Mechanism Of Adipsin In Aerobic Exercise Improving Bone Marrow Adipose Tissue Metabolism In Obese Mice

Object: Obesity is a worldwide pandemic related to diabetes、cardiac-cerebral vascular disease and musculoskeletal diseases.According to the epidemiological data,obese patients with type Ⅱ diabetes showed bone loss,osteoporosis,and expanding bone marrow adipose tissue.Distinct from general adipose tissue(brown adipose tissue and white adipose tissues),BMAT is located in the bone marrow cavity.Furthermore,it was shown that BMAT regulates bone mass,hematopoietic function,metabolic homeostasis,and bone remodeling.Exercise has been proven to decrease BMAT volume and improve bone quality in obese patients.However,the mechanism of exercise-induced bone remodeling is not well understood.In this study,we aim to investigate how exercise induces bone remodeling under diet-induced obesity in a comprehensive way.We employed unbiased RNA-Seq to investigate the effect of exercise on the bone marrow of diet-induced obese male mice.Moreover,bone mesenchymal stem cells(BMSCs)were isolated to explore the regulatory effects of exercise in vitro.These findings are likely to provide new strategies for treating obesity and diabetes with fewer side effects on bone health.Materials and methods: 1.Animals studies Obtained 8-week male C57BL/6 mice from the Model Animal Research Center.After the acclimation period,mice were provided a HFD for six weeks to induce obesity.Then the obese animals were randomly divided into two groups: The obesity group(Ob group,n=9)and the Obesity combined Exercise group(Ob+Exe group,n=9).The exercise group ran one hour of moderate intensity six days per week and maintained for 12 weeks.The obesity group had no interventions except for feeding HFD.Body composition was measured weekly by Echo magnetic resonance imaging(MRI).GTT and ITT were applied in mice at the beginning of the twelve weeks.2.Bone processing and analysis The micro CT was applied to isolated femurs to quantify bone density after the fixing.The distal bone growth plate’s cortical and trabecular bone structure was built after scanning.We employed the osmic acid staining method to fix the other side femur BMAT and quantified it by CT scanning.3.Bone marrow processing and analysis We isolated the bone marrow of the tibia and employed RNA-seq technology to detect whole genome variation of bone marrow before and after exercise intervention.RT-PCR detected the m RNA expression of bone metabolism genes.The Adipsin and related protein expression was estimated by Western blot.4.BMSCs culture and processing We isolated BMSCs from the mouse tibia and cultured the cells with a basal medium.Second or third-generation mesenchymal stem cells were collected and incubated with an adipogenic differentiation medium in the 6-well culture plates after the density up to 70%-80%.All cells were divided into the control group(Control,n=4)and the stretch group(Stretch,n=4),the control group’s culture conditions were consistent except for the stretch.Meanwhile,we employed the Flexcell FX-5000 Tension system to tension the membrane that imitated the exercise state.We collected all cells to test after the stretch intervention.Result: 1.12 weeks of aerobic exercise improved obese mice’s body composition and insulin resistance.Body weight data indicated that exercise could postpone excessive weight gain even under a HFD.The body weight of the Ob+Exe group mice was less than the Ob group,accompanied by reduced HFD-induced fat expansion significantly but not lean mass(p<0.01).At the 60 min time,the blood glucose concentration of mice in the Ob+Exe group was significantly lower than that in the Ob group(p<0.05).The AUC of ITT in the Ob+ Exe group was significantly lower than in the Ob group(p<0.05).2.Aerobic exercise improved bone density in obese mice at 12 weeks.From the micro-CT quantitative analysis,bone density was enhanced after 12 weeks of moderate-intensity treadmill exercise,especially in the trabecular bone.Trabecular bone area,volume,and mean density of the Ob+Exe group were significantly improved than the Ob group(p<0.05).3.12 weeks of aerobic exercise inhibited the excessive expansion of BMAT in obese mice.The micro-CT quantitative analysis showed that the BMAT volume of the Ob+Exe group was 28% lower than the Ob group after 12 weeks of moderate-intensity treadmill exercise.4.12 weeks of aerobic exercise significantly suppressed the expression of Adipsin and Spp1 in the bone marrow of obese mice.The results of RT-PCR in bone marrow showed that the m RNA expression levels of Atf4 and Sparc in the Ob+Exe group were significantly up-regulated compared with those in the Ob group(p<0.05),Rankl and Spp1 were down-regulated in Ob+Exe group(p<0.05).The results of Western blot in bone marrow showed that the levels of Alp in the Ob+Exe group were significantly up-regulated(p<0.01).In contrast,the levels of Spp1 and Adipsin were significantly down-regulated after exercise(p<0.01).5.12 weeks of aerobic exercise improved the bone marrow microenvironment in obese mice through a mechanical induction mechanism.The RNA-seq data showed that ECM-receptor interaction and Focal adhesions pathway were significantly inhibited in Ob+Exe group compared with the Ob group.The results of RNA-seq analysis showed that the bone remodeling-related factor Spp1 in Ob+Exe group was significantly lower than that in the Ob group(p<0.001);in addition,Spp1 was enriched in ECM-receptor interaction,Focal adhesion,and PI3K-AKT signaling pathways.Interactome analysis of Spp1 using the HINT database showed that Spp1 interacted with adipokine Adipsin.6.Mechanical tension inhibited the differentiation of BMSCs into mature adipocytes.RT-PCR results of BMSCs showed that stretch significantly reduced the m RNA expression of Pparg1 and Pparg2 after mechanical stretch(p<0.05).The expression of Adipsin protein in the Stretch group was significantly lower than that in the Control group(p<0.01).Conclusion: 1.Aerobic exercise retarded weight gain and improves insulin resistance in obese mice.2.Aerobic exercise can improve the BMD of obese mice,inhibit BMAT over-expansion of obese mice,and improve the bone marrow microenvironment of obese mice.3.Aerobic exercise remodeled the bone marrow microenvironment through mechanosensory mechanisms and may regulate dynamic equilibrium between bone and adipose tissue through the Adipsin-Spp1 axis.4.Mechanical stretch stimulation inhibited BMSCs differentiation into mature adipocytes.