二甲双胍通过LKB1/AMPKα2信号通路调节雏鸡的生长代谢

doi:10.11843/j.issn.0366-6964.2025.09.044

摘要/Abstract

摘要： 二甲双胍(MET)是一种临床常用的降血糖药，但其滥用或不当使用对动物健康具有不良影响。本文旨在探讨高剂量MET对健康雏鸡生长代谢的影响及其潜在调节机制。将80只1日龄雏鸡随机分为对照组(40只，雌雄各20只)和试验组(40只，雌雄各20只)，从7日龄开始，试验组雏鸡每天灌服0.6 g·kg^-1的MET-HCl(根据体重计算给药量，溶于1 mL生理盐水中)，连续灌服23 d，对照组每天灌服等量生理盐水，记录各组雏鸡的每日摄食量。在第0天(孵出后第1天)和第30天，称量禁食12 h后各组雏鸡的体重，并评估平均日摄食量(ADFI)、平均日增重(ADG)、饲料/增重比(F/G)。分析30日龄雏鸡血清中腺嘌呤核苷三磷酸(ATP)和生长相关激素的水平；检测肝、肾和肌肉中ATP水平、线粒体呼吸酶活性；利用RT-PCR和Western blot检测ATP合成酶、肝激酶B1(LKB1)/腺苷酸活化蛋白激酶α2(AMPKα2)通路及其下游因子的mRNA(在肝、肾和肌肉中)和蛋白表达水平(在肌肉中)，胰岛素(INS)与受体、胰岛素样生长因子1(IGF1)与受体及其下游因子的mRNA和蛋白表达水平。结果显示，高剂量MET可显著降低雏鸡的体重，减少平均日增重，提高料重比。MET减少了雏鸡血清中的ATP、INS、生长激素(GH)和IGF1水平。同时，MET降低了肝、肾和肌肉中ATP水平以及线粒体呼吸酶(NADH、细胞色素C氧化酶和ATP合酶)的活性。此外，MET降低了ATP5A的表达，激活了LKB1/AMPKα2信号通路，抑制了哺乳动物雷帕霉素靶蛋白(mTOR)、INS与受体、IGF1与受体、磷脂酰肌醇-3-激酶(PI3K)、蛋白激酶B(AKT)、细胞周期蛋白依赖性激酶2(CDK2)和细胞周期蛋白E1(cyclin E1)的表达，增加了p21和p27水平。本研究结果表明，高剂量MET可能通过激活LKB1/AMPKα2信号通路对雏鸡的生长代谢产生负面影响，为临床上避免滥用或不当使用MET提供了一定的支撑依据。

关键词: 二甲双胍, 生长代谢, 肝激酶B1, 腺苷酸活化蛋白激酶α2

Abstract: Metformin (MET) is a commonly used hypoglycemic drug in clinical practice, but its abuse or improper use has adverse effects on animal health. This article aims to explore the effects of high-dose MET on chicken, the growth metabolism of healthy chicks and its potential regulatory mechanisms. Eighty 1-day-old chicks were randomly divided into a control group (40 chicks, 20 males and 20 females) and an experimental group (40 chicks, 20 males and 20 females). Starting from 7 days old, the experimental group chicks were orally administered 0.6 g·kg^-1 MET-HCl (the dosage was calculated based on chicken weight, dissolved in 1 mL of physiological saline) daily for 23 consecutive days, the control group was orally administered an equal amount of physiological saline daily. The daily food intake of each group of chicks was recorded. On day 0 (the first day after hatching) and day 30, the weight of each group of chicks after fasting for 12 h was measured, and the average daily food intake (ADFI), average daily weight gain (ADG), and feed/gain ratio (F/G) were evaluated. We analyzed the levels of adenosine triphosphate (ATP) and growth related hormones in the serum of 30-day-old chicks; measured ATP levels and mitochondrial respiratory enzyme activities in the liver, kidneys, and muscles; detected the mRNA levels (in the liver, kidney, and muscle) and protein expressions (in muscle) of ATP synthase, liver kinase B1 (LKB1)/AMP-activated protein kinase α2 (AMPKα2) pathway and its downstream factors, as well as the mRNA and protein expressions of insulin (INS) and its receptor, insulin-like growth factor 1 (IGF1) and its receptor and their downstream factors using RT-PCR and Western blot. The results showed that high-dose MET can significantly reduce the chicken weight, decrease average daily weight gain, and improve the feed to weight ratio. MET reduced ATP, INS, growth hormone (GH), and IGF1 levels in the serum of chicks. Meanwhile, MET reduced ATP levels in the liver, kidneys, and muscles, as well as the activities of mitochondrial respiratory enzymes (NADH, cytochrome C oxidase, and ATP synthase). In addition, MET reduced the expression of ATP5A, activated the LKB1/AMPKα2 signaling pathway, inhibited the expressions of mammalian target of rapamycin (mTOR), INS and its receptor, IGF1 and its receptor, phosphatidylinositol-3-kinase (PI3K), protein kinase B (AKT), cyclin-dependent kinase 2 (CDK2), and cyclin E1, and increased the levels of p21 and p27. Our research findings suggest that high-dose MET may have a negative impact on the chicken growth metabolism by activating the LKB1/AMPKα2 signaling pathway, which provides some supporting evidence for avoiding the abuse or improper use of MET in clinical practice.

Key words: metformin, growth metabolism, LKB1, AMPKα2

中图分类号:

S859.794

石美, 位格格, 李怡涵, 王鲜忠, 张姣姣. 二甲双胍通过LKB1/AMPKα2信号通路调节雏鸡的生长代谢[J]. 畜牧兽医学报, 2025, 56(9): 4673-4685.

SHI Mei, WEI Gege, LI Yihan, WANG Xianzhong, ZHANG Jiaojiao. Metformin Regulates Chicken Growth Metabolism through LKB1/AMPKα2 Signaling Pathway[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(9): 4673-4685.

参考文献

[1] CHAN J C N, YANG A, CHU N, et al. Current type 2 diabetes guidelines: individualized treatment and how to make the most of metformin[J]. Diabetes Obes Metab, 2024, 26(Suppl 3): 55-74.
[2] YEREVANIAN A,SOUKAS A A. Metformin: mechanisms in human obesity and weight loss[J]. Current obesity reports, 2019, 8(2): 156-164.
[3] HUA Y, ZHENG Y, YAO Y, et al. Metformin and cancer hallmarks: shedding new lights on therapeutic repurposing[J]. J Transl Med, 2023, 21(1): 403.
[4] LAMOIA T E,SHULMAN G I. Cellular and molecular mechanisms of metformin action[J]. Endocr Rev, 2021, 42(1): 77-96.
[5] FONTAINE E. Metformin-induced mitochondrial complex I inhibition: facts, uncertainties, and consequences[J]. Front Endocrinol (Lausanne), 2018, 9: 753.
[6] WANG Y, AN H, LIU T, et al. Metformin improves mitochondrial respiratory activity through activation of AMPK[J]. Cell Rep, 2019, 29(6): 1511-1523.
[7] AMENGUAL-CLADERA E, MORLA-BARCELO P M, MORN-COSTOYA A, et al. Metformin: from diabetes to cancer-unveiling molecular mechanisms and therapeutic strategies[J]. Biology (Basel), 2024, 13(5): 302.
[8] JOHANNS M, HUE L,RIDER M H. AMPK inhibits liver gluconeogenesis: fact or fiction?[J]. Biochem J, 2023, 480(1): 105-125.
[9] ZHU H, JIA Z, LI Y R, et al. Molecular mechanisms of action of metformin: latest advances and therapeutic implications[J]. Clin Exp Med, 2023, 23(7): 2941-2951.
[10] FAURE M, GUIBERT E, ALVES S, et al. The insulin sensitiser metformin regulates chicken Sertoli and germ cell populations[J]. Reproduction, 2016, 151(5): 527-538.
[11] ZHAO J, YANG P C, YANG H, et al. Dietary supplementation with metformin improves testis function and semen quality and increases antioxidants and autophagy capacity in goats[J]. Theriogenology, 2022, 188: 79-89.
[12] HOLLSTEIN P E, EICHNER L J, BRUN S N, et al. The AMPK-related kinases SIK1 and SIK3 mediate key tumor-suppressive effects of LKB1 in NSCLC[J]. Cancer Discov, 2019, 9(11): 1606-1627.
[13] MIHAYLOVA M M,SHAW R J. The AMPK signalling pathway coordinates cell growth, autophagy and metabolism[J]. Nat Cell Biol, 2011, 13(9): 1016-1023.
[14] YANG L, GARCIA CANAVERAS J C, CHEN Z, et al. Serine catabolism feeds NADH when respiration is impaired[J]. Cell Metab, 2020, 31(4): 809-821.
[15] DUTTA N, PEMMARAJU D B, GHOSH S, et al. Alkaloid-rich fraction of ervatamia coronaria sensitizes colorectal cancer through modulating AMPK and mTOR signalling pathways[J]. J Ethnopharmacol, 2022, 283: 114666.
[16] GONZÁLEZ A, HALL M N, LIN S C, et al. AMPK and TOR: the yin and yang of cellular nutrient sensing and growth control[J]. Cell Metab, 2020, 31(3): 472-492.
[17] CHEN W L, WEI H W, CHIU W Z, et al. Metformin regulates hepatic lipid metabolism through activating AMP-activated protein kinase and inducing ATGL in laying hens[J]. Eur J Pharmacol, 2011, 671(1-3): 107-112.
[18] JIANG S, LUO Y, ZHAN Z, et al. AMP-activated protein kinase re-sensitizes A549 to paclitaxel via up-regulating solute carrier organic anion transporter family member 1B3 expression[J]. Cell Signal, 2022, 91: 110215.
[19] LI M, WEI X, XIONG J, et al. Hierarchical inhibition of mTORC1 by glucose starvation-triggered AXIN lysosomal translocation and by AMPK[J]. Life Metabolism, 2023, 2(3): 1-14.
[20] CICCARESE F, ZULATO E,INDRACCOLO S. LKB1/AMPK pathway and drug response in cancer: a therapeutic perspective[J]. Oxid Med Cell Longev, 2019, 2019: 8730816.
[21] LIANG J, SHAO S H, XU Z X, et al. The energy sensing LKB1-AMPK pathway regulates p27kip1 phosphorylation mediating the decision to enter autophagy or apoptosis[J]. Nat Cell Biol, 2007, 9(2): 218-224.
[22] YANG X, KORD-VARKANEH H, TALAEI S, et al. The influence of metformin on IGF-1 levels in humans: a systematic review and meta-analysis[J]. Pharmacol Res, 2020, 151: 104588.
[23] YOSHIDA T,DELAFONTAINE P. Mechanisms of IGF-1-mediated regulation of skeletal muscle hypertrophy and atrophy[J]. Cells, 2020, 9(9): 1970.
[24] SOLINI A,TRICÒ D. Clinical efficacy and cost-effectiveness of metformin in different patient populations: a narrative review of real-world evidence[J]. Diabetes Obes Metab, 2024, 26(Suppl 3): 20-30.
[25] CAI Z, WU X, SONG Z, et al. Metformin potentiates nephrotoxicity by promoting NETosis in response to renal ferroptosis[J]. Cell Discov, 2023, 9(1): 104.
[26] HURLEY-KIM K, VU C H, DAO N M, et al. Effect of metformin use on vitamin B12 deficiency over time (EMBER): a real-world evidence database study[J]. Endocr Pract, 2023, 29(11): 862-867.
[27] NASRI H,RAFIEIAN-KOPAEI M. Metformin: current knowledge[J]. J Res Med Sci, 2014, 19(7): 658-664.
[28] RIVERA D, ONISKO N, CAO J D, et al. High risk and low prevalence diseases: metformin toxicities[J]. The American Journal of Emergency Medicine, 2023, 72: 107-112.
[29] MU Y M, JI L N, NING G, et al. Chinese expert consensus on metformin in clinical practice: 2023 update[J]. Chin J Intern Med, 2023, 62(6): 619-630.
[30] DUPONT J, MTAYER-COUSTARD S, JI B, et al. Characterization of major elements of insulin signaling cascade in chicken adipose tissue: apparent insulin refractoriness[J]. Gen Comp Endocrinol, 2012, 176(1): 86-93.
[31] JI J, TAO Y, ZHANG X, et al. Dynamic changes of blood glucose, serum biochemical parameters and gene expression in response to exogenous insulin in arbor acres broilers and silky fowls[J]. Sci Rep, 2020, 10(1): 6697.
[32] PETRILLA J, MTIS G, MACKEI M, et al. Modulation of hepatic insulin and glucagon signaling by nutritional factors in broiler chicken[J]. Vet Sci, 2022, 25(9): 103.
[33] ASHWELL C M,MCMURTRY J P. Hypoglycemia and reduced feed intake in broiler chickens treated with metformin[J]. Poult Sci, 2003, 82(1): 106-110.
[34] TRIGGLE C R, MOHAMMED I, BSHESH K, et al. Metformin: is it a drug for all reasons and diseases?[J]. Metabolism, 2022, 133: 155223.
[35] ZHANG Y, MENG Q, SUN Q, et al. LKB1 deficiency-induced metabolic reprogramming in tumorigenesis and non-neoplastic diseases[J]. Mol Metab, 2021, 44: 101131.
[36] 赵玲，尹香花. LKB1-AMPK-mTOR信号通路在子宫内膜癌的研究现状[J]. 中华妇幼临床医学杂志, 2019, 15(2): 228-232.
ZHAO L,YIN X. Research status of LKB1-AMPK-mTOR signal pathway in endometrial cancer[J]. Chinese Journal of Obstetrics and Gynecology, 2019, 15(2): 228-232. (in Chinese)
[37] WESSELS B, CIAPAITE J, VAN DEN BROEK N M, et al. Metformin impairs mitochondrial function in skeletal muscle of both lean and diabetic rats in a dose-dependent manner[J]. PLoS One, 2014, 9(6): e100525.
[38] MOHAMMED I, HOLLENBERG M D, DING H, et al. A critical review of the evidence that metformin is a putative anti-aging drug that enhances healthspan and extends lifespan[J]. Front Endocrinol (Lausanne), 2021, 12: 718942.
[39] AGIUS L, FORD B E,CHACHRA S S. The metformin mechanism on gluconeogenesis and AMPK activation: the metabolite perspective[J]. Int J Mol Sci, 2020, 21(9): 3240.
[40] TULIPANO G. Integrated or independent actions of metformin in target tissues underlying its current use and new possible applications in the endocrine and metabolic disorder area[J]. Int J Mol Sci, 2021, 22(23): 13068.
[41] XIAO X, HE Q, LU C, et al. Metformin impairs the growth of liver kinase B1-intact cervical cancer cells[J]. Gynecol Oncol, 2012, 127(1): 249-255.
[42] AHN H K, LEE Y H,KOO K C. Current status and application of metformin for prostate cancer: a comprehensive review[J]. Int J Mol Sci, 2020, 21(22): 8540.
[43] HUME S, GROU C P, LASCAUX P, et al. The NUCKS1-SKP2-p21/p27 axis controls S phase entry[J]. Nat Commun, 2021, 12(1): 6959.
[44] PAN J, SHANG F, MA R, et al. Advances of the regulatory mechanism of cyclin, cyclin- dependent kinases and related kinase inhibitors in cell cycle progression[J]. Chin J Biotechnol, 2023, 39(4): 1525-1547.
[45] ZHOU M, YANG Z, WANG D, et al. The circular RNA circZFR phosphorylates Rb promoting cervical cancer progression by regulating the SSBP1/CDK2/cyclin E1 complex[J]. J Exp Clin Cancer Res, 2021, 40(1): 48.
[46] MARTIN M,MARAIS R. Metformin: a diabetes drug for cancer, or a cancer drug for diabetics?[J]. J Clin Oncol, 2012, 30(21): 2698-2700.
[47] MURAOKA T, ICHIKAWA T, TAURA N, et al. Insulin-induced mTOR activity exhibits anti-hepatitis C virus activity[J]. Mol Med Rep, 2012, 5(2): 331-335.
[48] QIU H, YANG J K,CHEN C. Influence of insulin on growth hormone secretion, level and growth hormone signalling[J]. Acta Physiologica Sinica, 2017, 69(5): 541-556.
[49] STRELECKIENE G, INCIURAITE R, JUZENAS S, et al. MiR-20b and miR-451a are involved in gastric carcinogenesis through the PI3K/AKT/mTOR signaling pathway: data from gastric cancer patients, cell lines and ins-gas mouse model[J]. Int J Mol Sci, 2020, 21(3): 877.
[50] HUNTER R W, HUGHEY C C, LANTIER L, et al. Metformin reduces liver glucose production by inhibition of fructose-1-6-bisphosphatase[J]. Nat Med, 2018, 24(9): 1395-1406.
[51] VáZQUEZ M J, NOVELLE M G, RODRÍGUEZ-PACHECO F, et al. AMPK-dependent mechanisms but not hypothalamic lipid signaling mediates GH-secretory responses to GHRH and ghrelin[J]. Cells, 2020, 9(9): 1940.