| Traditional view holds that adipose tissue plays an important role in controlling energy balance.However,recently,large numbers of researches have shown that adipose tissue is considered an endocrine organ which has impacts on many physiological and pathological processes,such as adipogenesis,lipometabolism,co-metabolism,immune response and vascular function,etc.Increasing evidences indicate that such progresses are mediated by signaling molecules,termed adipokines,e.g.,adiponectin,leptin,TNF-a,and IL-6,etc,secreted by adipocyte.Chemerin,as a noval adipokine,was highly expressed in placenta,white adipose tissue and liver.A series of studies have displayed that chemerin and its G protein-coupled receptor CMKLR1(or ChemR23)are involved in many physiological and pathology processes,such as,inflammatory response,lipid metabolism and vascular dysfunction.Epidemiologic survey has displayed that plasma chemerin levels are closely associated with obesity,body mass index and serum triglyceride levels.Mice with high-fat diet also show increased mRNA levels of chemerin in tissues,which has also been observed in the biological phenotype transformation from preadipocytes to mature adipocytes.CMKLR1 deficient mice show impaired adipogenesis and knockdown of chemerin/CMKLR1 seriously inhibits differentiation of preadipocytes into mature adipocytes and decreases the expression of lipid metabolic genes in these adipocytes.These studies reveal that chemerin plays an important role in adipogenesis.Chemerin participates in angiogenesis as well.Angiogenesis assays in vitro demonstrated that chemerin significantly induced the formation of capillary-like structures,increased the total tubule length,and the number of branches as well as total number of microtubules in these cultures.However,the underlying mechanism of chemerin promoting adipogenesis and/or angiogenesis remains elusive.In this study,we tried to investigate the molecular signaling mechanism of chemerin affecting the biofunctions of preadipocyte and neovascularization.The procession of adipogenesis is orchestrated by a successive activation of signaling molecule and transcription factors consolidating and transmitting information from extracellular factors to intracellular ones under suitable conditions for differentiation.Accumulating studies have validated an inseparable link between adipogenesis and angiogenesis during deposition of fat mass.The reciprocities between adipogenesis and angiogenesis may provide new therapeutic options for prevention and treatment of cardio-metabolic disorders by targeting the adipose vasculature.The procession is so complicated that an appropriate manipulable system(or model)should be carried out to investigate its mechanism.When subcutaneously injected into athymic mice,3T3-F442A preadipocytes could develop into fat pads that are indiscernible from normal adipose tissue.The model of fat pads can well demonstrate vascular adipogenesis,microenvironment and angiogenesis in the vivo.Regulation on transcription level is the most important regulation way of eukaryotic cell biological function.Gax gene,also known as Homobox gene,as a nuclear transcription factor,plays a significant role in regulating henogenesis,organofaction,in particular,cell growth,differentiation and migration in the cardiovascular system cells.Previous studies show that it participate in angiogenesis and adipocyte differentiation.However,whether Gax gene regulates effects of chemerin on adipogenesis and angiogenesis remains unclear.The similar research has not been reported at home and abroad.To sum up,we hypothesize that chemerin can activate some special signaling transduction network to promote adipogenesis and angiogenesis.Meanwhile,Gax can steadily express both in vitro and vivo,and block the signaling pathway,adipogenesis and angiogenesis which activated by chemerin.Part ≈ Effects of chemerin and Gax on proliferation and differentiation of 3T3-F442A preadipocyte and expression of VEGF in vitroMethods:1.Experimental grouping:According to the concentration of chemerin to divide the groups:In order to exame the preadipocytes biofuctional effects of the different concentration of chemerin,we divide six groups according the concentration of chemerin:0 ng/ml group(control group),20 ng/ml group,40 ng/ml group,60 ng/ml group,80 ng/ml group and 100 ng/ml group.2.According to whether there is LY294002(Akt inhibitor)and(or)chemerin,we divide four groups:PBS group(control group),LY294002 group,chemerin group and LY294002+ chemerin group.3.According to whether there is PD98059(ERK1/2 inhibitor)and(or)chemerin,we divide four groups:PBS group(control group),PD98059 group,chemerin group and PD98059+ chemerin group.4.According to whether there is Ad-Gax and(or)Ad-chemerin,we divide four groups:Ad-GFP group(control group),Ad-Gax group,Ad-chemerin group and Ad-Gax + Ad-chemerin group.5.MTT assay:The proliferation of 3T3-F442A cells was assessed by MTT assay.6.Flow cytometry:Cell cycle analysis was performed by using flow cytometry.7.Oil red O staining:to assess the differentiation of the different groups.8.Western blot:analysis:To exame expression of FABP4,VEGF,chemerin,CMKLR1,phosphorylated proteins of Akt/mTOR and ERK1/2 after different treatments.9.Cyto-immunofluorescence staining:To observe the expression of FABP4,VEGF,chemerin,CMKLR1,phosphorylated proteins of Akt/mTOR and ERK1/2 after different treatments.Results:1.The proliferation of 3T3-F442A cells was analyzed after treating the cells with recombinant mouse chemerin at various concentrations.MTT assays indicated that chemerin promoted 3T3-F442A cell proliferation in a concentration-dependent manner,with the 100 ng/mL concentration having the strongest effect.2.The effect of LY294002 on proliferation of preadipocyte:MTT assays indicated that,LY294002(20um/ml)inhibits 3T3-F442A cell proliferation and has an adverse effect on promotion of chemerin.3.The effect of PD98059 on proliferation of preadipocyte:MTT assays indicated that,PD98059(40um/ml)inhibits 3T3-F442A cell proliferation and has an adverse effect on promotion of chemerin.4.The effect of LY294002 on differentiation of preadipocyte:MTT assays indicated that,LY294002(40um/ml)inhibits 3T3-F442A cell differentiation and has an adverse effect on promotion of chemerin.5.The effect of PD98059 on differentiation of preadipocyte:MTT assays indicated that,PD98059(40um/ml)inhibits 3T3-F442A cell differentiation and has an adverse effect on promotion of chemerin.6.After 72 hours transfection of recombinant adenovirus:Immunofluorescence labeling showed that Gax was mainly overexpressed in nuclei and chemerin was highly expressed primarily in the cytoplasm.7.The effects of chemerin and Gax on cell cycle,cell proliferation and cell apoptosis:Compared with the Ad-GFP,Ad-Gax and Ad-chemerin+Ad-Gax groups,the Ad-chemerin group had a greater proportion of cells in the G2/M phase and a higher rate of cell proliferation.In contrast,Gax significantly inhibited 3T3-F442A cell proliferation,induced cell cycle arrest at the G0/G1 phase,and led toapoptosis.Meanwhile,the Ad-chemerin+Ad-Gax group exhibited moderate effects on cell-cycle distribution and proliferation,which indicated that the effects of chemerin on the cell cycle and proliferation were partially reversed by Gax.8.Oil Red O staining revealed that Gax blocked the differentiation of 3T3-F442A cells mediated by chemerin/CMKLR1.9.Compared with control group,chemerin group has increased levels of FABP4,VEGF,chemerin,CMKLR1,p-Akt,p-mTOR,p-p70S6K,p-4EBP1 and p-ERK1/2.Compared with control group,LY294002 group has dereased levels of FABP4,VEGF,p-Akt,p-mTOR,p-p70S6K and p-4EBP1.Compared with control group,PD98059 group has dereased levels of FABP4,VEGF,p-Akt,p-mTOR,p-p70S6K and p-4EBP1.Compared with control group,the expression of FABP4,VEGF,chemerin,CMKLR1,p-Akt,p-mTOR,p-p70S6K,p-4EBP1 and p-ERK1/2 of LY294002+chemerin group and PD98059+chemerin have no statistical difference.Compared with Ad-GFP group,Ad-chemerin group has increased levels of FABP4,VEGF,chemerin,CMKLR1,p-Akt,p-mTOR,p-p70S6K,p-4EBP1 and p-ERK1/2 while Ad-Gax group has decreased levels of FABP4,VEGF,chemerin,CMKLR1,p-Akt,p-mTOR,p-p70S6K,p-4EBP1 and p-ERK1/2.Compared with Ad-GFP group,the expression of FABP4,VEGF,chemerin,CMKLR1,p-Akt,p-mTOR,p-p70S6K,p-4EBP1 and p-ERK1/2 of Ad-Gax+Ad-chemerin group has no statistical difference.Conclusions:1.Chemerin stimulates proliferation and differentiation of preadipocyte via Akt/mTOR and ERK1/2 pathways.2.Chemerin accelerates cell cycle period.Gax enhances cell apoptosis,block cell cycle and weakens the effects of promotion of chemerin on cell cycle.3.Gax can inhibits the chemerin’s promotion of on cell differentiation.4.Gax can inhibits the chemerin’s activation on cell differentiation.Part Ⅱ Effects of chemerin and Gax on adipogenesis and angiogenesis in vivo Methods:1.In vivo animal model:To conduct de novo fat pad formation,2×1073T3-F442A preadipocytes were grown to 70%confluence and then injected subcutaneously into the hips of 4-to 6-week-old female athymic Balb/c nude mice.In the 1 week,4 week,7 week and 10 week,mice were anesthetized with 5%chloral hydrate,fat pads tissue formed by injected 3T3-F442A preadipocytes were excised,then the mice were killed by cervical dislocation.2.Experimental grouping:The mice were randomly divided into four groups:Ad-GFP group(control group),Ad-chemerin group,Ad-Gax group and Ad-chemerin+Ad-Gax group.To conduct de novo fat pad formation,2×1073T3-F442A preadipocytes were grown to 70%confluence and re-suspended in 0.2ml liquid(containing corresponding viral vectors)and then injected subcutaneously into the hips of 4-to 6-week-old female athymic Balb/c nude mice.3.Recombinant adenovirus transfect efficiency was assessed by using the FMT 4000TM fluorescence tomography imaging system(PerkinElmer,Hopkinton,MA)on the third days in vivo.4.Detection of microstructure and immunohistochemical:In the second week,mice were anesthetized with 5%chloral hydrate,fat pads tissue formed by injected 3T3-F442A preadipocytes were excised,then the mice were killed by cervical dislocation.The fat pads were fixed in 4%paraformaldehyde for subsequent microstructure and immunohistochemical analyses of FABP4,VEGF,CD31,p-ERK1/2,p-Akt,p-mTOR,p-p70S6K and p-4EBP1.5.Western blot:analysis:To exame activation of ERK1/2 and Akt/mTOR/p70S6K/4EBP1 signaling pathways in different treatment fat pads.Results:1.Microscopic examination revealed that subcutaneously implanted cells were still showed the morphological features of fibrocytes but out-of-order fibrous structure in 1 week after implantation.In 4 weeks some preadipocytes had transformed into adipocytes.In 7 weeks,almost all preadipocytes had transformed into adipocytes but the lipid droplets were not big enough.In 10 weeks,there was little difference in the appearance of fat pads from the epididymal adipose tissue.2.Fluorescence could be detected in vivo image system as early as the third day and GFP could be detected by immunohistochemistry in 2 weeks.3.H&E staining showed that there were lots of vessels in Ad-chemerin group compared with the other three groups(Ad-GFP group,Ad-Gax group and Ad-chemerin+Ad-Gax group).In contrast,the fat pad showed lower of vessels in the Ad-Gax group.4.There were lots of microvessels and more expression of VEGF and FABP4 in Ad-chemerin group compared with the other three groups(Ad-GFP group,Ad-Gax group and Ad-chemerin+Ad-Gax group).In contrast,the fat pad showed lowest of microvessels and more expression of VEGF and FABP4 in the Ad-Gax group.Immunohistochemical analysis demonstrated that over-expressed chemerin increased the expression of phosphorylated-ERK1/2,-Akt,-mTOR-p70S6K and-4EBP1 proteins,respectively,whereas over-expressed Gax could not only decrease that of these phosphorylated-proteins but also attenuate the effects of chemerin on the phosphorylation of these signaling proteins.Western Blotting analysis showed the same results of the immunohistochemical staining as mentioned above.Conclusions:1.After injection of 3T3-F442A cells,fad pat was clearly visible on the back of mouse and abundant in blood supply.The fat pad derived from 3T3-F442A cells was indistinguishable from normal adipose tissue in appearance.It provides an ideal model that could learn mechanisms of adipogensis and angiogenesis de novo through genetic intervention.2.Fluorescence could be detected in vivo image system as early as the third day and GFP could be detected by immunohistochemistry in 2 weeks.It showed that exogenous chemerin and/or Gax gene could be effectively transferred into fat pads and stably expressed.3.Chemerin enhanced adipogenesis and angiogenesis in fat pads.Inversely,Gax suppressed adipogenesis and angiogenesis in fat pads.In addition,Gax weakened the effects of chemerin on adipogenesis and angiogenesis.4.Chemerin over-expression activated ERK1/2 and Akt/mTOR/p70S6K/4EBP1 signaling pathways in fat pads.Conversely,Gax overexpression suppressed the activation of these pathways and partially attenuated the effects of chemerin in vivo. |
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