Study On Adipogenesis-related Gene Expression Profiles Of Intramuscular Preadipocyte From Japanese Black Cattle And Correlation With Intramuscular Fat

Japanese Black cattle are particularly characterized by a higher amount of marbling and unsaturated fatty acids (UFA), esp. monounsaturated fatty acids (MUFA) in adipose tissue than is observed in adipose tissue of other breeds. Beef marbling is considered as flecks of intramuscular fat within the cross section of longissimus muscle, consisting of the content and distribution of intramuscular fat (IMF). As the physical condition for marbling, IMF is one of significant carcass traits that affect tenderness, juiciness and taste of meat, which contributes directly to quality grading and economic value on especially Japanese market. The Major genes on IMF selected by DNA markers can be an effective method for improving carcass qualities, such as content of IMF and composition of fatty acids, due to the the impossibility of direct determination before slaughter, high cost and slow progress by using the conventional selection and breeding of meat qualities. This study is to investigate the temporal expression profiles of adipogenesis-related genes in bovine intramuscular preadipocyte (BIP) cells, to explore their correlation with IMF accumulation at slaughter by adding plasma of cattle at the growing stage into differentiation media, and to select the candidate gene of IMF desposition in Japanese Black Cattle, which will provide the theoretic basis for research on the mechanism of IMF accumulation in Japanese Black Cattle and improvement of beef quality. The main work is as follows:1. Isolation, in vitro culture and identification of bovine intramuscular preadipocyte (BIP) cells from Japanese Black CattleThe BIP cells were isolated from the6th and7th rib section of musculus longissimus thoracis of3Japanese Black cattle at the growing stage, followed by digestion with collagenase. The BIP cells were cultured in vitro and used for the generation of growth curve. The Oil red-O staining and RT-PCR were performed to examine the BIP cells induced by adipogenic differentiation media. The results showed that the separated BIP cells were highly homogenous and proliferative with a spindle shaped fibroblastic morphology. The growth curve of adherent BIP cell generated by cell number counting was in the typical shape of "S", according with a normal growth behavior of attachment-dependent cells in vitro. Based on the formula, the population doubling time of BIP cells was within43.3h. On8day of adipogenic stimulation, a large amount of ipid droplets were observed near the nucleus of the induced BIP cells with a red color after Oil red O staining. Based on RT-PCR, the glucose transporter1, GLUT-1, is detected in both BIP cells and the induced cells, while GLUT-4was low abundant expressed gene in BIP cells and not apparently affected by adipocyte differentiation. In this study, the isolated cells exhibited the typical morphological and expression characteristics of BIP cells, which could be recommended for further study.2. The temporal expression profiles of adipogenesis-related genes during the differentiation of BIP cellsThe frozen BIP cells were thawed, passeaged for2-3times, and induced in an differentiation medium supplemented with5mM octanate,50ng/ml Insulin,0.25mM Dex, lOmM Acetic acid and10%fetal bovine serum (FBS). Total RNA was extracted from the cells on Od,2d,4d,6d,8d after adipogenic stimulation and the quantitative real-time PCR was utilized to detect the mRNA expression of genes involved in adipogenesis. The results showed that dramatic increases in peroxisome proliferator-activated receptors-y (PPAR-y) and CCAAT/enhancer binding protein8(C/EBP-8) were detected after2d of induction. The expression of PPAR-y reached a peak at2d, decreased greatly on4d, and then remained a slight growth. Expression level of C/EBP-δ was peak at4d, down-regulated signigicantly at6d, and kept a steady increase subsequently. However, sterol regulatory element binding protein-1(SREBP-1) maintained a steady declining level without significant differenences between the control group and the experiment group treated with differentiation medium. Genes involved in fat synthesis including fatty acid synthase (FASN) and stearoyl-CoA desaturase (SCD) were showed a tendency to ascend first, descend later and ascend last, presenting their peaks on8d. Adipocyte fatty acid binding protein (aP2) exhibited an expression profile similar to that of fatty acid translocase (FAT/CD36) during the early and middle stages of differentiation, showing a rapid increase on2d and a marked reduction on4d. In the terminal differentiation, the expression of aP2was found to gradually up-regualte, reaching a maximum value of aP2on8d. On the other hand, the expression level of CD36continued a slight rise and kept a stable level. A little drop could be seen in the expression level of one of the fatty acid translocase, fatty acid transport protein-1(FATP-1). These results indicated that transactivation events seemed to be active in the early stage of adipocyte differentiation, while fatty acid synthesis and transmembrane activities play a more important role in the middle and the terminal stage of differentiation. Moreover, there might be species differences and lineage differences in the expression prolife of these genes.3. Correlation analysis between expression levels of adipogenesis-related genes in BIP cells and IMF performance at slaughter in Japanese Black Cattle47Japanese Black Cattle from4regions of Northeast Japan were assigned to one of two dietary treatments:a normal diet and a rice diet, respectively. Plasmas were obtained from blood samples of Japanese Black Cattle at the growing period (22month) and then used to prepare47differentiation media, respectively. The frozen BIP cells were thawed, passeaged for2-3times, cultured for2days in the growth medium and exposed to47kinds of differentiation media using2.5%inactivated plasma sample,7.5%FBS instead of10%FBS, respectively. The treatment was continued using the same differentiation medium plus2.5%plasma sample for8days. Total RNA was extracted from the cells on8d after adipogenic stimulation and the quantitative real-time PCR was used to detect the mRNA expression of genes involved in adipogenesis. The longissimus thoracis were collected at slaughter (27-31months) and scored according to Japanese Carcass Grading Standards, followed by the determination of fatty acid composition. The correlation analysis with gene expressions in BIP cells induced by differentiation medium was preformed. Based on the results, beef marbling standard (BMS) at slaughter ranging from27to31months displayed highly positive association with aP2(R=0.469, P＜0.01), SREBP-1(R=0.389, P＜0.01) and PPAR-y (R=0.533, P＜0.01) in BIP cells, respectively. The ratio of unsaturated fatty acids to saturated fatty acids (US/S) in longissimus muscle at slaughter was positively correlated with SREBP-1(R=0.398, P＜0.01). Using plasma from normal-fed cattle, BMS exhibited a significantly positive association with PPAR-y (R=0.428, P＜0.05). US/S of longissimus muscle at slaughter was highly positive correlated with gene expression of SREBP-1(R=0.600, P＜0.01) in the normal-fed cattle. As for the addition of plasma from rice-fed cattle, BMS displayed the highly positive association with aP2(R=0.469, P＜0.01), SREBP-1(R=0.389, P＜0.01) and PPAR-y (R=0.533, P＜0.01). However, there was no remarkable relation between US/S of longissimus muscle at slaughter and expression levels of these four genes. In conclusion, the finding in this study showed PPAR-y might be more reliable in predicting IMF content before slaughter, while SREBP-1could be considered as marker gene associated with fatty acid profile with high stability.