| Chapter1 IntroductionObesity is one of the most significant health problems plaguing humanity in recent years.Obesity can lead to a range of diseases,among which damage to the endothelium is one of the earliest and most important fatal risk factors in the obesity process.It is generally believed that endothelial damage is closely related to abnormal lipid metabolism.However,recent studies have shown that energy metabolism imbalance can be attributed to mitochondrial dysfunction.To adapt to energy metabolism homeostasis,the morphology,number,distribution,and function of mitochondria constantly change.In the obese state,the imbalance of energy homeostasis in the body causes changes in the adipose microenvironment composed of adipocytes,vascular cells,neurons,immune cells,and their secreted cytokines.Asprosin(ASP)is a newly discovered adipokine that plays an important role in regulating glucose and lipid metabolism homeostasis.ASP can promote the release of liver glucose by binding to the OLFR734 receptor on the liver and can also directly stimulate appetite by acting on the hypothalamus through the blood-brain barrier.In obese populations,its abnormally elevated levels are involved in the progression of cardiovascular disease(CVD)and the pathological and physiological processes of diseases such as diabetes.However,the role and mechanism of ASP in endothelial dysfunction in the obese state are not yet clear.Our study conducted a relatively in-depth and systematic investigation around the "relationship between abnormally elevated ASP in the obese state and endothelial dysfunction." We first used different levels of ASP to treat human umbilical vein endothelial cells(HUVECs)and rat thoracic aortic endothelial cells(RTAECs)to observe the effects of ASP on endothelial cell function and mitochondrial homeostasis.Secondly,we established a fat tissue-specific ASP-deficiency(ASPdeficiency)mouse model and a high-fat diet(HFD)-induced mouse obesity model to study the role and mechanism of ASP in the development of endothelial dysfunction at the overall level.Finally,we used small interfering RNA(sh RNA)technology to knock down DRP1 and the DRP1 pharmacological-specific inhibitor(MDIVI-1)to inhibit mitochondrial fission,and studied the changes of ASP on the insulin signaling pathway(PI3K/AKT/eNOS)of endothelial cells at the cellular level.Observing the effects of different ASP levels on arterial endothelial function,insulin signaling pathways,and mitochondrial dynamics at the animal,organ,cellular,and molecular levels provides new strategies for the prevention and treatment of obesity-related cardiovascular diseases and lays a solid experimental foundation for new drug development targeting ASP.Chapter2 The effect of ASP on the function of vascular endothelial cellsObjective: Recombinant ASP was employed to treat HUVECs and RTAECs to simulate the increase of ASP under obesity condition in vitro,and to observe the effect of ASP on the function of vascular endothelial cell.The expression and activation of PI3K/AKT/eNOS pathway were examined by Western blot,and the relationship between ASP and vascular endothelial cell function was extensively analyzed,which laid a foundation for the subsequent study on the role of ASP in the induction and development of vascular endothelial dysfunction.Methods: 1.Confirmation of ASP treatment concentration.A series of ASP concentration gradients(20 nmol/L,40 nmol/L,60 nmol/L,80 nmol/L and 100nmol/L)were added to HUVECs and RTAECs cell culture-medium for 48 h,respectively.MTT was used to detect the effect of ASP on vascular endothelial cell activity.2.A series of ASP concentration gradients(1 nmol/L,10 nmol/L,50 nmol/L,100 nmol/L)were added to the medium of HUVECs and RTAECs cell culture for 48 h,and insulin(100 nmol/L)was used to treat cells for 30 min.Detecting:(1)nitric oxide(NO)levels: NO assay kit(nitric reductase assay)was used to detect NO levels in HUVECs and RTAECs cell cultures.(2)Malondialdehyde(MDA)content:detected by MDA kit;(3)Insulin signaling pathway detection: The levels of PI3K/AKT/eNOS pathway protein and phosphorylated protein in the endothelial cells were detected by Western blot.Results:1.With the increase of ASP concentration(20 nmol/L,40 nmol/L,60 nmol/L,80 nmol/L,100 nmol/L),ASP had no significant effect on the activity of HUVECs and RTAECs cells.2.With the increase of ASP concentration(1 nmol/L,10 nmol/L,50 nmol/L,100 nmol/L),the content of NO released by HUVECs and RTAECs cells into the medium decreased.Meanwhile,the levels of MDA,which reflects the level of lipid oxidation increased gradually in HUVECs and RTAECs cells.3.With the increase of ASP concentration(1 nmol/L,10 nmol/L,50 nmol/L,100 nmol/L),the expression of insulin pathway related proteins p-PI3K/PI3 K,pAKT/AKT,p-eNOS/eNOS decreased gradually.Conclusions:(1)ASP does not exhibit obvious toxicity to vascular endothelial cells(HUVECs and RTAECs)under the condition of the concentration less than 100nmol/L,and the ASP concentration ranged from 1 to 100 nmol/L were used to the subsequent experiments.(2)At the cellular level,ASP leads to oxidative stress level increase and inhibited PI3K/AKT/eNOS signaling in a concentration-dependent manner,leading to vascular endothelial cell dysfunction.Chapter3 The effect of ASP on vascular endothelial function in miceObjective: The aims of this Chapter were to explore the effect of adipocytokine ASP on vascular endothelial function using mouse obesity model,and to study the role and mechanism of ASP in mice.Methods: 1.ASP or ASP neutralizing antibody(AASP)intervention experiment: ASP antibody AASP was successfully prepared.In this study,24 male C57BL/6 mice were randomly divided into 4 groups(6 mice in each group),namely Ctrl group,ASP group,HFD group and HFD+AASP group.The Ctrl group and HFD group were fed with normal diet and high fat diet for 24 weeks,respectively.After 24 weeks of normal diet,the mice in the ASP group were intraperitoneally injected with ASP(0.5 μg·g-1·day-1)for 4 consecutive weeks,while those in the HFD+AASP group were fed with HFD for 24 weeks,and intraperitoneally injected with AASP(0.5 μg·g-1·day-1)for 4 consecutive weeks.(1)The morphological indexes(body weight)of mice were monitored.(2)The serum ASP level and glycolipid metabolic indexes,including blood glucose,T-CHO,TG,HDL-C,ITT and GTT,and the contents of NO and MDA in aorta were detected.(3)The vasodilation function and hemodynamics were detected by ultrasound imaging,and the blood pressure of mice was detected by MRBP Non-Invasive Blood Pressure Apparatus.(4)The expressions of p-PI3K/PI3 K,p-AKT/AKT and p-eNOS/eNOS in aorta were detected by Western blot.2.ASP deficiency of C57BL/6 mice: we have successfully constructed adipose tissue ASP deficiency mice(ASP-deficiency).12 wild-type(WT)male C57BL/6 rats were randomly divided into WT group and HFD+WT group.The WT group was fed with a normal diet for 24 weeks and the HFD+WT group was fed with the HFD for 24 weeks.Additionally,12 ASP-deficiency male C57BL/6 mice were randomly divided into two groups: ASP-deficiency group and high fat group.The ASPdeficiency group was fed with a normal diet for 24 weeks,and the HFD+ASPdeficiency group was fed with the HFD for 24 weeks.(1)The morphological indexes(such as body weight,etc.)of mice were monitored.(2)The serum ASP level,glycolipid metabolic indexes(blood glucose,ITT,GTT,T-CHO,TG,HDL-C)and the contents of NO and MDA in aorta were detected.(3)The vasodilation function and hemodynamics of mice were detected by ultrasound,and the blood pressure of mice was detected by MRBP Non-Invasive Blood Pressure Apparatus.(4)The expressions of p-PI3K/PI3 K,p-AKT/AKT and p-eNOS/eNOS in aorta were detected by Western blot.Results: 1.Intervention experiment of ASP or AASP(1)Morphological indexes of mice: ASP was given to C57BL/6 mice on normal diet,and body weight increased significantly.In obese mice fed with HFD,the weight of the mice decreased significantly after AASP administration.(2)Changes of serum ASP level,glycolipid metobolic indices,NO and MDA levels in mice: compared with Ctrl group,serum ASP,blood glucose,T-CHO,TG and MDA levels in aortic tissue were significantly increased in HFD group,while serum HDL-C and NO levels in vascular tissue were significantly decreased,and glucose tolerance and insulin tolerance of mice were significantly impaired.The glycolipid metobolic indexes of ASP intervention group were similar to those of HFD intervention group.However,after 4 weeks of AASP intervention(HFD+ AASP),the biochemical indexes of mice basically returned to normal levels,and the abnormal glucose tolerance and insulin tolerance were also significantly improved.(3)Changes in vasodilation function,blood pressure and hemodynamics of mice: Compared with Ctrl group,non-endothelium-dependent vasodilation mediated by SNP in ASP,HFD and HFD+AASP groups was not significantly affected,while the endothelium-dependent vasodilation mediated by Ach or INS in ASP and HFD groups was significantly decreased.Compared with HFD group,vascular endothelium-dependent relaxation function was significantly improved in HFD+AASP group.In addition,there was no significant difference in carotid artery flow rate in ASP,HFD,and HFD+AASP groups compared with Ctrl group,although the blood pressure was increased in HFD group.There were no significant differences in blood pressure and carotid artery flow rate in ASP,HFD and HFD+AASP groups.(4)Changes in the expression of proteins related to the aortic endothelial insulin signaling: compared with the Ctrl group,ASP and HFD can negatively regulate the PI3K/AKT/eNOS pathway;compared with HFD group,high lipid induced inhibition of this pathway was reversed after administration of AASP.2.ASP deficiency C57BL/6 mice experiment(1)Morphological indexes of mice: compared with the WT group,the body weight of mice in the HFD+WT group was significantly increased,while the body weight of mice in the ASP-deficiency and HFD+ASP-deficiency groups was not significantly different.(2)Changes in serum ASP level,glycolipid metobolic indexes,and blood vessel NO and MDA levels of mice: compared with WT group,other indexes except NO and HDL-C levels were significantly increased in HFD+WT group,indicating that HFD leads to disorders of glucose and lipid metabolism and insulin resistance(IR)in mice.The serum ASP level of mice in ASP-deficiency and HFD+ASP-deficiency groups was decreased,but NO and MDA levels in serum glucose and lipid metabolism indexes and vascular tissues were not significantly different,but significantly lower than those in HFD+WT group.(3)Changes of vasodilation function,blood pressure and hemodynamics in mice: compared with WT group,the non-endothelium-dependent vasodilation function of ASP-deficiency,HFD+WT and HFD+ASP-deficiency groups was not significantly affected,and the endothelium-dependent vasodilation function of ASPdeficiency and HFD+ASP-deficiency groups was not significantly different,while the endothelium-dependent vasodilation function of HFD+WT group was significantly decreased.Compared with WT group,there was no significant difference in blood pressure between ASP-deficiency and HFD+ASP-deficiency group,blood pressure increased in HFD+WT group,and carotid artery flow velocity showed no significant difference between ASP-deficiency,HFD+WT and HFD+ASP-deficiency groups.After the administration of vascular dilators(SNP,Ach and INS),There were no significant changes in blood pressure and carotid artery flow rate in ASP-deficiency,HFD+WT and HFD+ASP-deficiency groups.(4)Changes in the expression of proteins related to aortic endothelial insulin signaling: compared with WT group,HFD+WT group negatively regulates PI3K/AKT/eNOS pathway,and ASP deficiency in C57BL/6 mice has no significant effect on PI3K/AKT/eNOS pathway,but ASP deficiency can reverse the inhibition of high fat on PI3K/AKT/eNOS pathway.Conclusions:(1)ASP can cause disorder of glucose and lipid metabolism,vascular endothelial dysfunction and insulin resistance IR in mice.(2)AASP can improve HFD-induced glucose and lipid metabolism disorder, vascular endodermal dysfunction and IR.(3)Adipose tissue ASP deficiency can also improve HFD-induced glucose and lipid metabolism disorder,vascular endothelial dysfunction and IR.Chapter 4 Effect of ASP on mitochondrial dynamic homeostasis of vascular endothelial cellsObjective: According to the data of Chapter 3,ASP can damage vascular endothelial function and lead to glycolipid disturbance and oxidative stress level increase in vivo.However,the oxidative stress level increase in vivo may be closely related to ROS level increase due to mitochondrial function impairment,and mitochondrial dynamics is the main manner to maintain mitochondrial homeostasis.Therefore,this chapter aims to study the effect of ASP on mitochondrial dynamic homeostasis,and further clarify the relationship between ASP-induced vascular endothelial dysfunction and mitochondrial dynamic homeostasis.Methods: 1.In vitro experiments: A series of ASP concentration gradients(1 nmol/L,10 nmol/L,50 nmol/L,100 nmol/L)were added to the medium of HUVECs and RTAECs for 48 h,and then insulin(100 nmol/L)was used to treat the cells for 30 min.Western blot was used to detect the phosphorylation protein levels of DRP1(mitochondrial fission protein),Ser-616 and Ser-637 of DRP1,and the expression level of MFN2(mitochondrial fusion protein).2.In vivo experiments:(1)Grouping of animal is the same as Method 1 in Chapter 3.The protein levels of DRP1 and MFN2 were detected respectively.(2)Grouping of animal experiments is the same as Method 2 in Chapter 3.The protein levels of DRP1 and MFN2 were detected in WT,ASP-deficiency,HFD+WT and HFD+ASP-deficiency groups mice.Results:1.In vitro experiments(1)With the increase of ASP concentrations,the DRP1 expressions in both HUVECs and RTAECs were up-regulated gradually,while the MFN2 levels were down-regulated gradually,leading to mitochondrial dynamic homeostasis imbalance.(2)With the increase of ASP concentrations,phosphorylation levels of Ser-616 and Ser-637 of DRP1 in HUVECs and RTAECs were increased significantly,like total DRP1.2.In vivo experiments(1)Treatment with ASP significantly increased the expressions of DRP1 and down-regulated the expressions of MFN2 in vascular tissue of mice,like HFD treatment,resulting in the imbalance of mitochondrial dynamic homeostasis.(2)ASP deficiency in adipose tissue did not affect the expression of DRP1 and MFN2 in vascular tissue under the condition of normal diet,but ASP-deficiency can reverse the HFD-induced up-regulation of DRP1 and down-regulation of MFN2 under HFD,and rescue mitochondrial dynamic homeostasis.Conclusions:(1)ASP can cause the imbalance of mitochondrial dynamic homeostasis in vascular endothelial cells.(2)Both AASP intervention and ASP deficiency can improve mitochondrial dynamics disorder in obesity mice.(3)Vascular endothelial dysfunction induced by ASP may be related to the imbalance of mitochondrial dynamic homeostasis.Chapter 5 Mitochondrial dynamic mechanism and relevance of the vascular endothelial dysfunction induced by ASPObjective: According to the results in Chapter 4,ASP can cause vascular endothelial dysfunction and induce the mitochondrial dynamic disturbance.However,it is unclear whether the mitochondrial dynamics disorder induced by ASP could lead to the vascular endothelial dysfunction.and whether the recovery of mitochondrial dynamical homeostasis disturbance by inhibiting the mitochondrial fission has such an effect on the improvement of vascular endothelial dysfunction induced by ASP.Therefore,to address these issues,pharmacological inhibitor of DRP1 or RNA interference targeted at DRP1 were used to inhibit DRP1 activity or down-regulate DRP1 expression.Methods: 1.Pharmacological inhibition experiments of DRP1: HUVECs and RTAECs were randomly allocated into 6 groups: Ctrl group,PA group(0.25 mmol/L)group,ASP(50 nmol/L)group,PA+ASP group,PA+ASP+MDIVI-1(10 μmol/L)group,PA+ASP+DMSO group.The cells were treated with PA and ASP for 36 h,followed by the treatment of MDIVI-1 for 12 h,and insulin(100 nmol/L)for 30 min.(1)the morphology and structure of mitochondria were observed by confocal microscopy;(2)the protein levels of DRP1 and MFN2 were detected by Western blot.(3)NO level(4)MDA content;(5)the insulin signaling proteins of PI3K/AKT/eNOS pathway and their phosphorylation levels were detected by Western blot in endothelial cells.2.DRP1 knockdown experiments: three Drp1-knock-down lentiviruses and a control one carrying Drp1-sh RNA1,Drp1-sh RNA2,Drp1-sh RNA3 and the Scrambled sh RNA,respectively,were infected with HUVECs and RTAECs and incubated for 72 h.Then Western blot was used to screen the silencing efficiency.HUVECs and RTAECs were randomly divided into the Scrambled sh RNA and DRP1-sh RNA groups,and each group was further subdivided into four subgroups,namely,Ctrl group,PA(0.25 mmol/L)group,ASP(50 nmol/L)group and PA+ASP group.The cells were treated with PA and ASP for 48 h,and then treated with insulin(100 nmol/L)for 30 min.Detection of the indexes was the same as described in the Methods 1.Results:1.Pharmacological inhibition experiments of DRP1(1)Mitochondrial morphological and structural changes: both PA and ASP treatment can cause the mitochondrial fission,evidenced as mitochondrial fragmentation and shortening of branches,and mitochondrial fission is more significant in the combination of PA and ASP treatment.MDIVI-1 administration can significantly counteract the mitochondrial fission(MT fission),the up-regulation of DRP1 and the down-regulation of MFN2 induced by ASP.(2)MDIVI-1 administration reversed the decrease of NO levels and the increases of MDA levels induced by ASP.(3)MDIVI-1 administration reversed the down-regulation of PI3K/AKT/eNOS signaling protein and improved vascular endothelial dysfunction induced by ASP.2.DRP1 knockdown experiments:(1)Screening of the silencing efficiency by western blot: compared with the Scrambled sh RNA group,DRP1-sh RNA1,DRP1-sh RNA2,DRP1-sh RNA3 significantly down-regulated the expression of DRP1,but the silencing efficiency of DRP1-sh RNA1 was the most significant.Therefore,DRP1-sh RNA1 was used for subsequent DRP1 knock-down experiments.(2)Mitochondrial morphology and structure: DRP1 knockdown can significantly improve the mitochondrial fragmentation inhibit the mitochondrial fission,up-regulate the MNF2 expression and down-regulate the DRP1 expression.(3)DRP1 knockdown significantly reversed the decrease of NO levels and the increases of MDA levels induced by ASP.(4)DRP1 knockdown significantly reversed the down-regulation of PI3K/AKT/eNOS signaling protein and improved vascular endothelial dysfunction induced by ASP.Conclusions:(1)Inhibition of DRP1 activity can rescue the mitochondrial dynamics disorder of vascular endothelial cells induced by ASP in the hyperlipidemic model,reduce the level of oxidative stress and thus improve vascular endothelial function.(2)DRP1 knockdown can correct the mitochondrial dynamics disorder of vascular endothelial cells induced by ASP in the hyperlipidemic model,reduce the level of oxidative stress and thus improve vascular endothelial function.(3)The vascular endothelial dysfunction caused by ASP is due to the imbalance of mitochondrial dynamic homeostasis.Therefore,rescue of the imbalance of mitochondrial dynamic homeostasis induced by ASP might be an effective way to prevent and treat the obesity-related cardiovascular diseases. |
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