Regulation Of Duck Preadoipocyte Differentiation And The Clone And Expression Patterns Of FAS And SCD1 Gene

As the main sites for lipid deposition, the adipose tissues play important roles in energy homeostasis. Preadipocytes are a kind of special precursor cells with the ability of generation and cell differentiation towards adipocytes which has been deeply researched. However, the biological characteristics and regulation mechanism of waterfowl preadipocytes differentiation remain unknown. In the current study, Peking duck was employed as material to investigate the biological characteristics and differentiation process of the preadipocytes in vitro. Also, we determined the effects of rosiglitazone (RSG), insulin, and dexamethasone (DEX) on duck preadipocyte differentiation using the primary preadipocytes isolated from duck subcutaneous and abdominal adipose tissues. Lastly, duck FAS and SCD1 gene were cloned and their expression patterns during preadipocyte differentiation were investigated. The main results of this study are as follows:(1) From 1 week old Nonghua meat duck subcutaneous adipose tissue we could isolate enough pure primary preadipocytes by the means of collagenase digestion. Primary preadipocytes began to grow quickly 3 days post seeding and maintained stable after day 7. After 8 days the preadipcoytes were induced to differentiate into mature adipocytes by the addition of insulin, DEX, IBMX, and oleate, which was stained red by oil red O. at that time, huge lipid droplets accumulated in the center of the adipocytes. In the process of preadipocyte differentiation, PPARymRNA was highly expressed at day 3, while expression of C/EBPa and FABP4 mRNA gradually increased and peaked at day 5 and 8, respectively.(2) Effect of RSG on duck preadipocyte differentiation was employed. RSG could promote lipid accumulation in the preadipocytes whether oleate is added in the medium. On one hand, RSG may significantly increase mRNA expression level of lipogenic genes including FAS, ACC, SCD 1, PLIN, and SREBP1c, as well as the protein contents of FAS and ACC (p<0.05). On the other hand, RSG could stimulate mRNA expression level and protein contents of ATGL (p<0.05), and promote free fatty acid release in the culture medium to enhance lipolysis (p<0.05). These results demonstrate that RSG could regulate duck preadipcoyte differentiation via influencing both lipogenesis and lipolysis pathway.(3) Effects of insulin and DEX on the preadipocyte differentiation isolated from subcutaneous and abdominal adipose tissue were investigated. Results showed that in the preadipocytes isolated from subcutaneous adipose tissue, DEX could increase expression level of several lipogenic genes(eg. FAS, SCD1, SREBPlc, GPAT, DGAT2), but insulin lead to decreased expression level of many lipogenic genes (FAS, ACC, SCD1, PPARy, ADRP, DGAT2). Insulin plus DEX treatment also resulted decreased expression level of most genes.In the preadipocytes isolated from abdominal adipose tissue, DEX would increase mRNA expression level of FAS, SCD1, GPAT, PPARy, and ADRP, however, insulin decreased expression level of FAS, SCD1, SREBPlc, ELOVL6, C/EBPa, and LPL. In the abdominal preadipocytes, insulin plus DEX exposure promoted most lipogenic gene expression above. No significant differences were observed for FAS and ACC protein contents in both subcutaneous and abdominal preadipocytes under DEX and insulin exposure. In subcutaneouspreadipocytes, DEX treatment would significantly increase ATGL protein contents(p<0.05), while in the abdominal preadipocytes insulin and DEX might enhance ATGL protein contents (p< 0.05).In addition, lipid accumulation in the subcutaneous preadipocytes was promoted by DEX and insulin(p< 0.05), while in the abdominal preadipocytes lipid accumulation could not been induced by insulin exposure alone. These results above indicate that effects of insulin and DEX on preadipocyte differentiation were site specific and further research is needed to investigate the molecular mechanisms.(4) Duck FAS and SCD1 gene were cloned and characterized, the CDS of duck them is 7545 bp and 1083 bp, coding 2515 and 361 amino acids, respectively. Both FAS and SCD1 gene are highly conserved among different poultry species, whose amino acids similarity is over 90%. During preadipocyte differentiation, expression level of duck FAS mRNA exhibited an increase-decrease-increase-decrease pattern, while expression of SCD1 gradually increased and peaked at the late differentiation period. Based on these results above, we can deduce that FAS and SCD1 gene may regulate duck preadipocyte differentiation at different phase.