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.
LIU Yue-Xia,LIU Zun-Hai,LIANG Jun-Tong, et al. DLD, NDUFA9 and Their Crotonylation Promote Browning of Primary Subcutaneous White Adipocytes in Piglets (Sus scrofa)[J]. 农业生物技术学报, 2024, 32(4): 820-832.
[1] 陈艳珍. 2012. 猪肉品质的评定及影响因素[J]. 中国畜牧兽医, 39(7): 4.
(Chen Y Z.2012. Factors assessing and influencing pork quality[J]. China Animal Husbandry and Veterinary Medicine, 39(7): 4.)
[2] Ahmad W.2018. Dihydrolipoamide dehydrogenase suppression induces human tau phosphorylation by increasing whole body glucose levels in a C. elegans model of alzheimer's disease[J]. Experimental Brain Research, 236(11): 2857-2866.
[3] Arrabal S, Lucena M A, Canduela M J, et al.2015. Pharmacological blockade of cannabinoid CB1 receptors in diet-induced obesity regulates mitochondrial dihydrolipoamide dehydrogenase in muscle[J]. PLOS ONE, 10(12): e0145244.
[4] Babot M, Birch A, Labarbuta P, et al.2014. Characterisation of the active/de-active transition of mitochondrial complex I[J]. Biochimica et Biophysica Acta-boenergetics, 1837(7): 1083-1092.
[5] Candek-Potokar M, Zlender B, Lefaucheur L, et al.1998. Effects of age and/or weight at slaughter on longissimus dorsi muscle: Biochemical traits and sensory quality in pigs[J]. Meat Science, 48(3-4): 287-300.
[6] Carruthers N, Strieder-Barboza C, Caruso A, et al.2021. The human type 2 diabetes-specific visceral adipose tissue proteome and transcriptome in obesity[J]. Scientific Reports, 11(1): 17394.
[7] Chen Y, Wang Z, Wang Y, et al.2021. nhKcr: A new bioinformatics tool for predicting crotonylation sites on human nonhistone proteins based on deep learning[J]. Briefings in Bioinformatics, 22(6): bbab146.
[8] Jeremic N, Chaturvedi P, Tyagi S C2017. Browning of white fat: Novel insight into factors, mechanisms, and therapeutics[J]. Journal of Cellular Physiology, 232(1): 61-68.
[9] Jiang N, Yang M, Han Y, et al.2022. PRDM16 regulating adipocyte transformation and thermogenesis: A promising therapeutic target for obesity and diabetes[J]. Frontiers in Pharmacology, 13: 870250.
[10] Kelly R D, Chandru A, Watson P J, et al.2018. Histone deacetylase (HDAC) 1 and 2 complexes regulate both histone acetylation and crotonylation in vivo[J]. Scientific Reports, 8(1): 14690.
[11] Kucha C, Liu L, Ngadi M.2018. Non-destructive spectroscopic techniques and multivariate analysis for assessment of fat quality in pork and pork products: A review[J]. Sensors (basel), 18(2): 377.
[12] Liang J, Jia Y, Yu H, et al.2022. 5-Aza-2'-deoxycytidine regulates white adipocyte browning by modulating miRNA-133a/prdm16[J]. Metabolites, 12(11): 1131.
[13] Liao W, Xu N, Zhang H, et al.2022. Persistent high glucose induced EPB41L4A-AS1 inhibits glucose uptake via GCN5 mediating crotonylation and acetylation of histones and non-histones[J]. Clinical and Translational Medicine, 12(2): e699.
[14] Lin C, He X, Chen X, et al.2022. miR-1275 inhibits human omental adipose-derived stem cells differentiation toward the beige phenotype via PRDM16[J]. Stem Cells and Development, 31(23-24): 799-809.
[15] Liu Y, Li Y, Liang J, et al.2022. Non-histone lysine crotonylation is involved in the regulation of white fat browning[J]. International Journal of Molecular Sciences, 23(21): 12733.
[16] Lonergan S M, Huff-Lonergan E, Rowe L J, et al.2001. Selection for lean growth efficiency in duroc pigs influences pork quality[J]. Journal of Animal Science, 79(8): 2075-2085.
[17] Lonergan S M, Stalder K J, Huff-Lonergan E, et al.2007. Influence of lipid content on pork sensory quality within pH classification[J]. Journal of Animal Science, 85(4): 1074-1079.
[18] Ni J, Zhang H, Wang X, et al.2022. Rg3 regulates myocardial pyruvate metabolism via P300-mediated dihydrolipoamide dehydrogenase 2-hydroxyisobutyrylation in TAC-induced cardiac hypertrophy[J]. Cell Death & Disease, 13(12): 1073.
[19] Shen H, He T, Wang S, et al.2022. SOX4 promotes beige adipocyte-mediated adaptive thermogenesis by facilitating PRDM16-PPARγ complex[J]. Theranostics, 12(18): 7699-7716.
[20] Sreekumar S, Vijayan V, Singh F, et al.2022. White to brown adipocyte transition mediated by apigenin via VEGF-PRDM16 signaling[J]. Journal of Cellular Biochemistry, 123(11): 1793-1807.
[21] Staretz-Chacham O, Pode-Shakked B, Kristal E, et al.2021. The effects of a ketogenic diet on patients with dihydrolipoamide dehydrogenase deficiency[J]. Nutrients, 13(10): 3523.
[22] Stroud D A, Formosa L E, Wijeyeratne X W, et al.2013. Gene knockout using transcription activator-like effector nucleases (TALENs) reveals that human NDUFA9 protein is essential for stabilizing the junction between membrane and matrix arms of complex I[J]. Journal of Biological Chemistry, 288(3): 1685-1690.
[23] Su C, Xiao Y, Zhang G, et al.2022. Exogenous nicotinamide adenine dinucleotide attenuates postresuscitation myocardial and neurologic dysfunction in a rat model of cardiac arrest[J]. Critical Care Medicine, 50(2): e189-e198.
[24] Suárez-Zamorano N, Chevalier C, Stojanović O, et al.2015. Microbiota depletion promotes browning of white adipose tissue and reduces obesity[J]. Nature Medicine, 21(12): 1497-1501.
[25] Tan M, Luo H, Lee S, et al.2011. Identification of 67 histone marks and histone lysine crotonylation as a new type of histone modification[J]. Cell, 146(6): 1016-1028.
[26] Wan J, Liu H, Chu J, et al.2019. Functions and mechanisms of lysine crotonylation[J]. Journal of Cellular and Molecular Medicine, 23(11): 7163-7169.
[27] Wang Q, Li H, Tajima K, et al.2022. Post-translational control of beige fat biogenesis by PRDM16 stabilization[J]. Nature, 609(7925): 151-158.
[28] Wang S, Mu G, Qiu B, et al.2021. The function and related diseases of protein crotonylation[J]. International Journal of Biological Sciences, 17(13): 3441-3455.
[29] Wankhade U D, Lee J H, Dagur P K, et al.2018. TGF-β receptor 1 regulates progenitors that promote browning of white fat[J]. Molecular Metabolism, 16: 160-171.
[30] Wei W, Liu X, Chen J, et al.2017. Class I histone deacetylases are major histone decrotonylases: Evidence for critical and broad function of histone crotonylation in transcription[J]. Cell Research, 27(7): 898-915.
[31] Wu Q, Ke L, Wang C, et al.2018. Global analysis of lysine 2-hydroxyisobutyrylome upon SAHA treatment and its relationship with acetylation and crotonylation[J]. Journal of Proteome Research, 17(9): 3176-3183.
[32] Wu Q, Li W, Wang C, et al.2017. Ultradeep lysine crotonylome reveals the crotonylation enhancement on both histones and nonhistone proteins by SAHA treatment[J]. Journal of Proteome Research, 16(10): 3664-3671.
[33] Yang H, Yang K, Gu H, et al.2020. Dynamic post-translational modifications in obesity[J]. Journal of Cellular and Molecular Medicine, 24(3): 2384-2387.
[34] Yang X, Song J,Yan L J.2019. Chronic inhibition of mitochondrial dihydrolipoamide dehydrogenase (DLDH) as an approach to managing diabetic oxidative stress[J]. Antioxidants (basel), 8(2): 32.
[35] Zhao Y, Hao S, Wu W, et al.2022. Lysine crotonylation: An emerging player in DNA damage response[J]. Biomolecules, 12(10): 1428.
