| Diabetes is a metabolic disease mainly characterized by hyperglycemia,and its pathogenesis is closely related to insulin resistance and pancreatic islet dysfunction.Insulin resistance(IR)is an important driving factor in the development of type 2 diabetes(T2DM)and its complications.As of 2021,Type 2 diabetic mellitus(T2DM)accounts for more than 96%of global diabetes cases.Sustained high blood sugar levels can cause damage to blood vessels throughout the body and can cause a series of complications such as diabetic nephropathy and diabetic retinopathy,seriously threatening patients’ health and reducing their quality of life.Therefore,the prevention,control and treatment of T2DM are urgent,and effective intervention strategies are urgently needed to prevent the increase in the number of diabetic patients.Studies have shown that T2DM is characterized by chronic low-grade inflammation and metabolic dysfunction(sub-inflammation).The inflammatory response causes T2DM by aggravating insulin resistance.The pathological process is accelerated during hyperglycemia,leading to diabetes-related complications.The above discussion shows that the development of inflammation and the improvement of insulin function may be important entry points for the prevention and treatment of diabetes.Research shows that regular exercise can effectively regulate abnormal glucose and lipid metabolism and insulin resistance in patients with T2DM,reduce the content of visceral adipose tissue,improve body composition and quality of life,and has significant effects in controlling blood sugar,reducing cardiovascular risk factors and death risks,and is a preventive effective intervention method for T2DM and its health management.Previous studies by the research team have shown that moderate-intensity exercise may improve T2DM by regulating the levels of exosomes derived from macrophages in adipose tissue.However,the specific mechanism by which exercise improves insulin sensitivity in T2DM through adipose tissue is not yet fully understood.Based on the above background,we intend to use exercise-induced macrophagederived exosomes as a starting point to explore its mechanism of action in improving insulin sensitivity in T2DM through exercise,in order to discover the effective targets of exercise in preventing and treating T2DM,and to provide experimental basis and clinical reference for scientific exercise in preventing and treating type 2 diabetes.Research 1:Exercise regulates macrophage polarization to improve insulin resistance in adipose tissue of T2DM micePurpose:To explore the effects of different-intensity treadmill exercise on improving adipose tissue inflammation,insulin sensitivity and lipid metabolism in T2DM mice,and to analyze the possible mechanisms of different-intensity exercise in improving T2DM.Methods:Thirty-two 8-week-old SPF-grade spontaneously obese type 2 diabetes male db/db(BSK.Cg-Dock7m+/+Leprdb/JNju)mice were randomly divided into type 2 diabetes group(M group),low-intensity treadmill exercise group(LE group),moderate-intensity treadmill exercise group(ME group),and high-intensity treadmill exercise group(HE group),with 8 mice in each group.Eight non-diabetic SPF-grade db/m mice of the same strain were used as the normal control group(NC group).The LE group,ME group,and HE group first performed treadmill exercise training with increasing intensity for one week,followed by 8 weeks of treadmill exercise training,5 times a week,30 minutes each time.During the 8-week intervention period,body weight,water intake,food intake,and fasting blood glucose were measured.After the intervention,the changes in IPGTT and IPITT of mice in each group were detected,and the weights of white adipose tissue and brown adipose tissue of mice in each group were weighed after sampling.PCR experiments were used to detect the gene expression levels of F4/80,Argl,iNOS,TNF-α,IL-6,IL-1β,MCP1,IL-10,PPARy,GLUT4,ADPN,UCP1,and IRS1 in white adipose tissue,and Western blot was used to detect the protein expression levels of Argl,iNOS,PPARy,GLUT4,ADPN,UCP1,TNF-α,IL-6,and IL-1β in white adipose tissue.HE staining was used to detect the morphological changes of adipocytes in white adipose tissue,and immunofluorescence was used to detect the proportion of M1 and M2 macrophages in white adipose tissue of each group.Results:After 8 weeks of intervention,compared with the M group,the body weight and fasting blood glucose of the LE group,ME group,and HE group was significantly reduced(P<0.01);compared with the LE group,the ME group’s body weight was significantly reduced(P<0.01).Compared with the M group,the average food intake and average water consumption of the ME group decreased significantly from the 2nd to the 8th week(P<0.05 or P<0.01).As the intervention time prolongs,the average daily food intake and average water consumption of the LE group,ME group and HE group generally shows a downward trend,and the ME group has the most significant effect.The results of the area under the IPGTT curve of mice in each group showed that compared with the M group,the area under the IPGTT curve of the ME group and the HE group decreased significantly(P<0.01).The results of the area under the IPITT curve of mice in each group showed that compared with the NC group,the area under the IPITT curve of mice in each group increased significantly(P<0.01).Compared with group M,the area under the IPITT curve of group LE,group ME and group HE was significantly decreased(P<0.01).The proportion of total macrophages in white adipose tissue was detected by flow cytometry.Compared with the NC group,the proportion of total macrophages in the M group was significantly increased(P<0.01),indicating that there is a large amount of inflammation in the body.Compared with group M,the proportion of total macrophages in group ME was significantly reduced(P<0.01).Elisa and biochemical results demonstrated that compared with the M group,the TC and LDL-C levels of the LE group,ME group,and HE group were significantly decreased(P<0.05 or P<0.01),and the TG level of the ME group was significantly decreased(P<0.05).The level of HDL-C increased significantly(P<0.05),the level of LDL-C in the HE group decreased significantly(P<0.01),and the level of HDL-C increased significantly(P<0.05).Compared with the M group,the serum free fatty acid(NEFA)level of the ME group was significantly lower(P<0.01).Compared with group M,the level of FINS in group ME was significantly lower(P<0.01).In addition,compared with the model group,HOMA-IR in the LE group,ME group and HE group was significantly reduced(P<0.01).Immunofluorescence results showed that compared with the M group,the number of CD206-positive cells in the ME group and HE group increased significantly(P<0.01).The results suggest that the ratio of M2-type anti-inflammatory macrophages in white adipose tissue decreased in group M,and treadmill exercise intervention of different intensities can increase the ratio of M2-type anti-inflammatory macrophages in white adipose tissue and inhibit the ratio of M2-type anti-inflammatory macrophages in white adipose tissue.inflammation,with moderate intensity showing the most significant improvement.The PCR results indicated that compared with the M group,the mRNA expression levels of F4/80,iNOS,TNF-α,and MCP1 in the LE group were significantly reduced(P<0.05 or P<0.01),and the ME group’s F4/80,iNOS,and TNFα,IL-6,IL-1β,and MCP1 significantly decreased(P<0.01),and the mRNA expression levels of Argl,PPARy,GLUT4,ADPN,UCP1,IL-10,IRS1 significantly increased(P<0.05 or P<0.01),the mRNA expression levels of F4/80,iNOS,TNF-α,IL-6,and MCP1 in the HE group were significantly reduced(P<0.05 or P<0.01),and the mRNA expression levels of Argl,PPARy,UCP1,and IL-10 in the HE group were significantly reduced(P<0.05 or P<0.01).levels increased significantly(P<0.01).WB results showed that compared with the M group,the protein expression level of IL-1β in the LE group was significantly reduced(P<0.01),and the protein expression levels of iNOS,TNF-α,IL-6,and IL-1β in the ME group were significantly reduced(P<0.05 or P<0.01),the protein expression levels of Argl,PPARy,GLUT4,ADPN,and UCP1 increased significantly(P<0.05 or P<0.01),and the protein expression levels of TNF-α,IL-6,and The levels of IL-1β in the HE group decreased significantly(P<0.05 or P<0.01).Research 2:The mechanism of action of exercise-induced adipose tissue macrophage exosomes in improving insulin sensitivity in T2DM mice Purpose:To explore the specific targets of adipose tissue macrophage-derived exosomes induced by different intensities of treadmill exercise in improving insulin sensitivity in adipose tissue of T2DM mice,and to analyze its possible pathways and mechanisms.Methods:Take mouse epididymal fat,rinse with saline,cut into pieces,grind and transfer to a homogenizer tube.Digest with collagenase II in a 37℃ constant temperature incubator for 15 minutes,then centrifuge for 10 minutes to remove the upper layer of fat and primary adipocytes on the top layer of the supernatant.Then add 500μL 0.1%type I collagenase,mix well by blowing,and digest in a 37℃ water bath.Incubate the suspension containing adipose tissue macrophages with red blood cell lysis buffer,add 10μL F4/80 magnetic bead antibody,incubate at 2-8℃ in the dark for 15 minutes,add 500μL PBS,and centrifuge for 10 minutes.Place the MACS sorting column on the magnetic bead sorter.After the magnetic bead sorting of the cell suspension was completed,the macrophage suspension that had been bound to the F4/80 magnetic bead antibody was collected and transferred to a 37℃ carbon dioxide incubator for culture.Subsequently,the exosome particle size was detected by NTA,the positive markers on the surface of the exosome were detected by Western blot,the differential miRNA in the exosomes of each group was detected by high-throughput sequencing,the miR-10a-5p content in the exosomes and adipose tissue was verified by PCR,and the dual luciferase experiment was used to detect the targeted binding of miR-10a-5p to ATF2.Results:Comprehensive electron microscopy,NTA,and WB results proved that the exosomes extracted were derived from adipose tissue macrophages.The results of highthroughput sequencing of miRNA showed that through PCR verification,we found that miRNA-10a-5p was the most significantly different miRNA and the trend was consistent with the sequencing results.miRNA heat map analysis found that compared with the M group,miR-10a-5p was significantly up-regulated in the ME group.The PCR verification results showed that compared with the M group,the expression of miR-10a-5p in the ME group and HE group was significantly increased(P<0.05 or P<0.01).Correlation analysis results showed that the expression level of exosome miR10a-5p was highly correlated with the expression change of adipose miR-10a-5p(r=0.7383,P<0.01).We performed pathway enrichment analysis on the target genes of miR-10a-5p of ATM-Exos.The KEGG enrichment results showed that 7 pathways were significantly enriched,including the PI3K-AKT signaling pathway and the synthesis and secretion of parathyroid hormone.And role,insulin secretion,glucagon signaling pathway,aldosterone synthesis and secretion,thyroid hormone synthesis,cortisol synthesis and secretion,comprehensive enrichment results ranking and significance results found that the PI3K-AKT signaling pathway has the highest relevance to research.GO enrichment results show that biological processes include pathways such as chromatin remodeling,skeletal system morphogenesis,regulation of neuroapoptotic processes,organ growth,liver development,JNK cascade,and hepatobiliary system development.Dual-luciferase results showed that the miR-10a-5p of TM-Exos can bind to the ATF2 sequence.Next,compared with the WT mmu-miR10a-5p mimic NC group,the gene expression level of WT miR-10a-5p mimic was significantly reduced(P<0.01).The above results prove that miR-10a-5p can specifically target and bind ATF2.Research 3:Regulation of exercise-induced adipose tissue macrophage exosomes on insulin resistance in 3T3-L1 adipocytesPurpose:To explore the improvement effect of adipose tissue macrophage-derived exosomes miR-10a-5p on adipocyte insulin resistance induced by treadmill exercise at different intensities,and to analyze its possible targets,pathways and mechanisms.Methods:3T3-L1 preadipocytes were resuscitated and passaged,and then induced to differentiate with 3-isobutyl-1-methylxanthine(IBMX),dexamethasone(DEX)and insulin,and the induced cells were identified by Oil Red O staining.Dexamethasone was used to establish an IR model and validate the model.CCK-8 method was used to detect cell proliferation,Transwell method was used to detect cell migration,GODPOD method was used to detect glucose consumption,and PCR method was used to detect the gene expression levels of cell miR-10a-5p and target gene ATF2,PI3K and AKT,macrophage-related indicators F4/80,iNOS and inflammatory factor-related indicators TNF-α,IL-6,IL-1β,IL-10,MCP1,and insulin sensitivity-related indicators PPARy,GLUT4,ADPN,UCP1,IRS1.Western blot method was used to detect the protein expression levels of ATF2,P-ATF2,PI3K,AKT and their phosphorylation,the protein expression levels of macrophage-related indexes F4/80,iNOS and inflammatory factor-related indexes TNF-α,IL-6,IL-1β,IL-10,MCP1,and the protein expression levels of insulin sensitivity-related indexes PPARy,GLUT4,ADPN,UCP1,and IRS1.Results:Compared with the M group,the cell activity of the LE-Exos group,ME-Exos group and HE-Exos group was significantly reduced(P<0.05 or P<0.01).Compared with the M group,the glucose consumption of the ME-Exos group and HE-Exos group was significantly increased(P<0.05 or P<0.01).Compared with the M group,the TG content of the ME-Exos group was significantly lower(P<0.01),while the TG content of the ME-Exos group was significantly lower than that of the LE-Exos group and HEExos group(P<0.05).Compared with the M group,the INS content in the cell supernatant of the ME-Exos group was significantly increased(P<0.05).PCR results showed that compared with the M group,the mRNA expression of miR-10a-5p in the ME-Exos group and HE-Exos group was significantly increased(P<0.01).Compared with the M group,the mRNA expression levels of ATF2,TNF-α,IL-6,IL-1β,and MCP1 in the LE-Exos group were significantly decreased(P<0.01),and the mRNA expression levels of Argl,PI3K,PPARy,GLUT4,ADPN,and IL-10,IRS1 were significantly increased(P<0.05 or P<0.01),and the mRNA expression levels of F4/80,iNOS,ATF2,TNF-α,IL-6,IL-1β,and MCP1 in the ME-Exos group were all significantly increased(P<0.05 or P<0.01).Significantly decreased(P<0.01),and the mRNA expression levels of Argl,PI3K,AKT,PPARy,GLUT4,ADPN,UCP1,and IL-10 were significantly increased(P<0.01).In the HE-Exos group,F4/80,iNOS,ATF2,TNF-α,IL-6,IL-1β,and MCP1 mRNA expression levels were significantly reduced(P<0.05 or P<0.01),while those of Argl,PI3K,PPARy,GLUT4,ADPN,UCP1,and IL-10 The mRNA expression levels increased significantly(P<0.05 or P<0.01).WB results showed that compared with the M group,the protein expression level of P-ATF2/ATF2 in the LE-Exos group was significantly reduced(P<0.01),and the protein expression levels of iNOS and P-ATF2/ATF2 in the ME-Exos group were significantly reduced.(P<0.05 orP<0.01),the protein expression levels of Argl,PPI3K/PI3K,P-AKT/AKT,PPARy,GLUT4,ADPN,and UCP1 all increased significantly(P<0.05 or P<0.01),HE-The protein expression levels of P-ATF2/ATF2 in the Exos group were significantly decreased(P<0.05 or P<0.01).Research 4:Regulatory effect of miR-10a-5p on insulin resistance in 3T3-L1 adipocytesPurpose:To explore the improving effect of miR-10a-5p on insulin resistance in adipocytes,and to analyze its possible targets,pathways and mechanisms.Methods:3T3-L1 adipocytes were divided into control group(NC group),IR model group(M group),IR model group+miR-10a-5p inhibitor group(inhibitor group),IR model group+miR-10a-5p inhibitor NC group(inhibitor NC group),IR model group+miR-10a-5p mimic group(mimic group),IR model group+miR-10a-5p mimic NC group(mimic NC group).At the same time,the concentration and time of miR-10a-5p transfection were explored by real-time fluorescence quantitative PCR detection method to ensure the timeliness of miR-10a-5p transfection.Cell proliferation was detected by CCK-8 method,cell migration was detected by Transwell,glucose consumption was detected by GOD-POD method,gene expression levels of miR-10a5p and target gene ATF2,PI3K and AKT,macrophage-related indexes F4/80,iNOS and inflammatory factor-related indexes TNF-α,IL-6,IL-1β,IL-10,MCP1,and insulin sensitivity-related indexes PPARy,GLUT4,ADPN,UCP1,IRS1 were detected by PCR.Protein expression levels of ATF2,P-ATF2,PI3K,AKT and their phosphorylation,protein expression levels of macrophage-related indexes F4/80,iNOS and inflammatory factor-related indexes TNF-α,IL-6,IL-1β,IL-10,MCP1,and insulin sensitivity-related indexes PPARy,GLUT4,ADPN,UCP1,IRS1 were detected by Western blot.Results:Compared with the M group,the cell activity of the mimic group and mimic NC group was significantly reduced(P<0.01).Compared with the M group,the glucose consumption of the inhibitor group,inhibitor NC group,mimic group and mimic NC group was significantly increased(P<0.01).Compared with the M group,the TG content in the mimic group was significantly reduced(P<0.01).Compared with the M group,the INS content in the cell supernatant of the mimic group was significantly increased(P<0.05 or P<0.01).Compared with the M group,the gene expression level of miR-10a-5p in the mimic group was significantly increased(P<0.05 or P<0.01).The PCR results showed that compared with the M group,the expression levels of F4/80,iNOS,ATF2,TNF-α,IL-6,and IL-1β and MCP1 were significantly reduced(P<0.01),and the mRNA expression levels of Arg3,PI3K,AKT,PPARγ,GLUT4,ADPN,UCP1,IL-10,IRS1 were significantly increased(P<0.01)in the mimic group.WB results indicated that compared with the M group,the protein expression levels of iNOS and P-ATF2/ATF2 in the mimic group were significantly decreased(P<0.01),the protein expression levels of Arg1,P-PI3K/PI3K,P-AKT/AKT,PPARγ,ADPN,GLUT4,and UCP1 increased significantly(P<0.01).Conclusion:Exercise can promote the release of miR-10a-5p in ATM-Exos,inhibit the expression of its target gene ATF2,and then activate the PI3K/AKT signaling pathway,regulate the polarization state of adipose tissue macrophages,reduce adipose tissue inflammation,and thus improve insulin resistance in T2DM. |
PDF Full Text Request