The Effects And Mechanisms Of AMPK Agonists Inhibiting Inflammation Associated With Perivascular Adipose Tissue And White Adipose Tissue

Objective To study the effect and mechanism of AMPK agonists on macrophage-related inflammation in perivascular adipose tissue and white adipose tissue.Methods C57BL/6 mice obese model was established by high-fat feeding,and AMPK direct agonist A769662 and indirect agonist Metformin were given for 12 weeks to investigate the expressions of inflammatory factors related to macrophages in adipose tissue.Oral glucose tolerance test(OGTT)was performed by mouse tail tip blood collection;blood was taken from eyelids,serum was separated,blood glucose and blood lipid levels were measured;detected the expression of macrophage markers and related inflammatory factors by Western Blot and qPCR in mice epididymal fat,analyzed polarization of pro-inflammatory M1 subtype and anti-inflammatory M2 subtype,and prepared epididymal adipose tissue and PVAT paraffin sections to make evaluation of cell size by HE staining;Immunofluorescence was used to detect the expression of macrophage inflammatory factors in PVAT;Mice abdominal aorta(with fat)and abdominal aorta(without fat)were seperated,then performed the measurement of vascular function to evaluate the vascular endothelial and smooth muscle functions between them.Results(1)C57BL/6 mice body weight changed over time during modeling,compared with the Control group,the weight of the Model group increased significantly(P<0.01);compared with the Model group,the A769662 group and the Metformin group showed significantly lower body weight(P<0.01).There was no difference in food intake between each group of mice.(2)Compared with the Control group,the weight of liver(P<0.05),subcutaneous fat(P<0.01)and epididymal fat(P<0.01)increased in Model group,but the weight of scapulat fat decreased(P<0.05).Compared with the Model group,the weight of liver and epididymal fat decreased in A769662 group(P<0.05),the weight of scapulat fat(brown adipose tissue)increased significantly(P<0.01).Compared with the Model group,the weight epididymal fat decreased(P<0.05),the weight of scapulat fat increased significantly in Metformin group(P<0.01).(3)Fasting blood glucose levels in the Model group were significantly higher than in the Control group under fasting conditions,(P<0.01).The OGTT experiments showed that high-fat-fed impaired islet function and glucose tolerance impaired caused by obese mice.After giving A769662 and Metformin,glucose tolerance and insulin resistance improved.(4)Compared with the Control group,the Model group had elevated triglyceride levels(P<0.05)and total cholesterol levels(P<0.01),high density lipoprotein cholesterol levels were significantly lower(P<0.01).Compared with the Model group,A769662 and Metformin reduced the triglyceride levels.(5)The white adipose tissue(epididymal fat): HE staining results showed that,compared with the Control group,the size of epididymal fat cells in Model group of high-fat C57BL/6 mice was significantly increased(P<0.01).After administration of AMPK agonist,mice epididymal fat cells size were significantly reduced(P<0.01).(6)Epididymal fat(white adipose tissue): qPCR results showed that compared with the Control group,the expression of macrophage markers F4/80 and CD68 increased significantly(P<0.01),the expression of M1 subtype markers MCP-1(P<0.01)and IL-6(P<0.05)increased,and the expression of M2 subtype marker CD163 and IL-10 decreased significantly in Model group(P<0.01).Compared with the Model group,the expression of macrophage marker F4/80 in the A769662 and Metformin groups decreased(P<0.05),the expression of NF-κB,IL-6 and MCP-1 in M1 subtype was decreased(P<0.05),and the expression of CD163 and IL-10 in M2 subtype was significantly increased in A769662 group(P<0.01).(7)Western Blot results showed that the expression of macrophage marker F4/80 and M1 subtype marker MCP-1 in Model group was higher than that in Control group(P<0.05).Compared with Model group,the expression of macrophage marker F4/80(P<0.01)and M1 subtype marker NF-κB(P<0.05)in A769662 group was lower,while the expression of M2 subtype marker CD163 was higher(P<0.05).Compared with Model group,the expression of macrophage marker F4/80 and M1 subtype markers NF-κB in Metformin group decreased(P<0.05).(8)The PVAT: HE staining results showed that compared with the Control group,the PVAT adipocytes in the abdominal aorta of the Model group were significantly increased(P<0.01).After administration of AMPK agonist,PVAT adipocytes were significantly reduced(P<0.01).(9)The PVAT immunofluorescence results showed that,the expression of the Model group PVAT macrophage marker F4/80 and M1 subtype markers MCP-1,NF-κB are significantly higher than that in Control group(P<0.01),but the expression of CD163 in the M2 subtype marker was significantly lower(P<0.01).Compared with the Model group,the expression of CD163 of the PVAT M2 subtype marker in the A769662 group and the Metformin group was significantly increased(P<0.01),the expression of macrophage marker F4/80 and M1 subtype markers MCP-1 and NF-κB were significantly decreased(P<0.01).(10)Compared with the Control group,the vascular with PVAT in Model group showed impaired endothelial function and smooth muscle function,while the vascular smooth muscle function with PVAT was improved after intervention with AMPK agonist A769662 and Metformin.Conclusion AMPK agonists inhibit the weight gain of obese C576L/6 mice,improve glucose tolerance,reduce the level of triglyceride,reduce the volume and fat content of adipocytes in white adipose tissue and PVAT,decrease the number of white adipose tissue and PVAT macrophages,and promote the transformation of macrophages from M1 pro-inflammatory subtype to M2 anti-inflammatory subtype,also improve vascular smooth muscle function through adventitia pathway.