Effects Of ZBTB16 Overexpression On Proliferation And Differentiation Of Bovine Intramuscular Preadipocytes

In the production of meat animals,the deposition and distribution of adipose tissue are closely related to the production efficiency and economic benefit The intramuscular fat content is a key factor that affects the quality of beef,and has important influence on the juiciness,tenderness and flavor of beef edible quality.Increasing intramuscular fat content is an important way to improve quality of beef.ZBTB16 gene plays an important role in animal ontogenesis,embryogenesis,reconstruction of the chromosome,and cell cycle regulation,etc.This study was to explore the effects of ZBTB16 on proliferation and differentiation of bovine intramuscular preadipocytes,providing a theoretical basis for regulating fat deposition by molecular breeding means.This study will be divided into the following four parts:The first part:The aims of this study were to establish the preadipocytes in vitro culture system of bovine intramuscular adipose tissue.Samples of longissimus dorsi muscle were collected to isolate intrairuscular adipose tissue pieces.Then preadipocytess were obtained from the adipose tissue by digestion with type ? collagenase.Morphological observation,growth curve,and Oil red O staining were performed,and mRNA expressions of PPAR?,C/EBP?,FABP4,ADIPOQ,ATGL,HSL,and LPL were measured during bovine preadipocytes differentiation by real-time fluorescent quantitative RT-qPCR.Results showed that bovine intramuscular preadipocytes began to adhere the wall after 6 hours and presented small spindle-shape and irregular triangle.The cells were highly homogeneous,adhered well and presented fibroblast-like morphology after subculturing.During 5th?11th day,the cells entered logarithmic growth phase and growth curve showed "S" shape.On 8th day of induced culture,the cells had a high differentiation rate and showed red by Oil red O staining.Fat content was significantly higher than the un-induced group(P<0.05),the cells presented typical characteristics of preadipocytess.PPAR?,C/EBP?,FABP4,ADIPOQ and ATGL mRNA levels gradually increased during preadipocytes differentiation(P<0.05).HSL mRNA level gradually decreased with preadipocytes differentiation significantly(P<0.05),while LPL mRNA level significantly increased in the early stage of preadipocytes differentiation and gradually decreased at the late stage of differentiation(P<0.05).In summary,the present study established a culture system and induced differentiation model for bovine intramuscular preadipocytes successfully,and provided a solid basis for further studying intramuscular fat deposition and beef quality.The second part:The aim of this study was to investigate effects of ZBTB16 overexpression on the proliferation and differentiation of bovine intramuscular preadipocytes.24 hours after transfection of adenovirus vector Ad-ZBTB16 and Ad-GFP,microscopic fluorescence photos were taken and morphological observation was performed.Results showed that the cells had normal morphology,showing a spindle and fibroblast-like shape,and were adherent firmly after transfection of adenovirus vector(MOI 280);transfection rate was over 90%by fluorescence observation.RT-qPCR and Western Blot were performed to detect the Ad-ZBTB16 overpression efficiency.Results showed that ZBTB16 mRNA level at 24 h post-transfection in Ad-ZBTB16 group was very significantly higher than that in blank control group and the Ad-GFP group(P<0.01).In addition,ZBTB16 protein level at 48 h post-transction in Ad-ZBTB16 group was very significantly higher than that in blank control group and Ad-GFP group(P<0.01).The data indicated ZBTB16 was successfully overexpressedin in bovine intramuscular preadipocytes,and Ad-ZBTB16 and Ad-GFP could be used for further studies.Moreover,to determine the cell proliferation activity,a CCK-8 kit was used.Results showed there was no significant difference among Ad-ZBTB16 group,Ad-GFP group and the blank control group(P>0.05).It revealed that ZBTB16 had no significant effect on bovine intramuscular preadipocutes proliferation.ZBTB16 mRNA and protein expressions during preadipocyte differentiation(-2,0.2,4,6,8 days)and ZBTB16 expressions in mature adipocytes were determined by RT-qPCR and Western Blot.Results showed that,with the bovine intramuscular preadipocytes differentiation,ZBTB16 mRNA and protein levels were significantly increased.Bovine intramuscular preadipocytes were transfected with adenovirus vector on Oth day during cell differentiation process and oil red O staining and intracellular GPDH activity determination were conducted on 8th day of cell differentiation.Results showed that,after oil red O staining,the lipid content in cells of Ad-ZBTB16 group was very significantly higher than that of blank control group and Ad-GFP group(P<0.01).GPDH activity test results showed that Ad-ZBTB16 cell activity was significantly stronger than the blank control group and the Ad-GFP group(P<0.01).In addition,mRNA expression levels of key adipogenic differentiation genes including PPAR?,CEBP/? and FABP4 mRNA expression levels were detected using RT-qPCR on the 1,2 and 8th day during cell differentiation,and FABP4 and PPAR? protein expressions on 8th day were detected by Western Blot.Results showed that PPAR?,CEBP/a,and FABP4 mRNA expressions of the Ad-ZBTB16 group were very significantly higher than that In blank control group and Ad-GFP group(P<0.01),and protein detection results were consistent with the testing results of mRNA.Thus,ZBTB16 had no significant effect on cell proliferation,but could significantly promote differentiation of bovine intramuscular preadipocytes.The third part:This study was to explore effects of ZBTB16 on the bovine intramuscular white preadipoceytes browning process and the related signaling pathways of preadipocytes differentiation.After transfection of the adenovirus vector,RT-qPCR was used to determinate mitochondrial ND1 expression level in cells on 8th day during cell differentiation process;changes of mitochondrias were observed by cell immunofluorescence staining.Results showed that Ad-ZBTB16 group had a significantly higher amount of mitochondrial ND1 expression than that in blank control group and Ad-GFP group(P<0.05),and it was consistent with immunofluorescence staining results.On the 8th day of cell differentiation process,UCP1 gene mRNA expression level and protein expression level in cells were detected by RT-qPCR and Western Blot;relative mRNA expression levels of the related genes of brown adipocyte browning including PRDM16,PGC-1?,CD137,Cox8b,TMEM26,Cidea and Tbxl were determinated by RT-qPCR.Results showed that UCP1 gene expression level in Ad-ZBTB16 group was very significantly higher than that in blank control group and Ad-GFP group(P<0.01),mRNA expressions of PGC-1?,TAEM26 and Tbxl in Ad-ZBTB16 group were very significantly higher than that in blank control group and Ad-GFP group(P<0.01),and mRNA expression leves of PRDM16,CD137,Cox8b and Cidea were significantly higher than that in blank control group and the Ad-GFP group(P<0.05).In order to further understand the regulatory mechanisms of ZBTB16 on the bovine intramuscular preadipocytes differentiation process,we extracted the total protein at 0 min,15 min,1 h and 24 h after the adenovirus transfection and determinated p38 MAPK,p-p38 MAPK,p44/42 MAPK,p-p44/42 MAPK protein expression levels in cells by Western Blot.According to the results,p-p38 MAPK protein expression level had no significant change in Ad-ZBTB16 group(P>0.05),and p-p44/42 MAPK protein expression level significantly increased in Ad-ZBTB16 group(P<0.01).In conclusion,we could preliminarily conclude that ZBTB16 overexpression of the bovine intramuscular preadipocytes could promote adipocyte browning,and p44/42 MAPK signalling pathway might involve in the adjustment process of ZBTB16 promoting bovine intramuscular preadipocytes differentiation.The fourth part:This study was to verify the previous test results and evaluate effects of ZBTB16 overexpression on the bovine subcutaneous preadipocytes differentiation and browning.After adenovirus vector transfection into cells,oil red O staining and determination of lipid content in cells were performed on the 8th day of the differentiation process.Results showed that the lipid content of Ad-ZBTB16 group was very significantly higher than that in blank control group and Ad-GFP group(P<0.01),which was consistent with the microscopic examination.The mRNA expressions of PPAR?,C/EBPa and FABP4 were measured by RT-qPCR on the 8th day of differentiation.Results showed that PPAR?,C/EBP? and FABP4 gene mRNA expressions in Ad-ZBTB16 group were very significantly higher than that in blank control group and the Ad-GFP group(P<0.01).After adenovirus vector transfection into cells,Mitochondrial ND1 gene expression was measured by RT-qPCR on the 8th day of differentiation,and mitochondrial changes were observed by cell immunofluorescence staining.Results showed that mitochondrial ND1 gene expression level of the Ad-ZBTB16 group was very significantly higher than that in the blank control group and Ad-GFP group(P<0.01),which was consistent with immunofluorescence staining results.The mRNA expressions of UCP1,PRDM16,PGC-1?,CD137,Cox8b and TMEM26 were measured during bovine preadipocytes differentiation by RT-qPCR on the 8th day.The results showed that,UCP1,CD137 and PRDM16 mRNA relative expression levels were very significantly higher in Ad-ZBTB16 group than that in blank control group and Ad-GFP group(P<0.01),and PGC-1 a,Cox8b and TMEM26 mRNA expression levels were significantly higher(P<0.05).Results showed that ZBTB16 could promote bovine subcutaneous preadipocytes differentiation and browning,which was consistent with the previous test results of ZBTB16 overexpression in the bovine intramuscular preadipocytes.