| Objective:(1)To observe the effects of high intensity interval training on glucose homeostasis of T2DM mice.(2)To identify the expression of lncRNAs in improving insulin resistance in T2DM mice by transcriptome sequencing and preliminarily study the possible mechanism of the lncRNA which involved in the regulation of improving insulin resistance by HIIT.(3)The function of the lncRNAs was studied in vitro and further determine its mechanismMethods:Part OneEffect of high intensity interval training on glucose homeostasis in type 2 diabetic mice.Six-week-old male C57BL/6J mice were randomly divided into normal diet group(CON group)and high-fat diet group(HFD group).HFD group mice were fed with a high-fat diet for 12 weeks and injected with STZ to induce type 2 diabetic mice Then fasting blood glucose(FBG),glucose tolerance(IPGTT)and insulin tolerance(ITT)were detected,and the mice with a fasting blood glucose concentration>13.8 mmol/l were considered diabetic mice.Then the T2DM mice were randomly assigned to two groups:a T2DM group without exercise(T2DM-SED,n=11)and a T2DM group with high-intensity interval training(T2DM-HIIT group,n=11).All mice in the T2DM-HIIT group performed the exercise training program on a mouse treadmill at 25° inclination five times a week for 8 weeks,the mice in the T2DM-HIIT group started with a warm-up at 5 m/min for 10min,in which the HIIT consisted of 10 rounds of 4 min of high-intensity treadmill running interspersed with 2min of complete rest.The pace during the HIIT increased gradually from 16 to 26 m/min over 8 weeks.The fasting blood glucose,glucose tolerance,insulin tolerance and body composition were measured at the end of the exercise.Then killed the mice and collected the blood and quadriceps.The levels of TC,TG,HHDL-C,LDL-C and serum insulin(FINS)were detected.Pathological sections were made to observe the content of lipid and glycogen in skeletal muscle.QRT-PCR and Western blot were used to detect the expression of glucose metabolism-related indicators(m-glut4,GS),lipid metabolism-related indicators(HMGCR,SCD1,CD36,CPT-1α),and mitochondrial biosynthesis and dynamics related indicators(mtDNA、PGC-1α、MFN2、FIS1、DRP1).Part TwoEffects of high intensity interval training on the expression of lncRNA and their downstream pathways in skeletal muscle of T2DM mice.Total RNA was extracted from the quadriceps of mice,and lncRNAs transcriptomics of quadriceps in T2DM-SED and T2DM-HIIT mice were sequenced by high-throughput sequencing technology.The data was analyzed by edger in R studio and Edger.The fold change≥2.0 with p<0.05 and an FPKM value≥ 0.1 in at least one sample from a group indicated differentially expressed lncRNAs.The expression of differentially expressed lncRNAs was verified by qRT-PCR and identified the key lncRNA and its target gene to be studied.The mRNA and protein expression levels of the target genes were detected by qRT-PCR and Western blot.Part ThreeRole and mechanism of lncRNA H19/miR-181a-5p axis in glucose and lipid metabolism of C2C12 cells.(1)The myotube cells were randomly divided into the CON group and PA group.The PA group was treated with 500μM palmitic acid,while the CON group was treated with 20%BSA for 16 hours.(2)Myotube cells were randomly divided into two groups and transfected with si-NC and si-H19 or empty plasmid vector and H19 overexpression plasmid or NC and miR-181a-5 mimic or inhibitor-NC and miR-181a-5p-inhibitor respectively.After 6 hours,the medium was changed.(3)The myotube cells were randomly divided into four groups and transfected with si-NC+inhibitor-NC,si-NC+inhibitor,si-H19+inhibitor-NC and si-H19+inhibitor respectively.The medium was changed after 6 hours transfected.The supernatant and cells were collected,and the cells were stained with Oil Red O and PAS,QRT-PCR and Western blot were used to detect the expression of H19,miR-181a-5p,PPARα,glycolipid metabolism-related indicators and mitochondrial biosynthesis and dynamics related indicators.Results:Part One1.Establishment of the T2DM mice.The FBG,ipgtt and ITT were damaged in T2DM group(p<0.01)after high fat diet and STZ injection,when compared with the CON group 2.The changes of glucose and lipid metabolism and body composition in T2DMmice after HIIT.(1)The body weight and body fat rate of T2DM-HIIT group were significantly decreased,and the percentage of lean body weight was significantly increased after HIIT(p<0.01).(2)The level of FBG(p<0.05),FINS and TG in T2DM-HIIT group were significantly decreased(p<0.01),and the IPGTT and ITT were improved after HIIT(p<0.05),but the level of TC,HDL-C and LDL-C in serum were not significantly changed after HIIT(p>0.05)(3)There were a small amount of orange lipid droplets and a large amount of glycogen in skeletal muscle of CON group,and a large number of orange lipid droplets and a small amount of glycogen in skeletal muscle of T2DM-SED group After HIIT,orange lipid droplets were decreased,and glycogen content was increased in skeletal muscle of T2DM-HIIT group3.The changes of glucose and lipid metabolism-related indexes in skeletal muscle after HIIT.Compared with CON group,the expression of m-GLUT4 and FATP1 in skeletal muscle of T2DM-HIIT mice were significantly decreased(p<0.05),and the phosphorylation levels of GS,HMGCR and SCD1 were significantly increased(p<0.01).The expression of m-GLUT4,CD36,CPT-1α and FATP1 were increased(p<0.05),and the phosphorylation level of GS,HMGCR and SCD1 were decreased after HIIT(p<0.05)4.The changes of mitochondrial biosynthesis and mitochondrial dynamic related indexes in skeletal muscle after HIIT.(1)Compared with CON group,the morphology of mitochondria in T2DM-SED group was more vacuolar,the cristae of mitochondria were destroyed or even completely disappeared,and the number of mitochondria decreased(p<0.01).After HIIT,the morphology of mitochondria in T2DM-HIIT group was partially restored,and the number of mitochondria increased(p<0.05)(2)Compared with CON group,the protein expression of mtDNA,PGC-1 α,Mfn2(p<0.01),FIS1,drp1(p<0.05)in skeletal muscle of T2DM-SED group were significantly decreased.After HIIT,the expression of these factors in skeletal muscle of T2DM-HIIT group were significantly increasedPart Two1.Transcriptome sequencing results.After HIIT,47 lncRNAs were up-regulated and 45 lncRNAs were down-regulated in T2DM-HIIT group,and H19 was located in the second position of significantly up-regulated expression2.Results of qRT-PCR.Compared with CON group,the expression of H19 in skeletal muscle of T2DM-SED group was significantly decreased,and the expression of H19 was significantly increased after HIIT3.Detection of H19 target genemiR-181a-5p,which were the target gene of H19 was significantly up-regulated when compared with CON group.PPARα was the target gene of miR-181a-5p,which was significantly down-regulated in T2DM-SED group(p<0.01).The expression of miR-181a-5p was significantly down-regulated and the expression of PPARα was significantly up-regulated after HIIT(p<0.01)Part Three1.Establishment of insulin resistance model in C2C12 cells.After PA intervention,the glucose uptake of C2C12 cells was decreased,the insulin signaling pathway was damaged,and the lipid deposition was increased,which indicated that the establishment of IR model of C2C12 cells was successful2.Expression of H19/miR-181a-5p axis in C2C12 cells with IRThe expression of H19 and PPARα were significantly down-regulated(p<0.05),and the expression of miR-181a-5p were significantly up-regulated(p<0.01)3.The effect of H19 inhibition/overexpression on its target gene.Compared with the si-NC group,the expression of miR-181a-5p was up-regulated(p<0.01)while the expression of PPARα was down-regulated in the si-H19 group(p<0.05).If H19 was overexpressed,the expression of miR-181a-5p was down-regulated and PPARα was up-regulated4.The effect of inhibition/overexpression of H19 on the expression of glucose and lipid metabolism-related indicators in C2C12 cells.Compared with the si-NC group,the lipid deposition was increased in the si-H19 group,while the glycogen content and glucose uptake were decreased in the si-H19 group.The expression of m-GLUT4(p<0.01),CD36,CPT-1α and FATP1 were significantly decreased(p<0.05).The phosphorylation of GS and the mRNA and protein expression of HMGCR were significantly higher than si-NC group(p<0.01),but the mRNA expression of SCD1 was not significantly changed(p>0.05)Overexpression of H19 has an opposite effect on the above factors5.The effect of inhibition/overexpression of H19 on the expression of mitochondrial biosynthesis and dynamics related indicators in each group.Compared with the si-NC group,the mtDNA of the si-H19 group was significantly decreased,the protein expression of PGC-1α,FIS 1,Mfn2 and DRP1 were also significantly decreased(p<0.05),while the expression of mitochondrial biosynthesis and dynamics related indexes were significantly upregulated after H19 overexpression.6.Effect of miR-181a-5p on the expression of its target geneThe gene and protein expression of PPARα were declined after miR-181a-5p mimic transfection(p<0.05),while the gene and protein expression of PPARα were increased after miR-181a-5p inhibitor transfection(p<0.05),but it has no significant effect on the expression of H19(p>0.05).7.The effect of inhibition/overexpression of miR-181a-5p on glucose and lipid metabolism in C2C12 cells.Compared with the NC group,the lipid content in the miR-181a-5p mimic group were increased,while the glycogen content and glucose uptake in the miR-181a-5p mimic group were decreased.The protein expression of m-GLUT4 and the gene and protein expression of CD36 and CPT-1α were decreased when compared with the NC group(p<0.05).The phosphorylation of GS and the expression of HMGCR were increased(p<0.01),while the gene expression of SCD1 and FATP1 were not affected after miR-181a-5p mimic transfection(p>0.05).If the expression of miR-181 a-5p was inhibited,the indexes related to glucose and lipid metabolism had opposite changes.8.The effect of inhibition/overexpression of miR-181a-5p on the expression of mitochondrial biosynthesis and dynamics related indexes in C2C12 cells.Compared with the NC group,the protein expression of PGC-1α and mtDNA were decreased in miR-181a-5p mimic group(p<0.01),and the protein expression of FIS1,Mfn2 and DRP1 were also decreased(p<0.05).If the expression of miR-181a-5p was inhibited,the expression of mitochondrial biosynthesis and dynamics related factors were significantly increased9.Effect of co-transfection of si-H19 and miR-181a-5p inhibitor on PPARαexpression.Compared with si-NC+inhibitor NC group,the gene and protein expression of PPARα were significantly decreased in si-H19+inhibitor NC group(p<0.05)Knockdown the expression of H19 and miR-181a-5p at the same time,and the gene and protein expression of PPARα were restored(p<0.05)10.The effects of co-transfection of si-H19 and miR-181a-5p inhibitor on the expression of glucose and lipid metabolism related indexes in C2C12 cells.Knockdown H19 caused lipid droplet content was increased,and the glycogen content and glucose uptake were decreased in C2C12 cells.It also decreased the protein expression of m-GLUT4,CPT-1α and CD36(p<0.05).Knockdown H19 also increased the protein expression of HMGCR and phosphorylation of GS in C2C12 cells(p<0.05).At the same time,interference with miR-181a-5p expression,which reduced lipid droplet content,increased glycogen content and glucose uptake,and also increased protein expression of GLUT4 and CPT-1α(p<0.05).The mRNA and protein expression of HMGCR and the phosphorylation of GS were also significantly decreased(p<0.01),but the expression of SCD1,CD36 and FATP1 were not significantly changed11.Effects of CO transfection of si-H19 and miR-181a-5p inhibitor on mitochondrial biosynthesis and dynamics.Compared with siNC+inhibitor NC group,the copy number of mtDNA(p<0.01)and the protein expression of PGC-1α,FIS1 and DRP1 were significantly decreased in si-H19+inhibitor group(p<0.05).When compared with si-H19+inhibitor NC group,the copy number of mtDNA and the protein expression of PGC-1α were significantly increased,while the protein expression of MFN2、DRP1 and FIS1 were not significantly changedConclusion:High intensity interval training improves insulin resistance in type 2 diabetic mice by upregulating the expression of H19 and negatively regulating the expression of miR-181a-5p to promote the expression of PPARα. |
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