The Function And Mechanism Of Complement-C1Q/tumor Necrosis Fator-related Protein 6 During Adipogenesis Of Porcine Adipocytes

The deposition of fat is a complex biological process, which regulated by many transcription factors and adipokines. And adipose tissue deposition and distribution are key factors for meat quality and flavor. Therefore, it is critically important to more fully understand the molecular mechanisms of adipokines in the adipogenesis and meat quality improvement in animal husbandry.C1q/tumor necrosis factor-related protein 6(CTRP6), an adipokine, belong to the C1 qTNF family. The structure of CTRP6 is similar to adiponectin. A recent study provides the evidence that CTRP6 was found in serum and its expression in fat tissues was enhanced in ob/ob and adiponectin null-mice. Rosiglitazone treatment decreased CTRP6 expression in adipose tissue. Moreover, in C2C12 cell, CTRP6 could stimulate fatty acid oxidation via the activation of AMPK. In our previous study has shown that CTRP6 expression abundance has changed during the process of 3T3-L1 cell differentiation. Based on the above findings, we provide a hypothesis that CTRP6 may be involved in the adipogenesis of porcine intramuscular adipocytes and subcutaneous adipocytes. To investigate the CTRP6 biological function, its expression was firstly examined in the process of proliferation and differentiation of porcine adipocytes. The effect of CTRP6 on adipocyte proliferation and differentiation were evaluated by flow cytometry, immunofluorescence, Oil Red O, real-time PCR and western blot. And the specific inhibitors of Erk1/2 and p38 MAPK signaling(U0126 and SB208530) were used to clarify the interaction between CTRP6 and MAPK signaling. Then the relationship between CTRP6 and adiponectin receptor(AdipoR) was detected by CoIP technology. Finally, the function of CTRP6 was also explored in vivo.The specific results are as follows:1. The expression of CTRP6 was decreased in the porcine adipocyte proliferation and was predominantly localized in the cytoplasm of intramuscular adipocytes and subcutaneous adipocytes. After inducing the differentiation, CTRP6 expression was gradually increased.2. Porcine CTRP6-shRNA lentivirus interfering vector was constructed. Virus titer was6.2×107 pfu/ml, then infected porcine intramuscular adipocytes and subcutaneous adipocytes.CTRP6 mRNA and protein expression was remarkably reduced. Knockdown of CTRP6 could promote the proliferation of adipocytes via regulating the expression of CyclinB and CyclinE.But during the process of adipocytes differentiation, CTRP6-shRNA suppresses the levels of adipogenic makers(P<0.01)..3. The data confirmed that adipocytes proliferation was inhibited by CTRP6 recombinant protein. However, the mRNA and protein expression of adipogenic marker genes PPARγ and aP2 were increased in adipocytes by CTRP6 recombinant protein(P<0.01).4. CTRP6 was targeted by miR-29 a in porcine intramuscular adipocytes and subcutaneous adipocytes. MicroRNA-29 a regulates the proliferation and differentiation of porcine intramuscular and subcutaneous adipocytes by target gene CTRP6.5. CTRP6 promoted differentiation of porcine intramuscular and subcutaneous adipocytes through Erk1/2(p-Erk1/2) and p38 MAPK signaling pathway. But during the proliferation of intramuscular adipocyte, CTRP6 play an major role in the p38 MAPK signaling pathway. Moreover, AdipoR1, not AdipoR2, was first identified as a receptor of CTRP6 in intramuscular adipocyte.6. Lentivirus-mediated downregulation of CTRP6(LC) in mice increased brown fat markers in iWAT, eWAT and promoted additional browning in BAT. LC mice were much thinner than control mice, independent of the quantity of food intake. These LC mice reduced the size of adipocyte, insulin resistance, and regulated the levels of serum leptin and adiponectin.In summary, the results indicated that CTRP6 which was targeted and regulated bymiR-29 a could inhibited the porcine adipocytes proliferation and promoted thedifferentiation of porcine adipocytes through MAPK signalling pathway. AdipoR1 wasidentified as a receptor of CTRP6 in porcine intramuscular adipocyte. In vivo, knockdownof CTRP6 expression could improve insulin sensitivity, glucose tolerance, and promotebrown adipogenesis.