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

摘要/Abstract

摘要：

目的探究环指蛋白20（ring finger protein 20，RNF20）对小鼠性腺脂肪组织（gonadal white adipose tissues，gWAT）及3T3-L1脂肪细胞中支链氨基酸（branched-chain amino acids，BCAAs）代谢的影响及调控机制。方法以雄性脂肪特异性Rnf20基因敲除小鼠（Rnf20^flox/flox；adiponectin-Cre⁺，ASKO）及同窝野生型小鼠（Rnf20^flox/flox；adiponectin-Cre^-，WT）为研究对象，通过靶向代谢组学检测小鼠gWAT和血清中BCAAs含量，结合实时荧光定量PCR分析gWAT、肝脏及腓肠肌中BCAAs分解代谢基因（Bcat2、Bckdha等）的表达情况。利用siRNA干扰3T3-L1前体脂肪细胞内源性Rnf20基因表达，并诱导细胞成脂分化，通过油红O染色评估siRNF20（干扰组）和siNC（对照组）细胞的成脂分化效率，检测成脂分化标志基因、脂肪水解基因及BCAAs分解代谢基因的表达情况以及脂肪细胞培养基中BCAAs的含量。结果与WT小鼠相比，ASKO小鼠gWAT中BCAAs（亮氨酸、异亮氨酸、缬氨酸）含量均极显著升高（P<0.01），且BCAAs分解代谢相关基因Bcat2、Acad5、Ehhand、Hibch表达量显著或极显著降低（P<0.05；P<0.01）；而血清中BCAAs含量及肝脏、腓肠肌中BCAAs分解代谢相关基因表达量均无显著变化（P>0.05）。与siNC组相比，体外敲除Rnf20基因可抑制3T3-L1细胞成脂分化，其中成脂标志基因Pparγ表达量显著降低（P<0.05），脂肪水解基因Adrb3表达量极显著升高（P<0.01）；成熟脂肪细胞中BCAAs分解代谢关键基因Bcat2、Bckdha表达量随Rnf20基因敲除而降低，且siRNF20组培养基中BCAAs含量极显著升高（P<0.01）。结论脂肪细胞Rnf20基因通过转录调控BCAAs分解代谢关键基因（Bcat2、Bckdha），维持细胞内BCAAs稳态；敲除Rnf20基因可抑制BCAAs分解代谢，导致BCAAs在脂肪组织内累积。研究结果为深入探究Rnf20基因功能提供了新的方向，为大动物脂肪沉积性状研究提供了新的基因素材。

关键词: Rnf20基因; 脂肪细胞; 支链氨基酸

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

中图分类号:

S852.16⁺3

张丽琼, 赵莹, 张留哲, 王彦芳, 郭慧慧. Rnf20基因对脂肪细胞中支链氨基酸含量的影响[J]. 中国畜牧兽医, 2026, 53(1): 287-298.

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.

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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