin.Palmitate inhibited irisin expression by affecting the expression of Smad3 instead of activating TGFβ signaling pathway,and the down-regulation of irisin mediated by Smad3 in response to palmitate could weaken insulin signaling pathway.These results provided vital theoretical basis for prevention and treatment of insulin resistance and type 2 diabetes. |
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