噻唑烷二酮通过脂联素介导的AMPK信号通路抑制鸡的生长

doi:10.11843/j.issn.0366-6964.2024.11.040

摘要/Abstract

摘要：

噻唑烷二酮(thiazolidinedione，TZD)是一种治疗胰岛素抵抗的口服糖尿病药物，滥用或不当使用TZD对动物和人类的健康具有不良影响。本研究探讨了TZD对健康雏鸡生长和代谢的影响以及这一过程的潜在机制。80只6日龄雏鸡分为对照组(雌雄各20只)、试验组(雌雄各20只)，对照组的鸡不灌服TZD，试验组的鸡连续14 d每日灌服25 mg·(kg·d)^-1TZD，检测雏鸡的生长情况；利用生化试剂盒检测腺嘌呤核苷三磷酸(ATP)和脂联素水平、生长代谢相关激素浓度、线粒体功能；通过RT-PCR和蛋白质印迹法检测调节细胞代谢和增殖相关基因的mRNA和蛋白质水平。结果发现，TZD显著降低了雏鸡的平均日增重以及血清ATP、胰岛素(insulin, INS)、生长激素(growth hormone, GH)和胰岛素样生长因子1(insulin-like growth factor 1, IGF1)的水平(P < 0.05)，而提高了脂联素和胰岛素样生长因子结合蛋白2(IGFBP2)的水平(P < 0.05)。TZD还降低了雏鸡肝、肾和肌肉的ATP水平及线粒体酶活性(P < 0.05)。此外，TZD的摄入使雏鸡脂联素及其受体、AMP活化蛋白激酶α2(AMPKα2)、p21和p27的mRNA及蛋白表达增强(P < 0.05)，而INS及其受体、IGF1及其受体、磷脂酰肌醇-3-激酶(PI3K)、AKT、哺乳动物雷帕霉素靶蛋白(mTOR)、细胞周期蛋白依赖性激酶2(CDK2)和细胞周期蛋白E1(cyclin E1)的mRNA和蛋白表达受到抑制(P < 0.05)。TZD通过脂联素介导的AMPK信号通路降低了雏鸡生长代谢相关激素水平和线粒体功能，该信号通路抑制其下游PI3K/AKT/mTOR，进一步导致p21/p27表达增加以及CDK2/Cyclin E1表达降低，从而抑制雏鸡生长和代谢组织中的细胞增殖。综上表明，TZD通过调节脂联素介导的AMPK信号通路对鸡的生长产生不利影响，这为临床实践与畜禽生产中避免滥用或不当使用TZD提供了一定的理论依据。

关键词: 噻唑烷二酮, 生长代谢, 脂联素, AMPK

Abstract:

Thiazolidinedione (TZD) is an oral anti-diabetic drug for the treatment of insulin resistance. Abuse or improper use of TZD has adverse effects on animal and human health. This study explores effects of TZD on the growth and metabolism of chicks and underlying mechanisms of this process. Eighty chicks at the age of 6-day-old were divided into a control group (20 females, 20 males) and an experimental group (20 females, 20 males). Chicks in the control group were administered without TZD, chicks in the experimental group were daily administrated with 25 mg·(kg·d)^-1 TZD by gavage for 14 days, and the growth of chicks was measured. The biochemical kits were used to detect the levels of adenosine triphosphate (ATP) and adiponectin, the concentrations of growth and metabolism-related hormones, and the function of mitochondria. The mRNA and protein expressions of genes which are associated with cell metabolism and proliferation were detected by RT-PCR and western blotting. Results showed that TZD treatment significantly decreased the average daily gain of chicks (P < 0.05). Serum ATP, insulin (INS), growth hormone (GH), and insulin-like growth factor 1 (IGF1) levels were reduced in TZD-treated group (P < 0.05), while adiponectin and insulin-like growth factor binding protein 2 levels was improved (P < 0.05). TZD also decreased ATP levels and mitochondrial enzyme activities in the liver, kidney and muscle of chicks. Moreover, mRNA and protein expressions of adiponectin and its receptors, AMP-activated protein kinase α2 (AMPKα2), p21 and p27 were enhanced in TZD-treated chicks (P < 0.05), whereas mRNA and protein expressions of INS and its receptor, IGF1 and its receptor, phosphatidylinositol-3-kinase (PI3K), AKT, mammalian target of rapamycin (mTOR), Cyclin-dependent kinase 2 (CDK2) and Cyclin E1 were inhibited (P < 0.05). The growth metabolism-related hormones and mitochondrial function in TZD-treated chicks were down-regulated by the adiponectin-mediated AMPK signaling pathway, which inhibited its downstream PI3K/AKT/mTOR, and further led to increased expressions of p21/p27 and decreased expressions of CDK2/Cyclin E1, this inhibiting the cell proliferation in growth and metabolic tissues of chicks. Our findings suggest that TZD has an adverse effect on chicken growth through regulating the adiponectin-mediated AMPK signaling pathway, this provides a certain theoretical basis for avoiding the abuse or improper use of TZD in clinical practice and animal production.

Key words: thiazolidinedione, growth metabolism, adiponectin, AMPK

中图分类号:

S859.793

唐瑶, 王涛, 薛梦晴, 张文芋, 石美, 王鲜忠, 张姣姣. 噻唑烷二酮通过脂联素介导的AMPK信号通路抑制鸡的生长[J]. 畜牧兽医学报, 2024, 55(11): 5247-5258.

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.

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基因 Gene	序列号 Sequence number	序列位置 Sequence position	序列长度/bp Product length	退火温度/℃ Annealing temperature	序列(5′→3′) Sequence
β-actin	NM_205518.1	625-818	194	58	F: GTGCGTGACATCAAGGAGAAGC R: CCACAGGACTCCATACCCAAGA
ADIPOQ	AY523637.1	309-473	165	58	F: TTCCTCCTTTGCTCACTGCT R: TCCATCTTTTCCATCCTTGC
ADIPOR1	NM_001031027	280-523	244	57	F: GTGATCCCCTACGATGTGCT R: TCCCAAACACAACCTTCTCC
ADIPOR2	NM_001007854	711-915	205	57	F: TGTGCAGGAGAAAGTGGTTG R: AGGCTGAGGGTTGCAGTAGA
AMPKα2	NM_001039605.1	726-943	218	57	F: GGAGGCGTGTTTTACATCCC R: AACTTCTCACAGACCTCCCG
mTOR	XM_417614.4	119-309	191	57	F: TGAAGGGGTCAAGGCAATCC R: GGCGAGCAGTGGTTGTGGAT
IGF1	NM_001004384.2	188-316	129	58	F: AGTTCGTATGTGGAGACAGAGGC R: CCAGCCTCCTCAGGTCACAAC
IGF1R	NM_205032.1	2 961-3 114	154	57	F: TTGTGCTCCCCATTGCTTTC R: GGAACGTACACATCCGAAGC
INS	NM_205222	126-308	183	59	F: CACTGCCTCTTCTGGCTCTC R: GCAAGGGACTGCTCACTAGG
INSR	XM_001233398	2 303-2 462	160	57	F: TGAGAGTGCAGAGGAGCAGA R: GGCACTGACATAAGCTGCAA
PIK3CA	NM_001004410	3 006-3 155	150	57	F: AACATCTGGCAAAACCAAGG R: CTGCAATGCTCCCTTTAAGC
AKT1	NM_205055.1	1 156-1 328	173	57	F: AACGGAGGGGAGTTGTTTTT R: ATGTGCCCGTCTTTATCCAG
p21	NM_204396	530-684	155	58	F: TTTCCCTGCCCTGTACTGTC R: AGTCCTCCTCAGTCCCTTCC
p27	NM_204256.2	922-1 102	181	58	F: AGGCCGAAAGACTGATGTTG R: CGATTTCTTGGGTGTTTGCT
CDK2	NM_001199857.1	386-562	177	58	F: AACCCCAGAACCTCCTCATC R: TCCAGATGTCCACAGCAGTC
Cyclin E1	NM_001031358.1	1 481-1 642	162	58	F: CGCCAGCCACTTAAAAGAAC R: TGTCAACAGGGGACAGCATA

抗体 Antibody	目标蛋白 Target	抗体名称 Name of antibody	来源 Source and reference	种属；克隆性 Species raised in; clonality	稀释度 Dilution used
一抗 Primary antibodies	p-AMPKα2	Anti-phospho-AMPK alpha2 (Thr172)	Bioss Antibodies Inc., Woburn, MA, USA	Rabbit; polyclonal	1∶300
	AMPKα2	Anti-AMPK alpha2	Abcam, Cambridge, MA, USA	Rabbit; polyclonal	1∶500
	p-mTOR	Anti-mTOR (phosphor S2448)	Abcam	Rabbit; monoclonal	1∶1 000
	mTOR	Anti-mTOR	Abcam	Rabbit; polyclonal	1∶1 000
	Adiponectin	Anti-Adiponectin	Abcam	Rabbit; polyclonal	1∶300
	IGF1R	Anti-IGF1R	Bioss	Rabbit; polyclonal	1∶500
	INSR	Anti-Insulin Receptor	Bioss	Rabbit; polyclonal	1∶500
	PIK3CA	Anti-PIK3CA	Bioss	Rabbit; polyclonal	1∶500
	AKT1	Anti-AKT1	Abcam	Rabbit; polyclonal	1∶500
	p21	p21 Polyclonal Antibody	Thermo Fisher Scientific, Waltham, MA, USA	Rabbit; polyclonal	1∶500
	p27	Anti-CDKN1B/p27 KIP 1	Bioss	Rabbit; polyclonal	1∶500
	CDK2	Anti-CDK2	Bioss	Rabbit; polyclonal	1∶500
	Cyclin E1	Anti-Cyclin E1	Bioss	Rabbit; polyclonal	1∶500
	Beta-actin	Beta-actin (AC-15)	Santa Cruz Biotechnology, Dallas, Texas, USA	Rabbit; polyclonal	1∶1 000
二抗 Secondary antibodies	Goat IgG	Anti-rabbit IgG H & L (HRP)	Abcam	Goat; polyclonal	1∶5 000
二抗 Secondary antibodies	Goat IgG	Anti-mouse IgG-HRP	Santa Cruz Biotechnology	Goat; polyclonal	1∶5 000

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