Rnf20基因对脂肪细胞中支链氨基酸含量的影响

doi:10.16431/j.cnki.1671-7236.2026.01.026

Abstract

Abstract:

Objective The aim of this study was to investigate the effects and regulatory mechanisms of ring finger protein 20 （RNF20） on the metabolism of branched-chain amino acids （BCAAs） in gonadal white adipose tissue （gWAT） in mice and 3T3-L1 adipocytes. Method Male adipocyte-specific Rnf20 gene knockout mice （Rnf20^flox/flox； adiponectin Cre⁺， ASKO） and littermate wild-type mice （Rnf20^flox/flox； adiponectin Cre^-， WT） were used in this study. Targeted metabolomics was used to detect the levels of BCAAs in gWAT and serum， and Real-time quantitative PCR was used to analyze the expression of BCAAs catabolic genes （Bcat2， Bckdha， etc.） in gWAT， liver， and gastrocnemius muscle from both groups of mice. To examine the effect of Rnf20 gene on BCAAs metabolism in vitro， siRNAs were designed to down-regulate the expression of Rnf20 gene in 3T3-L1 preadipocytes and differentiated into mature adipocytes. The adipogenic differentiation efficiency of interference group （siRNF20） and control group （siNC） was evaluated through oil red O staining， and the expression of adipogenic differentiation marker genes， lipolysis genes， and BCAAs catabolism genes were detected. The contents of BCAAs in adipocyte culture medium of siRNF20 and siNC groups were detected. Result Compared with WT mice， the contents of BCAAs （leucine， isoleucine and valine） in gWAT of ASKO mice were extremely significantly increased （P<0.01）， and the expression of BCAAs catabolism related genes Bcat2， Acad5， Ehhand and Hibch were significantly or extremely significantly decreased （P<0.05 or P<0.01）. However， there was no significant difference in the content of BCAAs in serum and the expression of BCAAs catabolism related genes in liver and gastrocnemius muscle （P>0.05）. Compared with siNC group， knockdown of Rnf20 gene in vitro could inhibit adipogenic differentiation of 3T3-L1 cells，with a significant decrease in the expression of the adipogenic marker gene Pparγ （P<0.05）， and an extremely significant increase in the expression of the lipolytic gene Adrb3 （P<0.01）. The expression of key genes Bcat2 and Bckdha involved in BCAAs catabolism in mature adipocytes decreased with the knockdown of Rnf20 gene， and the BCAAs content in siRNF20 group culture medium was extremely significantly increased （P<0.01）. Conclusion Rnf20 gene in adipocytes regulated the key genes Bcat2 and Bckdha involved in BCAAs catabolism through transcriptional regulation， maintaining the homeostasis of BCAAs within the cell. Knockdown of Rnf20 gene could inhibite BCAAs catabolism， leading to the accumulation of BCAAs in adipose tissue. The results provided a new direction for in-depth exploration of the function of Rnf20 gene and new genetic materials for genetic breeding research on fat deposition traits in large animals.

Key words: Rnf20 gene; adipocytes; branched-chain amino acids

CLC Number:

S852.16⁺3

ZHANG Liqiong, ZHAO Ying, ZHANG Liuzhe, WANG Yanfang, GUO Huihui. Effect of Rnf20 Gene on Branched-chain Amino Acid Content in Adipocytes[J]. China Animal Husbandry & Veterinary Medicine, 2026, 53(1): 287-298.

Figures/Tables 11

Table 1

Table 2

Table 3

Fig.1

Table 4

Fig.2

Fig.3

Fig.4

Fig.5

Table 5

Fig.6

References 30

[1]	WANG Y， ZHOU P， ZHOU X， et al. Effect of host genetics and gut microbiome on fat deposition traits in pigs［J］. Frontiers in Microbiology， 2022， 13： 925200.
[2]	SHEN L， BAI X， ZHAO L， et al. Integrative 3D genomics with multi-omics analysis and functional validation of genetic regulatory mechanisms of abdominal fat deposition in chickens［J］. Nature Communications， 2024， 15（1）： 9274.
[3]	TSENG Y H， CYPESS A M， KAHN C R. Cellular bioenergetics as a target for obesity therapy［J］. Nature Reviews. Drug Discovery， 2010， 9（6）： 465-482.
[4]	JIN W， PATTI M E. Genetic determinants and molecular pathways in the pathogenesis of type 2 diabetes［J］. Clinical Science， 2009， 116（2）： 99-111.
[5]	FUCHS G， SHEMA E， VESTERMAN R， et al. RNF20 and USP44 regulate stem cell differentiation by modulating H2B monoubiquitylation［J］. Molecular Cell， 2012， 46（5）： 662-673.
[6]	LIANG Q， XIA W， LI W， et al. RNF20 controls astrocytic differentiation through epigenetic regulation of STAT3 in the developing brain［J］. Cell Death and Differentiation， 2018， 25（2）： 294-306.
[7]	XU Z， SONG Z， LI G， et al. H2B ubiquitination regulates meiotic recombination by promoting chromatin relaxation［J］. Nucleic Acids Research， 2016， 44（20）： 9681-9697.
[8]	JEON Y G， NAHMGOONG H， OH J， et al. Ubiquitin ligase RNF20 coordinates sequential adipose thermogenesis with brown and beige fat-specific substrates［J］. Nature Communications， 2024， 15（1）： 940.
[9]	JEON Y G， LEE J H， JI Y， et al. RNF20 Functions as a transcriptional coactivator for PPARγ by promoting NCoR1 degradation in adipocytes［J］. Diabetes， 2020， 69（1）： 20-34.
[10]	ZHAO Y， PAN J， CAO C， et al. RNF20 affects porcine adipocyte differentiation via regulation of mitotic clonal expansion［J］. Cell Proliferation， 2021， 54（12）： e13131.
[11]	WANG T J， LARSON M G， VASAN R S， et al. Metabolite profiles and the risk of developing diabetes［J］. Nature Medicine， 2011， 17（4）： 448-453.
[12]	PATRICK M， GU Z， ZHANG G， et al. Metabolon formation regulates branched-chain amino acid oxidation and homeostasis［J］. Nature Metabolism， 2022， 4（12）： 1775-1791.
[13]	SHE P， REID T M， BRONSON S K， et al. Disruption of BCATm in mice leads to increased energy expenditure associated with the activation of a futile protein turnover cycle［J］. Cell Metabolism， 2007， 6（3）： 181-194.
[14]	ISLAM M M， WALLIN R， WYNN R M， et al. A novel branched-chain amino acid metabolon. Protein-protein interactions in a supramolecular complex［J］. The Journal of Biological Chemistry， 2007， 282（16）： 11893-11903.
[15]	GREEN C R， WALLACE M， DIVAKARUNI A S， et al. Branched-chain amino acid catabolism fuels adipocyte differentiation and lipogenesis［J］. Nature Chemical Biology， 2016， 12（1）： 15-21.
[16]	ARANY Z， NEINAST M. Branched chain amino acids in metabolic disease［J］. Current Diabetes Reports， 2018， 18（10）： 76.
[17]	SON S M， PARK S J， LEE H， et al. Leucine signals to mTORC1 via its metabolite acetyl-coenzyme A［J］. Cell Metabolism， 2019， 29（1）： 192-201.e7.
[18]	ZHAO Y， YANG S， WANG Y， et al. Molecular characterization， expression profiling， and SNP analysis of the porcine RNF20 gene［J］. Animals， 2020， 10（5）：888.
[19]	TIAN H， NI Z， LAM S M， et al. Precise metabolomics reveals a diversity of aging-associated metabolic features［J］. Small Methods， 2022， 6（7）： e2200130.
[20]	SONG J W， LAM S M， FAN X， et al. Omics-driven systems interrogation of metabolic dysregulation in COVID-19 pathogenesis［J］. Cell Metabolism， 2020， 32（2）： 188-202.e5.
[21]	LIANG X， TAO C， PAN J， et al. Rnf20 deficiency in adipocyte impairs adipose tissue development and thermogenesis［J］. Protein & Cell， 2021， 12（6）： 475.
[22]	MA Q X， ZHU W Y， LU X C， et al. BCAA-BCKA axis regulates WAT browning through acetylation of PRDM16［J］. Nature Metabolism， 2022， 4（1）： 106-122.
[23]	YONESHIRO T， WANG Q， TAJIMA K， et al. BCAA catabolism in brown fat controls energy homeostasis through SLC25A44［J］. Nature， 2019， 572（7771）： 614.
[24]	XU H， WANG X， GENG G， et al. Association of circulating branched-chain amino acids with cardiovascular diseases： A mendelian randomization study［J］. Nutrients， 2023， 15（7）： 1580.
[25]	ZHAO Y， CHEN C， PAN J， et al. Adipocyte Rnf20 ablation increases the fast-twitch fibers of skeletal muscle via lysophosphatidylcholine 16∶0［J］. Cellular and Molecular Life Sciences， 2023， 80（9）： 243.
[26]	HE A， CHEN X， TAN M， et al. Acetyl-CoA derived from hepatic peroxisomal β-oxidation inhibits autophagy and promotes steatosis via mTORC1 activation［J］. Molecular Cell， 2020， 79（1）： 30-42.e4.
[27]	BATRAN R AL， GOPAL K， CAPOZZI M E， et al. Pimozide alleviates hyperglycemia in diet-induced obesity by inhibiting skeletal muscle ketone oxidation［J］. Cell Metabolism， 2020， 31（5）： 909-919.e8.
[28]	LACKEY D E， LYNCH C J， OLSON K C， et al. Regulation of adipose branched-chain amino acid catabolism enzyme expression and cross-adipose amino acid flux in human obesity［J］. American Journal of Physiology-Endocrinology and Metabolism， 2013， 304（11）： E1175-E1187.
[29]	HSIAO G， CHAPMAN J， OFRECIO J M， et al. Multi-tissue， selective PPARγ modulation of insulin sensitivity and metabolic pathways in obese rats［J］. American Journal of Physiology. Endocrinology and Metabolism， 2011， 300（1）： E164-E174.
[30]	DENG Z， AI H， SUN M， et al. Mechanistic insights into nucleosomal H2B monoubiquitylation mediated by yeast Bre1-Rad6 and its human homolog RNF20/RNF40-hRAD6A［J］. Molecular Cell， 2023， 83（17）： 3080-3094.e14.

基因 Genes	引物序列 Primer sequences （5′→3′）		片段大小 Fragment length/bp
18S rRNA	F：GTAACCCGTTGAACCCCATT	R：CCATCCAATCGGTAGTAGCG	151
Rnf20	F：GTGAACCGGTACTGGAGCCAG	R：GAAAGCTGGCTCTTGCCCATC	192
Bcat2	F：ACAGACCACATGCTGATGGTG	R：CTGGGTGTAGCGTGAGGTTC	91
Bckha	F：CTCCTGTTGGGACGATCTGG	R：CATTGGGCTGGATGAACTCAA	258
Bckhb	F：CTTCCGATGCACTGTTGGTTT	R：GATTTCCGCAATAGCTGTAGCA	133
Acad8	F：TTTACATGGCGATGTTGCGG	R：TTAGCCCCAAGGAAGGATCG	123
Hibch	F：CCGGTGGAACCACTACACTC	R：TCTGAAGGAGCTGACCCTCG	100
Hibadh	F：ACCTTGGAATCAGGTCAGGC	R：GGGTTGTAAGTGTCGCTGGA	94
Pparγ	F：CGGAAGCCCTTTGGTGACTT	R：CCTCGATGGGCTTCACGTTC	154
Cebpα	F：CCCTTGCTTTTTGCACCTCC	R：GCTTTCTGGTCTGACTGGGG	118
Fas	F：GGGTCTATGCCACGATTC	R：GTGTCCCATGTTGGATTTG	272
Acc	F：GAGAGGGGTCAAGTCCTTCC	R：ACATCCACTTCCACACACGA	152
Scd1	F：CTGACCTGAAAGCCGAGAAG	R：GCGTTGAGCACCAGAGTGTA	172
Adrb3	F：TTTGATGGCTATGAAGGTGCG	R：CGTTGCTTGTCTTTCTGGAGG	169
Hsl	F：GACTCACCGCTGACTTCC	R：TGTCTCGTTGCGTTTGTAG	161
Atgl	F：CGTCACCTGTGCCTTACTC	R：GGGCTCAAACTCCCAAAC	407

项目 Items	含量 Contents/（μmol/kg）		ASKO/WT	P值 P-value
项目 Items	WT	ASKO	ASKO/WT	P值 P-value
异亮氨酸 Ilu	24.36±16.52	136.22±49.56	5.59	<0.01
亮氨酸 Leu	20.89±13.33	113.01±45.69	5.41	<0.01
缬氨酸 Vel	39.36±24.86	151.86±51.35	3.86	<0.01

项目 Items	含量 Contents/（μmol/L）		ASKO/WT	P值 P-value
项目 Items	WT	ASKO	ASKO/WT	P值 P-value
异亮氨酸 Ilu	0.12±0.03	0.12±0.02	1.04	0.78
缬氨酸 Vel	0.16±0.02	0.16±0.02	0.98	0.82
亮氨酸 Leu	0.08±0.02	0.08±0.01	0.96	0.77

项目 Items	含量 Contents/（μmol/L）		ASKO/WT	P值 P-value
项目 Items	siNC	siRNF20	ASKO/WT	P值 P-value
异亮氨酸 Ilu	0.48±0.05	0.62±0.05	1.28	<0.01
亮氨酸 Leu	0.25±0.02	0.29±0.02	1.18	<0.01
缬氨酸 Vel	0.47±0.02	0.52±0.01	1.11	<0.01

Effect of Rnf20 Gene on Branched-chain Amino Acid Content in Adipocytes

RichHTML

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 11

References 30

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	CHU Hongen, LIU Yuan, FENG Xue, BAI Xue, YANG Mengli, LI Juan, HE Lixia, LIU Shuang, FENG Lan, MA Yun. Cloning,Bioinformatics and Tissue Expression Analysis of Bovine TGFB1 Gene [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(6): 2506-2518.
[2]	ZHANG Qianwen, LI Jingping, YUE Chengguang, WANG Jiajun, LI Zhonghui, LI Wenrong. Study on the Proliferation and Differentiation Characterization of Precursor Adipocytes from Rump Adipose in Kazakh Sheep During Fetal Period [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(10): 4563-4574.
[3]	HAN Xu, PENG Yu, ZHANG Lilan, LIU Tianxia, LI Hongqiang, TAO Cong. Study on the Molecular Characteristics of Thermogenesis in Tibetan Pig Adipocytes [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(10): 4796-4808.
[4]	LIU Xiaotian, JIA Yuxin, GAO Feng, YUAN Xinxin, WANG Yongfa, XUE Yuheng, MENG Jun, ZENG Yaqi, LI Xiaobin. Effects of Supplemental Feeding of Branched-chain Amino Acids to Mares on Growth Performance and Fecal Flora of Lactating Foals [J]. China Animal Husbandry & Veterinary Medicine, 2024, 51(8): 3355-3364.
[5]	GUO Panpan, LI Qiang, LI Xiangzi, YAN Changguo, JIN Xin. Effect of Overexpression of DGAT2 Gene on Differentiation of Yanbian Bovine Preadipocytes [J]. China Animal Husbandry & Veterinary Medicine, 2023, 50(8): 3045-3055.
[6]	MENG Chaoqun, LI Chengping, ZHAO Wei, QIN Xuyong, ZHOU Guoli. Effect of miR-137-3p on Adipogenic Differentiation of Preadipocytes 3T3-L1 by Targeting MAX Gene [J]. China Animal Husbandry & Veterinary Medicine, 2023, 50(5): 1928-1937.
[7]	ZHANG Dianqi, MA Xinhao, YANG Zhimei, MEI Chugang, ZAN Linsen. Bioinformatics Analysis of PAI1 Gene and Its Effect on the Proliferation and Differentiation of Intramuscular Adipocytes in Qinchuan Beef Cattle [J]. China Animal Husbandry & Veterinary Medicine, 2023, 50(5): 1729-1741.
[8]	ZHANG Jiupan, MA Qing, WANG Jin, MA Lina, WEI Dawei, LIANG Xiaojun. Single-cell RNA Sequencing Technology and Its Application in the Study of Intramuscular Adipocytes [J]. China Animal Husbandry & Veterinary Medicine, 2023, 50(2): 479-489.
[9]	ZHANG Xueping, SHI Bingang, JIN Xiayang, WANG Xiangyan, LAN Lijuan, SHI Yu, QI Youpeng, ZHAO Shijie, LI Shaobin, HU Jiang. Expression Characterization Analysis of KLF7 Gene and Effects of Its Overexpression on Proliferation and Differentiation of Preadipocytes in Tibetan Sheep [J]. China Animal Husbandry & Veterinary Medicine, 2022, 49(8): 2920-2930.
[10]	GUO Panpan, JIN Xin, SUN Jianfu, LI Xiangzi, YIN Yunhou, YAN Changguo. Analysis of Differentially Expressed Genes in Differentiation of Yanbian Yellow Cattle Preadipocytes Induced by Cycloglitazone and Insulin Based on RNA-Seq Data [J]. China Animal Husbandry & Veterinary Medicine, 2021, 48(7): 2358-2368.
[11]	LI Anqi, JIANG Lei, SU Xiaotong, ZAN Linsen, WANG Hongbao. Expression Characteristics Analysis of ACTC1 Gene in Qinchuan Cattle [J]. China Animal Husbandry & Veterinary Medicine, 2020, 47(8): 2359-2367.
[12]	HUANG Huayun, LIANG Zhong, LIU Longzhou, ZHAO Zhenhua, WANG Qianbao, LI Chunmiao, HUANG Zhenyang, ZHANG Xin, LI Shoufeng. Mechanism of C-type Natriuretic Peptide Regulating Lipid Deposition of Intramuscular Adipocytes in Chickens [J]. China Animal Husbandry & Veterinary Medicine, 2020, 47(11): 3555-3562.
[13]	ZHAO Xinyan, GUO Yanting, CHEN Junzhen, LI Zeyu, SHI Huijun, FU Qiang. Isolation,Identification and Differentiation of Bovine Skeletal Muscle Satellite Cells [J]. China Animal Husbandry & Veterinary Medicine, 2020, 47(10): 3249-3258.
[14]	LYU Yang, CAO Yang, GAO Yi, LIU Lixiang, XUE Jiajia, ZHANG Guoliang. Cloning,Bioinformatics Analysis and Construction of Eukaryotic Expression Vector of AIDA Gene in Red Steppe Cattle [J]. China Animal Husbandry & Veterinary Medicine, 2019, 46(8): 2193-2202.
[15]	XIAO Cheng, JIN Haiguo, WEI Tian, CAO Yang. Study on the Expression of Genes Related to the Differentiation Process of Preadipocytes in Small-tail Han Sheep [J]. China Animal Husbandry & Veterinary Medicine, 2019, 46(7): 2030-2037.