CYP1B1 Deficiency Inhibits Insulin Resistance And Regulates Macrophages Polarization In Obese Mice

With the continuous improvement of the social economy, obesity is becoming a major public health problem. Obese people moves difficultly because of overweight, and has many health risks more associated with high blood sugar, high blood pressure, high cholesterol, cardiovascular disease and other chronic diseases, which take a serious threat to human health. There are so many researches about obesity and diabetes, and many related genes and drugs have been studied. There is a long-term low level inflammation in adipose tissue of obesity associated with insulin resistance and type II diabetes and other metabolic syndrome, and local macrophages in adipose tissue involves in the regulation of inflammation. Macrophages play an important role in tissue regeneration and repair, healing and pathogen removal which needs inflammatory signals to involve in, but it will cause tissue damages if the inflammation exits for a long time. Macrophages accumulation happens in the white adipose tissue during obesity and other organs such as the liver, pancreas, intestine, brain, etc., but the process in white adipose tissue is most importance. Macrophages polarization contains mainly two types:the classic polarization (M1) and alternative activation (M2). The M1 macrophages produce TNFa, IL-6 and monocyte chemoattractant protein (MCP)-1, and can promote insulin resistance in adipose tissue. In contrast, the M2 macrophages produce anti-inflammatory cytokines, such as arginase (arginase-1), Mgll and IL-10 and so on. The M1 macrophage markers include CD11c, TNFa, IL6, and MCP1, and the M2 macrophage markers include CD206 and IL10. Macrophages in lean adipose tissue are mainly M2 which contributs to the organization repair and remodeling, and plays an important role in maintenance of insulin sensitivity. Cytochrome P450, family 1, subfamily B, polypeptide 1 (CYP1B1) is a core element of the CYP superfamily of enzymes, and mainly expresses in extrahepatic tissues, such as fat, ovary, placenta, testis, and so on. Since CYP1B1 highly expresses in cancer tissues, such as breast cancer and bladder cancer, and plays a key role in hormone carcinogenic responsed by catalyzing hydroxylation of the estrogen, researches about CYP IB 1 primarily focus on the field of cancer.The present study explores the role of CYP IB 1 gene in obesity and insulin resistance by CYP1B1 deficiency. And thus, we focus on epididymal adipose tissue macrophages and research the function of macrophages. The results demonstrate that CYP1B1 deficiency inhibits macrophages to migrate from the blood vessels to epididymal adipose tissue, promotes M2 polarization, reduces inflammation in epididymal adipose tissue and improves insulin resistance, in turn to resist obesity and improve insulin sensitivity. Then we studies the functional changes of bone marrow macrophage and peritoneal macrophage and find that the influence of CYP1B1 deficiency can not only change the function of macrophages existing in epididymal adipose tissue, but also affect the function of peritoneal macrophages and bone marrow macrophages. The effect may be continuous, as epididymal tissue macrophages and peritoneal macrophages are derived from bone marrow macrophages. The study contains three parts as follows:Part one:CYP1B1 deficiency improved obesity and insulin resistance induced by HFD in micePURPOSE:investigating the role of CYP1B1 deficiency in the high-fat diet-induced obesity and related insulin resistanceDESIGN and METHODS:① Used genotyping to screen CYP1B1-/- mice and breeds for subsequent experiments. ② Selected 12 CYP1B1-/-mice(KO) and 12 C57BL/6J wild-type mice (WT), which were all 4-weeks old male mouse and then were randomly divided into high-fat-diet group or low-fat-diet group:the KO-LFD group, WT-LFD group, KO-HFD group and WT-HFD group, and fed 11 weeks. ③ After fast overnight, took tail blood to test fasting blood glucose levle, and then gave 5% glucose(2g/kg) by ip. to detect the glucose tolerance and insulin tolerance. ④ Removed eyeballs to collect bloods, which were standed at room temperature for 30min, and then centrifuged at 12000rpm for 15min to separate serum to detect the serum triglycerides and insulin. ⑤Broke the vertebra to kill mice, then took tissues containing heart, liver, kidney, epididymal fat, mesenteric fat, inguinal fat and perirenal fat and weighed them, then saved these tissues in-80℃.RESULTS:①elected CYP1B1-/- mice successfully and stably breeded. ②After 11 weeks of feeding, KO-HFD goup had less weight-gain and epididymal adipose tissue weight than WT-HFD group, which was statistically significant. ③The fasting blood glucose, serum triglycerides and insulin levels of WT-HFD and KO-HFD group increased significantly compared with WT-LFD group, but KO-HFD group was significantly lower than WT-HFD group.④ By GTT and ITT test, the AUC area of WT-HFD and KO-HFD group increased significantly compared with WT-LFD group, but KO-HFD group was significantly lower than WT-HFD group, and the insulin homeostasis evaluation index (HOMA-IR) showed a same trend.CONCLUSIONS:High fat diet can induce different degrees of obesity and insulin resistance in WT and KO mouse, but CYPIBI dificiency can inhibit nutritional obesity and insulin resistance.Part two:CYP1B1 dificiency adjusted macrophages accumulation and polarization in epididymal adipose tissuePURPOSE:To study the inlfuence of CYP1B1 dificiency on macrophages accumulation and polarization in epididymal adipose tissue.DESIGN and METHODS: ① Isolated stromal vascular cells from epididymal adipose tissue, and labeled with PE-F4/80 antibody for flow cytometry. ②Extracted total RNA from epididymal adipose tissue to detect the M1 and M2 phenotype factor containing CCL-2, CD11c, CD206, Dectin-1 and Arg-1 and detect inflammatory cytokines such as TNFα, IL-6, MCP-1 by QRT-PCR.③Took three-color flow cytometry, which using PE-F4/80 to lable macrophages in SVCs, APC-CDllc to lable M1 macrophages and FITC-CD206 to lable M2 macrophages, to sort out M1 and M2 and then extracted RNA from sorted cells to detect CD11c, TNFa, IL-6, IL-10 and Dectin-1 with QRT-PCR.RESULTS:① The proportion of macrophages was different:KO-LFD goup was 13.1%, WT-LFD goup was 27.89%, KO-HFD goup was 28.54% and WT-HFD goup was 40.86%. QRT-PCR showed the mRNA expression of F4/80 group was significantly up-regulated in both KO-HFD group and WT-HFD, and the WT-HFD group raised more than KO-HFD group. ②QRT-PCR showed that the mRNA expression of CD11c, CCL-2 and CD206 significantly increased both in KO-HFD group and WT-HFD group, and the WT-HFD group raised more than KO-HFD group; and the mRNA expression of Arg-1 and Dectin-1 significantly increased in KO-HFD group compared with WT-HFD group.③ The mRNA expression of TNFα, IL-6 and MCP-1 were significantly increased in KO-HFD group and WT-HFD group, and the WT-HFD group raised more than KO-HFD group.④ Flow sorting results showed that M1 was 14.11% and M2 was 4.06% in WT LFD group, while in KO-LFD group M1 was 7.19% and M2 was 19.45%, suggesting that in CYP1B1 dificiency made residented macrophages in epididymal adipose tissue M2 polarization mainly; Compared with WT-HFD group, KO-HFD group had less M1 and more M2, and the trend is consistent with the low-fad diet fedding, suggesting that high-fat diets induced macrophages M1 polarization while CYP1B1 dificiency reduced M2 polarization. ⑤Results of QRT-PCR about sorting cells showed that:In Ml, CD11c, TNFα and IL-6 expressed significantly higher in WT-HFD group than KO-HFD group, while CD206, IL-10 and Dectin-1 also expressed higher, indicating that M1 macrophages secreted large amounts of proinflammatory cytokines and anti-inflammatory cytokines; In M2, CD11c, TNF a and IL-6 almost had no expression, and KO-HFD group expressed more CD206, IL-10 and Dectin-1 than WT-HFD group, suggesting that CYP1B1 dificiency reduced higher mRNA expression of M2-related markers and anti-inflammatory cytokine secretion.CONCLUSIONS:High fat diet induced many macrophages accumulation in epididymal adipose tissue, which mainly were M1 polarization, while KO mouse expressed less M1 and more M2 than WT mice both in protein and gene level, indicating that CYP1B1 dificiency reduced macrophages accumulation and M1 polarization by down-regulating pro-inflammatory cytokines such as TNFa and IL-6. In addition, M1 expressed more proinflammatory cytokines and some anti-inflammatory cytokines, while M2 mainly expressed anti-inflammatory cytokine.Part three:CYP1B1 dificiency influenced the polarization and funciton of bone marrow-derived macrophages and peritoneal macrophagesPURPOSE:Explored the origins where CYP1B1 dificiency influenced macrophages migration and polarization functions.DESIGN and METHODS:① Using 2 WT mice and 2 KO mice, and half of them were injected intraperitoneally with 100ul PKH26, the other half were not.48h later, killed mouse and extracted peritoneal macrophages, bone marrow macrophages and epididymal adipose tissue macrophages, then cells which had not injected with PKH26 were labled with PE-F4/80 antibody.② Using 4-week-old weaned C57/BL6J male mouse, WT and KO were 8 each and half were fed with LFD, the other half were fed with HFD. They were injected intraperitoneally with 100ul PKH26. After 28 days of continuous feeding, mice were double marked with PKH26 and FITC-F4/80 for Flow cytometry. ㏒eparated PMs and BMDMs from WT and KO mouse that induced 11 weeks by HFD to detect the phenotypic characteristics by 3-corlor flow cytometry and detect the expression inflammatory cytokines by QRT-PCR.④ Separated PMs and BMDMs from the normal three-month-old WT and KO mice, seeded in 12-well plate for cultures, cultured cells were maintained at 37℃,5% CO2 under humid conditions. 6h after the medium was changed to remove non-adherent cells. Then adding M-CSF(10ng/ml) to stimulate bone marrow and other osteoclast precursor cells to differentiate into monocyte-macrophage cells, medium was changed after 4 days, and then stimulated respectively with LPS (lOng/ml) or IL-4(40ng/ml). Cells were collected after 48h to detect polarization phenotype by flow cytometry, and RNA was extracted to detect the mRNA expression by QRT-PCR.⑤ Separated PMs from the normal three-month-old WT and KO mouse and seeded PMs in 24-well transwell chamber, and added respectively 500ul normal medium, conditioned medium from WT-HFD Group’s primary adipocytes, and MCP-1(100ng/ml) to incubate 3h at 37℃,5% CO2 under humid conditions, then staining with DAPI about lOmin, then observed results under fluorescent microscope.RESULTS:①In PMs, the PKH26+cells was 1.46% and PE-F4/80+cells was 4.23% in WT mice, while in KO mice the PKH26+cells was 29.07% and PE-F4/80+cells was 29.42%. In BMDMs, we didn’t find PKH26+cells in both WT and KO mice, and PE-F4/80+cells was 14.10% in WT, while in KO the PE-F4/80+cells was 10.77%. In addition, due to low body weight, we failed to extract SVCs in WT mice, while in KO mice the PKH26+SVCs was 2.28% and the F4/80+SVCs was 2.64%, suggesting that PKH26 had already labeled macrophages successfully in adipose tissue after 48h. ② With HFD-induced 28 days, PKH26+PE-F4/80+cells of PMs and ATMs in KO mice were reduced while they had no significant changes in WT mice. And besides PKH26+ cells, WT mice’s PMs and ATMs were hiher than KO mice. ③Extracting PMs and inducing with M-CSF, the proportion of M1 had no significant differences between WT and KO mice but KO’s M2 expressed higher than WT’s; QRT-PCR showed that WT highly expressed CD11c and IL-6, while KO highly expressed CD206, IL-10, Arg-1 and Dectin-1. ④Extracting BMDMs and inducing with M-CSF, the proportion of M1 and M2 had no significant differences between WT and KO mice; RT-PCR showed that WT highly expressed TNF α, while KO highly expressed CD206, Arg-1 and Dectin-1.⑤ Detecting PMs by flow cytometry, the proportion of M1 and M2 had no significant differences between WT and KO mice with LPS-inducing, while with IL-4 induction, the proportion of Ml had no significant change and the proportion of M2 increased significantly higher and particularly evident in KO mice; and QRT-PCR showed that WT highly expressed CD11c, TNF a and MCP-1 with LPS-inducing, and KO highly expressed CD206, IL-10 and Arg-1 with IL-4-inducing. ⑥ Detecting BMDMs by flow cytometry, the proportion of M1 had no significant differences between WT and KO mice with LPS-inducing, while with IL-4 induction, the proportion of M1 was up-regulated and the proportion of M2 increased significantly higher and particularly evident in KO; and QRT-PCR showed that WT highly expressed CD11c, TNF α and IL-6 with LPS-inducing, and KO highly expressed CD206, IL-10 and Arg-1 with IL-4-inducing.⑦Detecting PMs from 11 weeks HFD induced mice by flow cytometry, the M1 expression of WT was raised significantly higher than KO, but the M2 expression of WT was lower s than KO; while in BMDMs, the change was similar. ⑧ PMs from WT migrated more than that from KO by vitro migration assay, which had significant difference and suggested taht CYP1B1 gene knockout inhibited the migration ability of PMs.CONCLUSIONS:CYP1B1 dificiency reduced the migration and gathering activities of both PMs and BMDMs, and tended them to M2 polarization, which might cause by the down-regulation of M1 Phenotype and proinflammatory cytokinesand containing CD11c, TNF α and IL-6 and and the up-regulation of M2 phenotype and anti-inflammatory cytokine containing CD206, IL-10 and Arg-1. The impact to macrophages function may be derived from bone marrow, where macrophages were produced and derived.