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

Abstract

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

CLC Number:

S828.2

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.

Figures/Tables 10

Table 1

Table 2

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

Fig. 6

Fig. 7

Fig. 8

References 36

1	BERG F , GUSTAFSON U , ANDERSSON L . The uncoupling protein 1 gene (UCP1) is disrupted in the pig lineage: A genetic explanation for poor thermoregulation in piglets[J]. PLoS Genet, 2006, 2 (8): e129. doi: 10.1371/journal.pgen.0020129
2	陈华. 藏猪在青藏高原独特生态环境下的养殖适应性研究[J]. 畜牧业环境, 2023 (20): 99- 101.
	CHEN H . Study on Tibetan pig breeding adaptability in Qinghai-Tibet Plateau's unique ecology[J]. Animal Industry and Environment, 2023 (20): 99- 101.
3	LIN J , CAO C , TAO C , et al. Cold adaptation in pigs depends on UCP3 in beige adipocytes[J]. J Mol Cell Biol, 2017, 9 (5): 364- 375. doi: 10.1093/jmcb/mjx018
4	李倩文, 杨榛, 杨雅楠, 等. 冬夏两季环境温度对藏猪生理特征及相关基因表达的影响[J]. 中国畜牧兽医, 2021, 48 (3): 873- 881.
	LI Q W , YANG Z , YANG Y N , et al. Effects of environmental temperature in winter and summer on physiological characteristics and related gene expression of Tibetan pigs[J]. China Animal Husbandry & Veterinary Medicine, 2021, 48 (3): 873- 881.
5	STRAUB L G , SCHERER P E . Metabolic messengers: Adiponectin[J]. Nat Metab, 2019, 1 (3): 334- 339. doi: 10.1038/s42255-019-0041-z
6	ACHARI A E , JAIN S K . Adiponectin, a therapeutic target for obesity, diabetes, and endothelial dysfunction[J]. Int J Mol Sci, 2017, 18 (6): 1321. doi: 10.3390/ijms18061321
7	OKADA-IWABU M , YAMAUCHI T , IWABU M , et al. A small-molecule AdipoR agonist for type 2 diabetes and short life in obesity[J]. Nature, 2013, 503 (7477): 493- 499. doi: 10.1038/nature12656
8	IWABU M , OKADA-IWABU M , TANABE H , et al. AdipoR agonist increases insulin sensitivity and exercise endurance in AdipoR-humanized mice[J]. Commun Biol, 2021, 4 (1): 45. doi: 10.1038/s42003-020-01579-9
9	ZHANG S , WU X , WANG J , et al. Adiponectin/AdiopR1 signaling prevents mitochondrial dysfunction and oxidative injury after traumatic brain injury in a SIRT3 dependent manner[J]. Redox Biol, 2022, 54, 102390. doi: 10.1016/j.redox.2022.102390
10	SONG N , XU H , WU S , et al. Synergistic activation of AMPK by AdipoR1/2 agonist and inhibitor of EDPs-EDP interaction recover NAFLD through enhancing mitochondrial function in mice[J]. Acta Pharm Sin B, 2023, 13 (2): 542- 558. doi: 10.1016/j.apsb.2022.10.003
11	XU H , ZHAO Q , SONG N , et al. AdipoR1/AdipoR2 dual agonist recovers nonalcoholic steatohepatitis and related fibrosis via endoplasmic reticulum-mitochondria axis[J]. Nat Commun, 2020, 11 (1): 5807. doi: 10.1038/s41467-020-19668-y
12	HAN Y , SUN Q , CHEN W , et al. New advances of adiponectin in regulating obesity and related metabolic syndromes[J]. J Pharm Anal, 2024, 14 (5): 100913. doi: 10.1016/j.jpha.2023.12.003
13	HERZIG S , SHAW R J . Ampk: Guardian of metabolism and mitochondrial homeostasis[J]. Nat Rev Mol Cell Biol, 2018, 19 (2): 121- 135. doi: 10.1038/nrm.2017.95
14	REISMAN E G , HAWLEY J A , HOFFMAN N J . Exercise-regulated mitochondrial and nuclear signalling networks in skeletal muscle[J]. Sports Med, 2024, 54 (5): 1097- 1119. doi: 10.1007/s40279-024-02007-2
15	STEINBERG G R , HARDIE D G . New insights into activation and function of the AMPK[J]. Nat Rev Mol Cell Biol, 2023, 24 (4): 255- 272. doi: 10.1038/s41580-022-00547-x
16	WU G , BAUMEISTER R , HEIMBUCHER T . Molecular mechanisms of lipid-based metabolic adaptation strategies in response to cold[J]. Cells, 2023, 12 (10): 1353. doi: 10.3390/cells12101353
17	IMBEAULT P , DéPAULT I , HAMAN F . Cold exposure increases adiponectin levels in men[J]. Metabolism, 2009, 58 (4): 552- 559. doi: 10.1016/j.metabol.2008.11.017
18	MENGEL L A , SEIDL H , BRANDL B , et al. Gender differences in the response to short-term cold exposure in young adults[J]. J Clin Endocrinol Metab, 2020, 105 (5): dgaa110.
19	HUI X , GU P , ZHANG J , et al. Adiponectin enhances cold-induced browning of subcutaneous adipose tissue via promoting M2 macrophage proliferation[J]. Cell Metab, 2015, 22 (2): 279- 290. doi: 10.1016/j.cmet.2015.06.004
20	唐妮, 王书瑶, 齐锦雯, 等. 脂联素调控脂质代谢的研究进展[J]. 畜牧兽医学报, 2018, 49 (12): 2550- 2557. doi: 10.11843/j.issn.0366-6964.2018.12.003
	TANG N , WANG S Y , QI J W , et al. Research progress on adiponectin regulating lipid metabolism[J]. Acta Veterinaria et Zootechnica Sinica, 2018, 49 (12): 2550- 2557. doi: 10.11843/j.issn.0366-6964.2018.12.003
21	WANG Y , LIANG B , LAU W B , et al. Restoring diabetes-induced autophagic flux arrest in ischemic/reperfused heart by ADIPOR (adiponectin receptor) activation involves both AMPK-dependent and AMPK-independent signaling[J]. Autophagy, 2017, 13 (11): 1855- 1869. doi: 10.1080/15548627.2017.1358848
22	BAUZÁ-THORBRVGGE M , VUJIČI Ć M , CHANCLÓN B , et al. Adiponectin stimulates Sca1⁺CD34^--adipocyte precursor cells associated with hyperplastic expansion and beiging of brown and white adipose tissue[J]. Metabolism, 2024, 151, 155716. doi: 10.1016/j.metabol.2023.155716
23	NGUYEN T M D . Adiponectin: Role in physiology and pathophysiology[J]. Int J Prev Med, 2020, 11, 136. doi: 10.4103/ijpvm.IJPVM_193_20
24	GHADGE A A , KHAIRE A A , KUVALEKAR A A . Adiponectin: A potential therapeutic target for metabolic syndrome[J]. Cytokine Growth Factor Rev, 2018, 39, 151- 158. doi: 10.1016/j.cytogfr.2018.01.004
25	KADOWAKI T , YAMAUCHI T , KUBOTA N , et al. Adiponectin and adiponectin receptors in insulin resistance, diabetes, and the metabolic syndrome[J]. J Clin Invest, 2006, 116 (7): 1784- 1792. doi: 10.1172/JCI29126
26	ZHAO Y , SUN N , SONG X , et al. A novel small molecule AdipoR2 agonist ameliorates experimental hepatic steatosis in hamsters and mice[J]. Free Radic Biol Med, 2023, 203, 69- 85. doi: 10.1016/j.freeradbiomed.2023.04.001
27	PAHLAVANI H A , LAHER I , WEISS K , et al. Physical exercise for a healthy pregnancy: The role of placentokines and exerkines[J]. J Physiol Sci, 2023, 73 (1): 30. doi: 10.1186/s12576-023-00885-1
28	KHORAMIPOUR K , CHAMARI K , HEKMATIKAR A A , et al. Adiponectin: Structure, physiological functions, role in diseases, and effects of nutrition[J]. Nutrients, 2021, 13 (4): 1180. doi: 10.3390/nu13041180
29	CHEN S , LIU X , PENG C , et al. The phytochemical hyperforin triggers thermogenesis in adipose tissue via a Dlat-AMPK signaling axis to curb obesity[J]. Cell Metab, 2021, 33 (3): 565- 580.e7. doi: 10.1016/j.cmet.2021.02.007
30	LIU J , WANG Y , LIN L . Small molecules for fat combustion: Targeting obesity[J]. Acta Pharm Sin B, 2019, 9 (2): 220- 236.
31	DENG Y , HAN Y , GAO S , et al. The physiological functions and polymorphisms of type Ⅱ deiodinase[J]. Endocrinol Metab (Seoul), 2023, 38 (2): 190- 202.
32	ZHOU Z , YON TOH S , CHEN Z , et al. Cidea-deficient mice have lean phenotype and are resistant to obesity[J]. Nat Genet, 2003, 35 (1): 49- 56.
33	OLIVERAS-CAñELLAS N , MORENO-NAVARRETE J M , LORENZO P M , et al. Downregulated adipose tissue expression of browning genes with increased environmental temperatures[J]. J Clin Endocrinol Metab, 2023, 109 (1): e145- e154.
34	JASH S , BANERJEE S , LEE M J , et al. CIDEA transcriptionally regulates UCP1 for britening and thermogenesis in human fat cells[J]. iScience, 2019, 20, 73- 89.
35	KIM M , PAIK J H , LEE H , et al. Ancistrocladus tectorius extract inhibits obesity by promoting thermogenesis and mitochondrial dynamics in high-fat diet-fed mice[J]. Int J Mol Sci, 2024, 25 (7): 3743.
36	GOTTSCHALK B , KOSHENOV Z , MALLI R , et al. Implications of mitochondrial membrane potential gradients on signaling and ATP production analyzed by correlative multi-parameter microscopy[J]. Sci Rep, 2024, 14 (1): 14784.

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

[1]	YAO Boyuan, YANG Zhiwen, SUN Yapeng, YANG Yanan, ZHANG Yaru, WANG Xinrong. Analysis of Novel Transcripts, Alternative Splicing, and SNP in Porcine Heart Tissue Based on RNA-Seq Technology [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(4): 1664-1675.
[2]	HOU Wanchen, XU Tong. Cannabidiol Antagonizes BPA-induced Apoptosis and Autophagy in Porcine Intestinal Epithelial Cells through the BRD4/AMPK/mTOR Signaling Pathway [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(4): 1919-1933.
[3]	YANG Yuting, CHEN Guoliang, CHANG Qiaoning, BAO Wu, LIU Jingchao, JI Mengting, RONG Xiaoyin, GUO Xiaohong, YANG Yang, LI Bugao. miR-375-3p Targets Fam229a to Regulate Porcine Precursor Adipocyte Differentiation [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(3): 1120-1133.
[4]	ZHAO Gangkui, GAO Haixu, YIN Siqi, SUN Honghong, XIN Yiran, ZAN Linsen, ZHAO Chunping. The Effects of the SFRP4 Gene on Bovine Preadipocyte Differentiation [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(2): 611-620.
[5]	Yi WANG, Juan GAO, Yuemin HU, Yuefei YANG, Bojun FAN, Huiming JU. Effect of Transient Serum Starvation on Metabolism and Autophagy of Porcine Skeletal Muscle Satellite Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(8): 3408-3417.
[6]	Yao LI, Rui JIA, Jie LI, Shuangbao GUN, Qiaoli YANG, Longlong WANG, Pengxia ZHANG, Xiaoli GAO, Xiaoyu HUANG. Effects of Low Temperature on Adipose Tissue Morphology, Lipid Metabolism-Related Gene Expression and Enzyme Activities, and AMPK/PGC-1α Pathway in Hezuo Pigs [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(8): 3418-3426.
[7]	Xiaojuan LIANG, Yushuang LI, Zhou FU, Duo TANG, Yingying LI, Shouwei WANG. Isolation, Culture and Adipogenic Differentiation of Pigeon Preadipocytes [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(8): 3482-3492.
[8]	Ya’nan LI, Tianwen MA, Yuhui MA, Chengwei WEI. Bilobalide Regulates Mitochondrial Biogenesis Mediated by AMPK-SIRT3 Positive Feedback Loop and Improves Inflammatory Damage of ATDC5 Chondrocytes [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(8): 3714-3724.
[9]	Xiaojuan LIANG, Yushuang LI, Yingying LI, Shouwei WANG. Isolation, Culture and Adipogenic Differentiation of Beijing Black Pig Preadipocytes [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(7): 2877-2889.
[10]	SUN Wenli, WANG Haoqi, ZE Licuo, GAO Yufan, ZHANG Feifan, ZHANG Jian, DUAN Mengqi, SHANG Peng, QIANG Bayangzong. Polymorphism of Pro-Inflammatory Factors (IL-1β, IL-6, TNF-α) in Tibetan Pigs and Its Association Analysis with Immune Traits [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 1958-1969.
[11]	Weiyu ZHANG, Jing CHENG, Jiabao XU, Jing WANG, Xinyan TAO, Bo LI, Yawei ZHANG, Dandan ZHANG, Ning ZHANG, Zhenkai HAO, Chenbo ZHOU, Yuanqing ZHANG. Regulation of Preadipocyte Differentiation by SREBP1 Gene in Jinnan Cattle [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(11): 5003-5017.
[12]	Yao TANG, Tao WANG, Mengqing XUE, Wenyu ZHANG, Mei SHI, Xianzhong WANG, Jiaojiao ZHANG. Thiazolidinedione Inhibits Chicken Growth via Adiponectin-mediated AMPK Signaling Pathway [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(11): 5247-5258.
[13]	Lan FENG, Xue FENG, Yulin MA, Lingkai ZHANG, Yanfen MA, Dawei WEI, Fen LI, Lupei ZHANG, Runjun YANG, Yun MA, Bei CAI. Study on the Function of PPP5C Gene in Regulating the Proliferation and Differentiation of Bovine Adipocytes [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(10): 4391-4402.
[14]	TANG Yinmei, LI Qi, LI Haiyang, LIN Yaqiu, WANG Yong, XIANG Hua, HUANG Lian, ZHU Jiangjiang. Cloning of the Goat FATP2 Gene and Its Regulatory Effect on Lipid Deposition in Precursor Adipocytes [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(9): 3642-3652.
[15]	SHAO Peng, TANG Yinmei, LIN Yaqiu, WANG Yong, XIANG Hua, HUANG Lian, ZHU Jiangjiang. Regulation Effect of PSMD9 on Lipid Deposition in Goat Precursor Adipocytes [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(9): 3653-3663.

The Effect of Interfering with AdiopR2 on the Thermogenesis of Subcutaneous Inguinal Adipocytes in Tibetan Pigs

RichHTML

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 10

References 36

Related Articles 15

Recommended Articles

Metrics

Comments