干扰AdiopR2对藏猪皮下腹股沟脂肪细胞产热的影响

doi:10.11843/j.issn.0366-6964.2025.06.011

摘要/Abstract

摘要：

旨在探究干扰Adiopr2基因对藏猪皮下腹股沟脂肪细胞产热的影响，揭示AdiopR2-AMPK通路活性对脂质合成和ATP生成影响的分子机制。本研究采集20日龄冬季藏猪(TW组，平均环境温度-15℃)、夏季藏猪(TS组，平均环境温度25℃)和夏季长白猪(LS组，平均环境温度25℃)的皮下腹股沟脂肪组织，每组5头。Adipor2的小干扰RNA (small interfering RNA, siRNA)用于转录后沉默Adipor2的表达。采用实时荧光定量聚合酶链反应(reverse transcription-quantitative polymerase chain reaction，RT-qPCR)检测脂肪组织中脂联素受体基因、siRNA-AdipoR2的干扰效率、以及AMPK下游信号、脂质合成相关基因、产热相关基因和线粒体复合物Ⅰ-Ⅲ基因的mRNA水平，细胞计数试剂盒(cell counting kit-8，CCK-8)分析藏猪脂肪细胞的增殖能力，蛋白印迹(Western blot，WB)方法检测AMPK及其下游信号的表达，油红O染色用于检测藏猪脂肪细胞的脂质沉积能力，JC-1线粒体膜电位和ATP生成试剂盒检测藏猪脂肪细胞的产热。结果表明：在体内，长期冷暴露下藏猪皮下腹股沟脂肪组织Adipor1/2表达显著升高(P < 0.01或P < 0.000 1)；在体外，藏猪诱导成熟脂肪细胞的第4、8、12天Adipor2的表达显著高于长白猪，Adipor1的表达显著低于长白猪(P < 0.01或P < 0.000 1)。siRNA-AdipoR2显著抑制了Adipor2的表达，促进脂质合成基因Fasn的表达，抑制产热基因(Dio2和Cidea)和线粒体复合物Ⅰ Ndufa的表达(P < 0.05，P < 0.001或P < 0.000 1)。此外，干扰Adipor2后48 h时细胞扩增效率显著降低，显著抑制AMPK的磷酸化，促进脂质沉积(P < 0.05，P < 0.01或P < 0.001)。JC-1聚合物线粒体膜电位和ATP生成分析发现，siRNA-AdipoR2显著抑制线粒体膜电位和ATP生成(P < 0.01或P < 0.001)。本研究结果显示，干扰Adipor2能够抑制AMPK信号，促进脂质沉积并抑制藏猪脂肪细胞的产热作用，为探究藏猪独特的耐寒机制提供分子依据和理论基础。

关键词: 藏猪, AdipoR2, 脂肪细胞, AMPK, 产热

Abstract:

This study aimed to investigate the effects of Adipor2 gene interference on thermogenesis in subcutaneous inguinal adipocytes of Tibetan pigs and to elucidate the molecular mechanisms by which AdipoR2-AMPK pathway activity influences lipid synthesis and ATP production. Subcutaneous inguinal adipose tissues were collected from 20-day-old Tibetan pigs in winter (TW group, average environmental temperature -15 ℃), Tibetan pigs in summer (TS group, average environmental temperature 25 ℃), and Landrace pigs in summer (LS group, average environmental temperature 25 ℃), with 5 individuals per group. Small interfering RNA (siRNA) targeting AdipoR2 was used to post-transcriptionally silence Adipor2 expression. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was employed to measure mRNA levels of adiponectin receptor genes, siRNA-AdipoR2 interference efficiency, AMPK downstream signaling, lipid synthesis-related genes, thermogenesis-related genes, and mitochondrial complex Ⅰ-Ⅲ genes in adipose tissues. Cell proliferation capacity was analyzed using a cell counting kit-8 (CCK-8), while Western blot (WB) was used to detect the expression of AMPK and its downstream signaling. Oil Red O staining was performed to assess lipid deposition capacity, and JC-1 mitochondrial membrane potential and ATP production kits were used to evaluate thermogenesis in Tibetan pig adipocytes. In vivo, long-term cold exposure significantly increased the expression of Adipor1/2 in subcutaneous inguinal adipose tissues of Tibetan pigs (P < 0.01 or P < 0.000 1). In vitro, the expression of Adipor2 in induced mature adipocytes from Tibetan pigs was significantly higher than that in Landrace pigs on days 4, 8, and 12, while Adipor1 expression was significantly lower (P < 0.01 or P < 0.000 1). siRNA-AdipoR2 significantly inhibited the expression of Adipor2, promoted the expression of the lipid synthesis gene Fasn, and inhibited the expression of thermogenesis-related genes (Dio2 and Cidea) and mitochondrial complex I gene Ndufa (P < 0.05, P < 0.001, or P < 0.000 1). Additionally, Adipor2 interference significantly reduced cell expansion efficiency at 48 h, inhibited AMPK phosphorylation, and promoted lipid deposition (P < 0.05, P < 0.01, or P < 0.001). JC-1 aggregate mitochondrial membrane potential and ATP production analyses revealed that siRNA-AdipoR2 significantly suppressed mitochondrial membrane potential and ATP production (P < 0.01 or P < 0.001). This study demonstrates that Adipor2 interference inhibits AMPK signaling, promotes lipid deposition, and suppresses thermogenesis in Tibetan pig adipocytes, providing molecular insights and a theoretical basis for understanding the unique cold adaptation mechanisms of Tibetan pigs.

Key words: Tibetan pig, Adipor2, adipocyte, AMPK, thermogenesis

中图分类号:

S828.2

柳思奇, 杨榛, 杨雅楠, 蔡原, 赵生国. 干扰AdiopR2对藏猪皮下腹股沟脂肪细胞产热的影响[J]. 畜牧兽医学报, 2025, 56(6): 2649-2660.

LIU Siqi, YANG Zhen, YANG Yanan, CAI Yuan, ZHAO Shengguo. The Effect of Interfering with AdiopR2 on the Thermogenesis of Subcutaneous Inguinal Adipocytes in Tibetan Pigs[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(6): 2649-2660.

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基因名 Gene symbol	引物序列(5′→3′) Primers sequence	产物长度/bp Products length	退火温度/℃ Temperature
β-actin	F: CAGTCGGTTGGATGGAGCAT	147	59.57
	R: AGGCAGGGACTTCCTGTAAC
Adipor1	F: GGACAACGACTACCTGCTGCA	82	61.41
	R: GTGGATGCGGAAGATGCTCT
Adipor2	F: TCCTTCCGGGCCTGTTTTAAG	233	59.46
	R: GAATGGCAGTAGACTGTGTGG
Cpt1-a	F: ATGCGCTACTCCCTGAAGGTG	77	63.54
	R: GTGGCGCGGCTCATCTTGC
Fasn	F: CCGAGACCCTCGTGGGCTA	209	63.28
	R: CTTCAGCAGGACGTTGAGGCC
Dio2	F: ATGGGCATCCTCAGCGTAGAC	151	63.89
	R: ACTCCCCGCGGGTGGACTT
Cidea	F: TTCCGAGTTTCCAACCACAA	97	57.64
	R: CGATAACCAGGGCATCCAG
Ucp3	F: AAGGTTCGATTCCAGGCCAG	86	60.32
	R: GCGATGGTCCTGTAGGCATC
Atp2a2	F: GGAACCCCTGCCACTTATCT	190	59.67
	R: ATCGGTACATGCCGAGAACG
Atp5a1	F: CGCCATTGATGGAAAGGGTC	97	59.65
	R: TGGTTCCCGCACAGAGATTC
Ndufa1	F: GCTCACATCCACAAGTTCACT	90	57.32
	R: GCACCTATCTCTTTCCATCAAA
Sdha	F: TGGCATTTCTACGACACTGTG	77	58.47
	R: GCCTGCTCTGTCATGTAGTG
Uqcrb	F: GGATGACGATGTAAAAGAAGCCA	212	58.54
	R: TCTTTGCCCATTCTTCTCTTTCT

siRNA名称 siRNA name	序列(5′ →3′) Sequence
siRNA-AdipoR2	S: CUGACUGGCUCAAGGAUAATT AS: UUAUCCUUGAGCCAGUCAGTT
siRNA-NC	S: UUCUCCGAACGUGUCACGUTT AS: ACGUGACACGUUCGGAGAATT

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