Abstract：Dihydrolipoyl dehydrogenase (Dld) and nicotinamide adenine dinucleotide (NADH) dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 9 (Ndufa9) gene were found to be related to energy metabolism and mitochondrial function. It was suggested that Dld and Ndufa9 may be intrinsically linked to white adipocytes browning. This study aimed to investigate the effect of Dld, Ndufa9 and their crotonylation modifications on the browning of primary subcutaneous white adipocytes in piglets (Sus scrofa). To investigate this intrinsic linkage, this study was conducted with primary subcutaneous white adipocytes from piglets, and induced their differentiation into mature adipocytes. Interference and overexpression treatment of Dld and Ndufa9, detection of expression of lipid metabolism genes using real-time quantitative PCR (qPCR) and Western blot. The results revealed that lipogenesis-related genes fatty acid binding protein 4 (Fabp4) and fatty acid synthase (Fasn) were significantly lower (P<0.05) and lipolysis-related genes adipose triglyceride lipase (Atgl) and hormone sensitivelipase (Hsl), browning of white fat-related genes peroxisome proliferator-activated receptor gamma coactivator 1 alpha (Pgc-1α) and PR domain containing 16 (Prdm16) and thermogenesis-related genes uncoupling protein 2 (Ucp2), bone morphogenetic proteins 4 (Bmp4), bone morphogenetic proteins 7 (Bmp7) and cell death-inducing DNA fragmentation factorα-like effector A (Cidea) were significantly up-regulated (P<0.05) after overexpression of Dld and Ndufa9 compared with the control group, which indicated that Dld and Ndufa9 promoted browning of subcutaneous white adipocytes in piglets. Next, mature adipocytes were treated with vorinostat (SAHA) and sodium crotonate (NaCr) to give the cells a crotonylation-modified environment. It was found that crotonylation modification of Dld and Ndufa9 similarly promoted browning of subcutaneous white adipocytes in piglets. And through co-immunoprecipitation (Co-IP), it was further discovered that DLD combined with NDUFA9. These results suggested that DLD, NDUFA9 and their crotonylation modifications promoted primary subcutaneous browning of white adipocytes in piglets. Browning of piglet subcutaneous white adipocytes was critical to lipid metabolism in pigs. Therefore, it also indicated that Dld and Ndufa9 were key factors in regulating pig lipid metabolism. The results of this study provide an experimental basis and rationale for further studies on pork quality improvement.
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