Studies On Differential Regulation Mechanism Of T10, C12-CLA On Lipid Deposition Between Subcutaneous And Longissimus Muscle Tissues In Growing Pigs

In this thesis, subcutaneous(SC) adipose and longissimus muscle tissue culture and preadipocyte culture techniques, cell biology and molecular biology techniques were employed to investigate the effects of t10,cl2-CLA on regulation of lipid deposition between SC adipose and longissimus muscle tissues in pig. There are three parts in this study:Part1:In vitro tissue culture system of SC adipose and longissimus muscle, and preadipocyte culture system taken from 30-days pig were constructed firstly,0.1 mmol/L bovine serum albumin(BSA, as control),100μmol/L t10,cl2-CLA were added to the cultures for 10 d, six replicates were performed for each treatment. At the end of a 10-d treatment period, cells were harvested and stored at-70℃. MTT assay(using preadipocyte culture system), GPDH activity assay and oil red O staining(using tissue culture system) were used to characterize the effects of t10,cl2-CLA on the proliferation and differentiation of pig SC and intramuscular(IM) preadipocytes in primary cultures. The results showed that:Proliferation and differentiation of SC preadipocytes were inhibited and differentiation of IM preadipocytes were promoted by adding 100μmol/L tl0.cl2-CLA(P<0.05), but no effect on proliferation of IM preadipocytes was observed(P>0.05). These results demonstrated that the effects of t10,cl2-CLA on proliferation and differentiation of SC preadipocytes differed from that on IM preadipocytes at cell morphology point of view.Part2:In this trial, in vitro tissue culture system and experiment design were same to partl. Real-time RT-PCR, enzyme activities assays and ELISA assays were used to study the effects of t10,cl2-CLA on the preadipocyte differentiation transcription factors, lipogenic and lipolytic enzymes, regulation hormones, lipogenic and lipolytic rates in SC and longissimus muscle tissue. The results showed that:the mRNA abundance of PPARγ, FAS and INSR, HSL activities, INSR protein level, lipolytic rates,TG content in SC adipose tissue and the mRNA abundance of HSL and LPL, LPL activities,GHR protein level in longissimus muscle tissue were decreased by adding t10,c12-CLA(P<0.05); by contrast, HSL mRNA abundance in SC adipose tissue and the mRNA abundance of PPARγ, ADD1 and INSR, ME activities, INSR protein level, lipogenic rates, TG content in longissimus muscle tissue were increased by adding t10,c12-CLA(P<0.05). These results illustrated that the effects of t10,cl2-CLA on lipid deposition of SC adipose tissue differed from that on longissimus muscle tissue at the lipid metabolism point of view and confirmed t10,cl2-CLA inhibited the lipid deposition of SC adipose and promoted the lipid deposition of longissimus muscle in pig further.Part3:In this trial, in vitro tissue culture system and experiment design were also same to partl. Real-time RT-PCR and ELISA assays were used to study the effects of t10,c12-CLA on the cytokines secreted by adipose and muscle tissue which exert the regulation action on both two tissues. The results showed that:the mRNA abundance and protein level of TNF-a, leptin and IL-15 in longissimus muscle tissue and IGF-1 protein level in SC adipose tissue were decreased by adding t10,c12-CLA(P<0.05); by contrast, the mRNA abundance and protein level of adiponectin, MSTN in longissimus muscle tissuee and TNF-a, IL- 6, leptin in SC adipose tissue were increased by adding t10,c12-CLA(P<0.05); But no effect on protein level of TNF-a in SC adipose tissue was observed (P>0.05). These results demonstrated that TNF-a, leptin, adiponectin, IL-15 and IL-6 play the important roles in molecular dialogue between adipocyte and myocyte, and these cytokines probably served as the mediator of tlO,cl2-CLA to exert different regulation effect on lipid deposition in SC and longissimus muscle tissue of pig.The general conclusion:In this study, the different mechanisms of effects of 100μmol/L t10,c12-CLA on regulation of lipid metabolism and deposition between SC adipose and longissimus muscle tissues in pig by affecting proliferation and differentiation of preadipocytes, differentiation transcription factors, lipogenic and lipolytic enzymes, regulation hormones, and cytokines secreted by two tissues were revealed, the inhibition effects of t10,cl2-CLA on lipid deposition in SC adipose and opposite effect on lipid deposition in longissimus muscle tissues were also confirmed further.