| Chapter1 IntroductionAsprosin（ASP）is a new adipocytokine recently discovered to regulate glucose and lipid metabolism,which is closely related to the occurrence of obesity.Our previous work showed that ASP can promote autophagy of white adipocytes but inhibit their browning.However,the causal relationship between ASP promoting autophagy and anti-browning and its regulatory mechanism has not been reported yet.Therefore,this project is the first to explore the mechanism of ASP regulation of white fat browning in obesity from the perspective of autophagy.Firstly,the mouse adipose tissue-specific ASP knockout(ASP-/-)model and adipocyte ASP knockout model was established to observe the effects of different ASP levels on the browning of white fat,autophagy,and mitochondrial homeostasis in a multi-dimensional and multi-level system.Then,by adjusting the level of autophagy,we investigated whether the inhibition of ASP on browning of white fat and the promotion of mitochondrial degradation was caused by autophagy.Finally,pharmacologic inhibitors were used to explore the signaling pathway that ASP promotes autophagy to regulate the browning of white fat and participates in obesity.It aims to enrich and improve the regulation mechanism of white fat browning induction network,provide new biomarkers and new therapeutic targets for the diagnosis and treatment of obesity and obesity-related diseases,and also lay a solid foundation for the research and development of subsequent biological drugs（such as ASP neutralizing antibody）or ASP small molecular inhibitors.With the support of national natural science funds we through synthetic ASP proteins,antibodies,and build knockout cells and animal models of the ASP,and at cell and animal level,respectively,to explore the ASP to white fat browning and the influence of the steady-state of mitochondria and finally explore the ASP by promoting autophagy of white fat cells and inhibiting browning mechanism,to provide a new therapeutic target for the treatment of obesity.Chapter 2 Construction of ASP protein,antibody,and ASP knockout modelObjective:To investigate the effects of ASP on autophagy and browning of adipocytes,we prepared ASP protein and antibody,and constructed 3T3-L1adipocyte lines with ASP deletion(ASP-/-)and C57BL6 transgenic mice（CKO）with ASP knockout(ASP-/-).This provides an experimental basis for exploring the effects of ASP on autophagy and browning of adipose cells at both cellular and global levels.Methods:1.The constructed expression vector p ET28a-ASP was transformed into BL21（DE3）,and the ASP protein was induced,expressed,and purified by conventional methods.2.KLH polypeptide coupling（coupling agent sulfo-SMCC）was used as an immune antigen.Two New Zealand rabbits were immunized with antigen at an interval of 14 days.On the 53rd day of immunization,blood was collected from the carotid artery.The rabbit blood was stored overnight at 4℃,centrifuged for 30 min,and the supernatant was collected for purification and identification of antibodies to prepare ASP antibodies.3.A single lentivirus carrying the sg RNA sequence expression frame of Cas9protein and target protein ASP was infected with CRISPR/Cas9 technology to induce mature 3T3-L1 cells with 90%fusion.After 3 days of infection,puromycin-resistant cells were screened and monoclonal culture was performed on resistant cells.Pass through more than 10 generations.By detecting the expression levels of ASP m RNA and protein,the target gene of ASP was knocked out,and the ASP knockout(ASP-/-)3T3-L1 fat cell line was established.4.Cas9 m RNA and g RNA were obtained by in vitro transcription.The donor vector is constructed by the in-fusion cloning method,which contains 3.0KB 5’homologous arm,2.3KB FLOx region,and 3.0KB 3’homologous arm.Cas9 m RNA,g RNA,and Donor vector were microinjected into the fertilized eggs of C57BL/6J mice to obtain F0 generation mice.After F0 generation mice were obtained,homozygous mice with ASP deletion were further screened.Results:1.ASP protein with 85%purity at a concentration of 1.6 mg/m L was synthesized.2.100μL of 1.86 mg/m L and 2.21mg/m L of ASP antibody was synthesized,and2 m L of ASP antibody was purified later.3.The ASP-/--3T3-L1 fat cell line was successfully established;4.C57BL6 mice with ASP conditional knockout（CKO）of adipose tissue were successfully constructed.Conclusion:1.ASP-/--3T3-L1 adipose cell line and CKO mice were successfully constructed.2.The purity and quantity of ASP protein and antibody met the requirements of the experiment,which laid a foundation for the subsequent experiments.Chapter 3 Effects of ASP intervention on browning and autophagy of white adiposeObjective: We used ASP protein synthesized in the previous stage to intervene3T3-L1 cells and C57BL/6 mice to explore its effects on white fat browning and autophagy at cellular,molecular and global levels.Methods 1.Cell experiment: Mature 3T3-L1 white adipocytes were induced with β3 receptor agonist CL-316243（1 μM）for 2 days.During the induction period,human recombinant ASP（1 μM）was administered,and the control group was treated with bovine serum albumin（BSA）.（1）The expression level of ASP in the cell culture medium was detected by ELISA.（2）Double fluorescence labeling LC3 method was used to detect the effect of ASP on autophagy of adiposity cells during browning induction.（3）Oil red O staining was used to observe the lipid size and aggregation of adipocytes.（4）Mitochondrial oxygen consumption rate（OCR）was measured by the hippocampal apparatus.（5）Western blot was used to detect the expression levels of PGC1-α,UCP1,PRDM16,Beclin,and ULK1 in cells.2.Animal Experiments: Wild-type normal mice（WT）and HFD-induced obese mice（DIO）were fed with normal diet（ND）and high-fat diet（HFD）for 24 weeks,and then placed in thermal neutral（30℃）and low temperature（4℃）environment for 8 weeks,respectively.During this period,the experimental group was subcutaneously injected with ASP protein（1 μg/kg body weight）,the control group was given the same amount of PBS subcutaneously,once every 3 days for 8weeks.The changes in body weight,body length,and metabolic indexes were observed.After 8 weeks,the mice were sacrificed.Morphological,histopathological,biochemical,and molecular biological indicators were detected to reflect the changes of glucose and lipid metabolism level,white fat browning level,autophagy level,and mitochondrial homeostasis.Results: 1.Cell experiment:（1）The results of oil red O showed that ASP intervention could promote the accumulation and formation of lipid in adipocytes;（2）Hippocampal detection results showed that ASP intervention significantly reduced mitochondrial OCR and impaired mitochondrial respiratory function.（3）Western blot results showed that ASP intervention could significantly reduce the expression levels of PGC1-α,UCP1,and PRDM16,while the expression levels of Beclin and ULK1 were significantly increased.2.Animal experiment:（1）Biochemical indexes: The results showed that there was no significant difference in the body length of mice in each experimental group.Compared with the ND group,body weight,serum levels of FBG,INS,HOMA-IR,T-CHO,and TG were significantly increased in ND+ASP and HFD groups,while HDL,GTT,and ITT were significantly decreased.Compared with the HFD group,body weight and serum levels of FBG,INS,HOMA-IR,T-CHO,and TG were significantly increased in high-fat diet combined with ASP,while HDL,GTT,and ITT were significantly decreased.In addition,compared with thermal neutral environment of 30°C,body weight and serum levels of FBG,INS,HOMA-IR,T-CHO,and TG of mice in each experimental group at cold exposure of 4°C were significantly decreased,while HDL,GTT,and ITT were significantly increased.（2）HE staining and histomorphology showed that compared with the ND group,the average cross-sectional area of adipocytes and the wet weight of white adipose tissue in ND+ASP and HFD groups were significantly increased.Compared with HFD group,the average cross-sectional area of adipocytes and the wet weight of white adipose tissue in HFD+ASP group increased significantly.At the same time,compared with thermal neutral environment of 30°C,the average cross-sectional area of adipocytes and the wet weight of white adipose tissue were significantly decreased.（3）Autophagic protein expression level: Western blot results showed that compared with the ND group,the expression levels of ULK1 and Beclin autophagic proteins in normal diet combined with ASP group and HFD group were significantly up-regulated.Compared with the HFD group,a high-fat diet combined with ASP treatment can increase the expression levels of ULK1 and Beclin autophagy proteins.At the same time,compared with thermal neutral environment of 30°C,the expression levels of ULK1 and Beclin autophagy proteins were significantly downregulated in all experimental groups at 4° C.（4）Browned protein expression level: Compared with the ND group,the browned protein expression levels of UCP1,PGC1-α,and PRDM16 in normal diet combined with ASP and HFD group were significantly down-regulated.Compared with the HFD group,a high-fat diet combined with ASP treatment could significantly down-regulate the expression levels of browned proteins UCP1,PGC1-α,and PRDM16.At the same time,compared with thermal neutral environment of30°C,,the expression levels of brown protein UCP1,PGC1-α,and PRDM16 in all experimental groups at cold exposure of 4°C were significantly up-regulated.Conclusion:1.ASP can promote the autophagy of fat cells and inhibit the browning of white fat,and participate in the occurrence and development of obesity in mice.2.ASP can reduce glucose and insulin resistance in mice,resulting in glucose and lipid metabolism disorder.3.Moderate cold stimulation increased glucose and insulin tolerance and improved HFD and ASP-induced fat "whitening" levels in mice.Chapter 4 Effects of ASP knockout on browning of white fat and autophagyObjective: We further explored the effects of ASP on browning and autophagy of white fat by conditional knockout of ASP.Methods: 1.Cell experiment: 3T3-L1 white adipocytes with ASP knockout and mature differentiation were taken and induced by β3 receptor agonist CL-316243（1 μM）for 2 days.The control group was changed to PBS with equal capacity.（1）The expression of ASP in the cell culture medium was detected by ELISA.（2）Double fluorescence labeling LC3 method was used to detect the effect of ASP knockout on the autophagy of adipocytes during browning induction.（3）Oil red O staining was used to observe the lipid droplet size and aggregation of lipid.（4）Mitochondrial oxygen consumption rate（OCR）was measured by the Seahorse XFe24 Extracellular Flux analyzer.（5）Western blot was used to detect the expressions of PGC1-α,UCP1,PRDM16,Beclin,and ULK1 in cells.2.Animal experiment: Wild-type normal mice（WT）and adipose tissue specific ASP-knockout transgenic mice（CKO）were fed to ND and HFD for 24 weeks,respectively,and then exposed to thermal neutral（30℃）and low temperature（4℃）environments for 8 weeks.The changes in body weight,body length,and metabolic indexes were observed.After 8 weeks,the mice were sacrificed.Morphological,histopathological,biochemical,and molecular biological indicators,energy metabolism,and other indicators were detected to reflect the changes of glucose and lipid metabolism,white fat browning,autophagy,and autophagy.Results: 1.Cell experiment:（1）Oil red O staining showed that knockout ASP could inhibit lipid accumulation and lipid droplet formation in adipocytes.（2）OCR results showed that ASP deletion significantly increased mitochondrial OCR and enhanced mitochondrial respiratory function.（3）Western blot results showed that ASP deletion significantly increased the expressions of PGC1-α,UCP1,and PRDM16,while the expression levels of Beclin and ULK1 were significantly decreased.2.Animal experiment:（1）Biochemical indices: Compared with WT+ND group,body length of WT+HFD group did not change significantly.Body length of ASP-/-+ND group was significantly reduced.Compared with ASP-/-+ND group,body length of ASP-/-+HFD increased.Compared to 30℃ thermal neutral,there was no significant change in body length in 4°C cold exposure group.Compared with WT+ND group,the body weight,serum levels of FBG,INS,HOMA-IR,T-CHO and TG of WT+HFD group were significantly increased,HDL,GTT and ITT were significantly decreased.while serum levels of FBG,INS,HOMA-IR,T-CHO and TG of ASP-/-+ND group were significantly decreased,HDL,GTT and ITT were significantly increased.Only compared with ASP-/-+ND group,body weight and serum levels of FBG,INS,HOMA-IR,T-CHO and TG were significantly increased in ASP-/-+HFD group,while HDL,GTT and ITT were significantly decreased.（2）HE staining and histomorphological results showed that: Compared with WT+ND group,the mean cross-sectional area and weight of adipocyte in WT+HFD group were increased,while the mean cross-sectional area and weight of adipocyte in ASP-/-+ND group were decreased.Compared with ASP-/-+ND group,the mean cross-sectional area and weight of adipocyte in ASP-/-+HFD group were increased.At the same time,compared with 30℃ thermal neutral,the average cross-sectional area of adipose cells and wet weight of white adipose tissue of mice in 4°C cold exposure groups were significantly reduced.（3）Metabolic cage experiment results showed that: VCO2,VO2,activity level and activity distance of mice in WT+HFD group were significantly lower than those in ASP-/-+HFD group,these results indicated that ASP knockout increased the overall energy consumption and metabolic rate of mice,but had no significant effect on food and water intake.（4）Autophagy protein expression results showed that: Compared with WT+ND group,the expression levels of ULK1 and Beclin in adipose tissues of WT+HFD group were significantly increased,while the expression levels of ASP-/-+ND group was significantly decreased.Compared with WT+HFD group,the expression levels of ULK1 and Beclin in adipose tissues of ASP-/-+HFD group was significantly decreased.At the same time,compared with 30℃ thermal neutral,the expression levels of ULK1 and Beclin in adipose tissues in 4°C cold exposure groups were significantly reduced.（5）Brown-specific protein expression results showed that: Compared with WT+ND group,the brown-specific protein expression levels of UCP1,PGC1-α and PRDM16 in adipose tissues of WT+HFD groups were significantly decreased,the brown-specific protein expression levels of ASP-/-+ND group was significantly increased.Compared with WT+HFD group,the brown-specific protein expression levels of UCP1,PGC1-α and PRDM16 in adipose tissues of ASP-/-+HFD groups were significantly decreased.At the same time,compared with 30℃ thermal neutral,the expression levels of browning proteins UCP1,PGC1-α and PRDM16 were also significantly up-regulated in 4°C cold exposure groups.Conclusion:1.Knockout of ASP can inhibit HFD-induced excessive autophagy of adipocytes.2.Knockout of ASP can induce browning of white fat.3.ASP knockout can improve glucose and insulin tolerance and improve glucose and lipid metabolism disorder in mice.4.Appropriate cold stimulation can enhance the effect of ASP knockdown on browning of white fat to improve metabolism.Chapter 5 Effects of ASP antibody intervention on browning and autophagy of white fat and glucose and lipid metabolism homeostasisObjective: To explore the effects of ASP antibody intervention on browning and autophagy of white fat and glucose and lipid metabolism homeostasis.Methods: 1.Cell experiment:The mature differentiated 3T3-L1 white fat cells were induced by β3 receptor agonist CL-316243（1μM）for 2 days.During the induction period,ASP antibody（1 μM）was administered.The control group was changed to Ig G.（1）The expression level of ASP in the cell culture medium was detected by ELISA.（2）Double fluorescence labeling LC3 method was used to detect the effect of ASP antibody intervention on adiposity autophagy flow.（3）Oil red O staining was used to observe lipid droplet size and lipid aggregation.（4）Mitochondrial oxygen consumption rate（OCR）was measured by the hippocampal apparatus.（5）Western blot was used to detect the expression levels of PGC1-α,UCP1,PRDM16,Beclin,and ULK1 in cells.3.Animal Experiments: Wild-type mice（WT）and HFD-induced obese mice（DIO）were fed with normal diet（ND）and high-fat diet（HFD）for 24 weeks,and then exposed to thermal neutral（30℃）and low temperature（4℃）environment for 8weeks,respectively.During this period,the experimental group was subcutaneously injected with ASP antibody（1 μg/kg body weight）,the control group was given the same amount of Ig G subcutaneously,once every 3 days for 8 weeks.The changes in body weight,body length,and metabolic indexes were observed.After 8 weeks,the mice were sacrificed.Morphological,histopathological,biochemical,and molecular biological indicators were detected to reflect the changes of glucose and lipid metabolism level,white fat browning level,autophagy level,and mitochondrial homeostasis.Results: 1.Cell experiment:（1）Oil red O showed that ASP antibody significantly reduced lipid accumulation and lipid droplet formation in adipocytes.（2）Hippocampal detection results showed that ASP antibody significantly increased mitochondrial OCR and improved mitochondrial respiratory function.（3）Western blot results showed that ASP antibody intervention significantly increased the expression levels of PGC1-α,UCP1 and PRDM16,but decreased the expression levels of Beclin and ULK12.Animal experiment:（1）Biochemical indexes: There was no significant difference in the body length of mice in each experimental group.Compared with the ND group,body weight,serum levels of FBG,INS,HOMA-IR,T-CHO and TG of HFD group were significantly increased,while HDL,GTT,and ITT were significantly decreased,however,there was no significant difference between ND group and ND+Anti-ASP group.Compared with the HFD group,body weight,serum levels of FBG,INS,HOMA-IR,T-CHO and TG of HFD +Anti-ASP group were significantly decreased,while HDL,GTT,and ITT were significantly increased.ASP neutralizing antibody was more effective in resisting weight gain and glycolipid metabolism disorder in HFD-induced mice.At the same time,compared with thermal neutral environment of30°C,the body weight and serum levels of FBG,INS,HOMA-IR,T-CHO and TG of mice in the cold exposure environment of 4°C groups were significantly decreased,while HDL,GTT and ITT were significantly increased.（2）HE staining and histomorphology showed that compared with ND group,the average cross-sectional area of adipocytes and the wet weight of white adipose tissue in HFD group were significantly increased.while there was no significant difference between ND group and ND+ Anti-ASP group.Compared with HFD group,the average cross-sectional area of adipocytes and the wet weight of white adipose tissue in HFD+Anti-ASP group decreased significantly.At the same time,compared with thermal neutral environment of 30°C,the average cross-sectional area of adipocytes and the wet weight of white adipose tissue in 4°C cold exposure group were significantly decreased.（3）Autophagy protein expression results showed that Compared with ND group,the expression levels of ULK1 and Beclin autophagy proteins in HFD group were significantly up-regulated,while there was no significant difference between ND group and ND+ anti-ASP group.Compared with HFD group,the expression levels of ULK1 and Beclin autophagy proteins in HFD+ anti-ASP adipose tissue were down-regulated.At the same time,compared with the thermal neutral environment of 30°C,the expression levels of ULK1 and Beclin autophagic proteins were significantly decreased in all experimental groups of 4°C cold exposure,and the down-regulation effect of ASP neutralizing antibody on autophagic protein expression was more obvious in 4°C cold exposure.（4）Browned protein expression level: Compared with ND group,UCP1,PGC1-α and PRDM16 browning protein expression levels in HFD group were significantly down-regulated.Compared with HFD group,UCP1,PGC1-α and PRDM16 browning protein expression levels were significantly up-regulated in HFD+ antiASP group.Meanwhile,compared with 30°C thermal neutral environment,the expression levels of UCP1,PGC1-α and PRDM16 in 4°C cold exposure groups were significantly up-regulated,and the up-regulated effect of ASP neutralizing antibody on browning protein expression was significantly enhanced in 4°C cold exposure environment.Conclusion:1.The intervention of ASP neutralizing antibody can inhibit HFD-induced excessive autophagy of adipose cells.2.The intervention of ASP neutralizing antibody can induce the browning of white fat.3.The intervention of ASP neutralizing antibody can improve the glucose and insulin tolerance of mice,and help to maintain glucose ester metabolism homeostasis.4.Appropriate cold stimulation can enhance the metabolic improvement of white fat browning induced by ASP neutralizing antibody intervention.Chapter 6 Study on the mechanism of ASP inhibiting white fat browning by promoting adipocyte autophagyObjective: Our previous study showed that ASP can promote the autophagy of adipocytes and inhibit the browning of white fat.Meanwhile,ASP antibody intervention and knockout can reduce the level of ASP in vivo,inhibit the autophagy and promote the browning.However,whether there is a causal relationship between ASP-mediated autophagy and browning and the signaling pathway that ASP inhibits browning will be the focus of this chapter.Therefore,this chapter will explore whether ASP inhibits browning by promoting autophagy and the signal pathways involved in its inhibition of browning.Methods:1.To detect the dynamic changes of autophagosomes and autolysosomes through the fluorescence intensity of RFP and GFP,3T3-L cell lines treated with β3 receptor agonist CL-316243（1 μM）for 2 days were employed to observe the autophagy in adipocytes and the experimental groups were as follows:Control,ASP（1 μM）group,ASP+Anti-ASP（1 μM）group and ASP+3MA（2.5μM）group.2.To regulate the level of autophagy and explore whether the inhibition of ASP on the browning of white fat and the promotion of mitochondrial degradation in mice are caused by autophagy.Fat cell lines WT 3T3-L were induced with β3receptor agonist CL-316243（1 μM）for 8 days to establish browning model.The cells were treated with PBS as control.On the basis of model group,the experiment was divided into 6 groups,which were BSA group（1 μM,control）,ASP group（1μM）,RP group（10 μM）,3-MA group（2.5 μM）,ASP+RP group and ASP+3-MA group.During browning induction,all groups were treated with ASP（1 μM）,BSA（1μM）,RP（10 μM）,3-MA group（2.5 μM）,ASP+RP and ASP+3-MA respectively.There were 7 groups including PBS group.3.Explore the signaling pathway that ASP participates in obesity by promoting autophagy and regulating the browning of white fat.The fat cell lines WT 3T3-L were browned by β3 agonist CL-316243（1 μM）for 8 days to establish browning model.The cells were treated with PBS as control.On the basis of model group,the experiment was divided into 8 groups,including BSA group（1 μM,control）,ASP group（1 μM）,TLR4 antibody group（1 μM）,SQ-22536 group（10 μM）,Compound C group（10 μM）,ASP+TLR4 antibody group,ASP+SQ-22536 group and ASP+Compound C group.During induction,all groups were treated with ASP（1μM）,BSA（1 μM）,TLR4 antibody group（1 μM）,SQ-22536（10 μM）,Compound C（10 μM）,ASP+TLR4 antibody,ASP+SQ-22536 and ASP+Compound C were used for intervention.There were 9 groups including PBS group.The detection index is the same as before.Results:1.The results showed that Asprosin increased the number of autophagosomes and autophagolysosomes in lipid cells.2.Compared with PBS group,the autophagy indexes including p-AMPK,ULK1 and LC3 B in CL+BSA group were not significantly different,but the levels of PGC1-α,UCP1 and PRDM16 in CL+BSA group were significantly increased,indicating that CL induced the browning but did not affect autophagy.Compared with CL+BSA group,p-AMPK,ULK1 and LC3 B were significantly increased in CL+ASP group and CL+RP group,while PGC1-α,UCP1 and PRDM16 levels were significantly decreased,while P-AMPK in CL+3MA group,The expression of PGC1-α,UCP1 and PRDM16 were significantly increased,indicating that ASP and RP could induce autophagy but inhibit browning,while 3-MA inhibited autophagy and promoted browning.Compared with CL+3MA group,the above indexes were reversed in CL+3MA+ASP group.Compared with CL+RP group,the expression of above indexes increased in CL+RP+ASP group.These results suggest that ASP inhibits the browning of white fat by promoting autophagy.3.The autophagy indexes including P-AMPK,ULK1 and LC3 B in CL+BSA group have no significant difference compared with PBS group,but the levels of PGC1-α,UCP1 and PRDM16 in CL+BSA group were significantly increased.Compared with CL+BSA group,the autophagy indexes including P-AMPK,ULK1 and LC3B were significantly increased in CL+ASP group,while the levels of PGC1-α,UCP1 and PRDM16 were significantly decreased.Compared with CL+ASP group,the levels of p-AMPK,ULK1 and LC3 B in CL+ Anti-TLR4,CL+SQ-22536 and CL+Compound C groups were significantly decreased,while the expressions of PGC1-α,UCP1 and PRDM16 were significantly increased.Compared with CL+Anti-TLR4,CL+SQ-22536 and CL+Compound C groups,the above indexes were reversed in CL+ASP+ Anti-TLR4,CL+ASP+SQ-22536 and CL+ASP+Compound C groups.Conclusion:1.ASP inhibits white fat browning and promotes mitochondrial degradation caused by autophagy.2.ASP promotes adipocyte autophagy through TLR4-c AMP-AMPK-ULK1 pathway and inhibits the white fat browning and participates in the process of obesity. |
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