PAI-1 Exacerbates White Adipose Tissue Dysfunction And Metabolic Dysregulation

Objective:Plasminogen activator inhibitor 1（PAI-1）is a member of the serine protease inhibitor family.Its main role in vivo is to rapidly inhibit tissue plasminogen activator（t-PA）and urokinase type plasminogen（u-PA）,which is not only related to thrombosis and fibrosis,but also to insulin resistance and metabolic syndrome.Studies showed that the expression of PAI-1 was high in white adipose tissue（WAT）of obese people and mice,while other tissues,such as heart,kidney,liver and muscle tissue were low.Therefore,it suggested that WAT was the main source of PAI-1.The latest study revealed that the presence of PAI-1caused accumulation of individual fat.After PAI-1 was knocked out,individual weight gain will be greatly slowed down,even if mice were fed with a high-fat diet（HFD）,weight gain was not obvious.In addition,white adipocytes appeared hypertrophy and hyperplasia in the condition of HFD.However,after PAI-1 was knocked out,the adipocytes became smaller,suggesting that PAI-1 may be a cause of obesity.Besides,the metabolism of wild type（WT）mice fed with HFD was in disorder,while metabolism of mice without PAI-1 was normal in the same condition,suggesting that PAI-1 may be involved in the function and metabolism of WAT.The mechanism of PAI-1 over-expression in obese people is very complex,and it is not clear at present.It needs further study and explanation.Macrophage can be divided into two sub-types according to the difference of function and activation state,namely M1 type（classically activated macrophage）and M2 type（alternatively activated macrophage）.M1 type mainly promotes inflammatory reaction and M2type is mainly involved in anti-inflammatory effects.The study found that proportion of M2 type was more than that of M1 type in WAT of lean mice,namely,anti-inflammatory effect was greater than the pro-inflammatory effect so that the metabolism of mice was in the normal state.On the contrary,the proportion of M1 type in WAT of obese mice increased significantly,and was more than that of M2 type,thereby breaking the proportional balance between M1 type and M2 type.The pro-inflammatory effect was greater than the anti-inflammatory effect leading to disorder metabolism of mice.Recent studies showed that macrophages in WAT of obese individuals increased and dispersed around white adipocytes,which look like"crowns"leading to the dysfunction of WAT and imbalance of metabolism.Both PAI-1 and macrophages infiltration can aggravate the dysfunction of WAT and metabolic disorder.Is there a certain relationship between the two?In addition,glucose metabolism is closely related to glucose transporter（Glucose Transporter,GLUT）,whose main function is associated with glucose transport.GLUT4 mainly existed in adipocytes is responsible for the glucose transport of adipocytes.Studies have found that low density lipoprotein receptor-related protein-1（LRP-1）could mediate GLUT4 of adipocytes,thereby affecting glucose transport in adipocytes.Is there a relationship between PAI-1 and the process?Therefore,the aim of this article is to study the mechanism that PAI-1 exacerbate WAT dysfunction and metabolic disorders through investigating effect of PAI-1 on polarization and infiltration of macrophage,expression of LRP-1 and GLUT4 in adipose tissue,to further identify the relationship between PAI-1 and macrophages,PAI-1 and LRP-1 so as to provide new therapeutic targets and theoretical basis for clinical prevention and treatment of metabolic diseases.Methods:1.Experimental preparation:Breeding and identifying C57/BL6 mice and Pai-1^-/-mice,and selecting several mice at the same age.2.Establishment of diabetic mice model:A high-fat diet（HFD:45%fat by kcal）was given to C57/BL6 mice and Pai-1^-/-mice called HFD-WT mice and HFD-Pai-1^-/-mice,respectively.Meanwhile,C57/BL6 mice and Pai-1^-/-mice were fed normal diet（ND）called ND-WT mice and ND-Pai-1^-/-mice,respectively.The four groups of mice were fed for about 14weeks in the same condition.Body weight and blood glucose values of all mice were measured at fixed time per week.3.q RT-PCR was used to detect the expression of PAI-1 m RNA:The epididymal adipose tissue（e WAT）were obtained from the above 4 groups of mice(ND-WT,HFD-WT,ND-Pai-1^-/-and HFD-Pai-1^-/-mice)respectively.Total RNA were extracted by using Trizol reagent,and the quality of total RNA was detected by RNA agarose electrophoresis.The m RNA expression of PAI-1,LRP-1,TNF-α,CD11c,MCP-1,IL-1β,CD206,IL-10,Fn1,and TGF-β1 was analyzed by q RT-PCR,respectively.4.PAI-039 treatment:The dose of PAI-039 was 2 mg/kg/day,and the sterile water was used as the solvent including 0.5%methylcellulose and 2%Tween 80.HFD-WT mice were treated with PAI-039.PAI-039 is a small molecule inhibitor of PAI-1.HFD-WT mice were selected to gavage with PAI-039 for 30 d called HFD-WT+PAI-039 mice.The body weight and blood glucose of five groups(HFD-WT+PAI-039,ND-WT,HFD-WT,ND-Pai-1^-/-and HFD-Pai-1^-/-mice)were regularly measured during the 30 d.5.The blood lipid indexes were measured.Mice were anesthetized using intraperitoneal sodium pentobarbital（60 mg/kg body weight）or isoflurane（5%by inhalation）.A subcutaneous dose of buprenorphine hydrochloride（0.1 mg/kg）was administered for analgesia.Additional sodium pentobarbital（12 mg/kg body weight）or 5%isoflurane was given as needed to maintain anesthesia.The blood of inferior vena cava was collected,and the serum was obtained by centrifugation.The blood lipid indexes of 5 groups were determined.6.Histological Assessment:e WAT were collected from the above five groups.e WAT with 4%（w/v）of formalin fixed,were embedded by paraffin and made pathological sections.HE staining experiment was performed to observe adipocyte size and morphology by microscope and image acquisition.Anti-F4/80was used to visualize the macrophages by using immunofluorescence staining.The F4/80-positive cells were counted and the expression of macrophages was analyzed.7.Glucose tolerance test（GTT）:5 groups of mice(ND-WT,HFD-WT,ND-Pai-1^-/-HFD-Pai-1^-/-mice and HFD-WT+PAI-039)were fasted overnight,and the mice were intraperitoneally injected with D-（+）-glucose for 2 g/kg on the next morning.The blood glucose values of 0,30,60,90 and 120 min were measured,respectively.Insulin tolerance test（ITT）:The above 5 groups of mice(ND-WT,HFD-WT,ND-Pai-1^-/-,HFD-Pai-1^-/-mice and HFD-WT+PAI-039)were fasted for 6 h,and the mice were intraperiton-eally injected with 0.75U/kg of insulin.The blood glucose values of 0,30,60,90 and 120 min were measured,respectively.8.Detection of the expression of macrophages polarization genes with q RT-PCR:The total RNA was transcribed to c DNA.q RT-PCR was used to detect and analyze the expression of the target genes（PAI-1,LRP-1,TNF-α,CD11c,MCP-1,IL-1β,CD206,IL-10,Fn1 and TGF-β1）.9.Statistical method:Single factor analysis of variance was carried out by SPSS 17.0 software,and P<0.05 was statistically significant.Results:1.HFD-WT mice fed for 14 weeks was up to 11.1 mmol/L of random blood glucose and obviously obtained more weight gain than ND-WT,the latter has only a slight increase.2.After 14 weeks,level of PAI-1 m RNA in WAT of HFD-WT mice increased significantly.3.After treatment with PAI-039 for 30 d,the weight of WT+PAI-039 mice decreased significantly.4.HFD induced hypertrophy of WAT.After knocking out PAI-1 and treatment with PAI-039,size of the adipocytes became smaller.5.HFD induced WAT metabolism disorder,and after knocking out PAI-1 and treatment with PAI-039,the metabolism was improved.6.HFD induced insulin resistance and increased insulin sensitivity after knocking out PAI-1 and treatment with PAI-039.7.HFD induced the increase of macrophages infiltration in WAT,which decreased after knocking out PAI-1 and treatment with PAI-039.8.Expression of M1 macrophage marker genes increased because of HFD induce.After knockout of PAI-1 and treatment with PAI-039,the expression of related genes decreased significantly,and the expression of M2 macrophage related marker genes increased.9.In the condition of ND,the expression of LRP-1 was no significant difference in adipose tissue between WT and Pai-1^-/-mice.In the condition of HFD,the expression of LRP-1 in adipose tissue of Pai-1^-/-mice showed significantly lower than that of in WT mice.Conclusions:Our findings provided support for PAI-1 contributing to the development of inflammation in adipose tissue and glucose transport process,and playing a role in the disordered metabolism in HFD-induced obesity.