Ndufa8 Regulates Animal White Adipose Tissue Browning By Increasing The Activity Of Respiratory Chain Complex Ⅰ

The high quality development of the livestock industry requires green and healthy development while providing people with better quality livestock products.Promoting the white adipose tissue browning in meat producing animals can help to increase the leanness of livestock and poultry and further improve the quality of livestock products,thus making them more suitable for people’s needs.Mammalian adipose tissue consists of white adipose tissue（WAT）and brown adipose tissue（BAT）.When the body is stimulated by external cold or adrenergic agonists,brown-like adipocytes are produced within WAT and have a similar thermogenic function to BAT,a process known as white adipose tissue browning.White adipose browning helps animals to consume excess energy in the WAT and is important for improving obesity and maintaining a healthy energy homeostasis.As the central energy metabolism of cells,mitochondria are extensively involved in a variety of metabolic processes including white adipose tissue browning,and as the site of cellular respiration,the respiratory chain complex has been shown to play an important role in white fat browning,Ndufa8 has been found to be involved in various physiological processes such as reproduction,embryonic development and oxidative stress by affecting the activity of respiratory chain complex Ⅰ.However,the mechanism of Ndufa8 regulation of white adipose browning in animals has not been reported.In this study,a model of white adipose browning was constructed in vivo and in vitro,and RT-q PCR,immunoblotting and fluorescent probes were used to investigate the role and mechanism of Ndufa8 in regulating white fat browning in animals by improving the activity of respiratory chain complex Ⅰ.This study provides a new theoretical basis for the involvement of respiratory chain complexes in regulating the mechanism of action of white adipose browning in animals,and provides new ideas for pig healthy breeding and genetic improvement of meat quality of livestock products.The following results were obtained:1.Ndufa8 enhances adipocyte respiratory chain complex Ⅰ activity and improves mitochondrial function.Based on preliminary proteomic sequencing results in the laboratory,Ndufa8 was screened for significant high expression in a white adipose browning model and Ndufa8 overexpression and interference vectors were successfully constructed.Overexpression of Ndufa8 significantly increased respiratory chain complex Ⅰ activity in a CL316,243（CL）-treated white adipose browning cell model（P<0.01）.Overexpression of Ndufa8 caused a significant increase in adipocyte ATP content（P<0.01）and NAD⁺/NADH levels（P<0.01）.The mitochondrial membrane potential of adipocytes was further examined using JC-1 staining and overexpression of Ndufa8 significantly upregulated the mitochondrial membrane potential（P<0.01）.overexpression of Ndufa8 was shown to reduce reactive oxygen species（ROS）in adipocytes by ROS fluorescence probes（P<0.05）.At the molecular level,Ndufa8 significantly promoted the expression of the respiratory chain complex functional genes Ndufa9,Uqcrc2,Cox4i and Atp5a1（P<0.05）.On porcine primary adipocytes,Ndufa8 similarly promoted the expression of mitochondrial functional genes（P<0.05）.This demonstrates that Ndufa8 enhances respiratory chain complex activity and improves mitochondrial function.2.Ndufa8 promotes white adipose browning in adipocytes by ameliorating mitochondrial oxidative stress.Using H₂O₂ to induce oxidative stress model in adipocytes,cellular MDA levels were significantly increased（P<0.05）,SOD activity was significantly decreased（P<0.01）and ROS content was significantly increased（P<0.01）after H₂O₂treatment,and the oxidative stress model was successfully constructed.Overexpression or interference vectors of Ndufa8 were transfected under the oxidative stress model,and Ndufa8 significantly reduced MDA levels（P<0.01）,significantly increased SOD activity（P<0.01）and significantly decreased cellular ROS levels（P<0.01）under the oxidative stress model.A model of complex Ⅰ damage was constructed by treating adipocytes with Rotenone（Rot）,a specific inhibitor of complex Ⅰ.Total ROS and mitochondrial ROS（mt ROS）levels in adipocytes were significantly increased after Rot treatment,while complex Ⅰ activity was significantly reduced（P<0.01）.Transfection with Ndufa8overexpression or interference vector under the complex Ⅰ injury model,Ndufa8 cleared the excessive accumulation of total cellular ROS and mt ROS caused by complex Ⅰ injury,while significantly increasing cellular ATP content and NAD⁺/NADH levels（P<0.05）,demonstrating improved mitochondrial function.Further co-treatment of adipocytes with CL and Rot,transfected with Ndufa8 overexpression or interference vector.Rot led to impaired complex Ⅰ function and prevented CL activation of the thermogenic protein UCP-1,and overexpression of Ndufa8 reversed the inhibitory effect of Rot on UCP-1 protein expression.Overexpression of Ndufa8 reversed the inhibitory effect of Rot on UCP-1protein expression and significantly promoted the m RNA expression of the thermogenic genes Ucp-1,Pgc1αand Prdm16（P<0.05）.The above experiments demonstrate that Ndufa8promotes white lipid browning by enhancing respiratory chain complex function and by improving cellular oxidative stress.3.Ndufa8 improves mitochondrial function and thus promotes lipolysis and white lipid browning in animal adipose tissue.On CL-treated browning models,transfected with Ndufa8 overexpression or interference vectors.Overexpression of Ndufa8 significantly reduced MDA content（P<0.01）and significantly increased SOD levels（P<0.01）.Ndufa8significantly inhibited m RNA expression of the lipid synthesis genes Fabp4 and Fasn（P<0.05）,while promoting the expression of the lipolysis genes Lipe and Pnpla2（P<0.05）,with the same effect obtained at the protein level.Oil Red O and Bodipy staining showed that Ndufa8 significantly reduced the level of lipid accumulation in adipocytes（P<0.01）.Ndufa8 significantly upregulated the expression of the thermogenic genes Ucp-1 and Prdm16（P<0.05）,while significantly promoting the expression of the thermogenic proteins UCP-1 and PGC1α（P<0.01）.immunofluorescence of UCP-1 showed that Ndufa8significantly promoted the expression of UCP-1 protein（P<0.01）.Meanwhile,overexpressed or interfered with Ndufa8 in CL-treated browning model mice.Ndufa8promoted m RNA expression of the mitochondrial functional genes Ndufa9,Cidea and Ucp-2 in mice inguinal white adipose tissue（i WAT）（P<0.05）.Ndufa8 significantly inhibited protein expression of FASN in mice i WAT（P<0.01）and promoted ATGL protein expression（P<0.05）.The expression of the thermogenic proteins UCP-1 and PGC1αwas significantly promoted（P<0.01）.On porcine primary adipocytes,Ndufa8 similarly promoted the expression of lipolysis and thermogenic genes（P<0.05）.This demonstrates that Ndufa8 promotes lipolysis and white adipose browning in animals by improving mitochondrial function in animal adipose tissue.This study demonstrates that Ndufa8 enhances respiratory chain complex Ⅰ activity and improves mitochondrial function,which in turn promotes lipolysis and white adipose browning in animal adipose tissue.It provides a new molecular pathway for the study of white adipose browning in animals and a new research idea for the genetic improvement of livestock and poultry meat quality in livestock production.