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Volume 42 Issue 4
Apr.  2024
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XU Fei, CHEN Jin, LI Zhiyong. The increasement of blood METRNL protein by insulin sensitizer rosiglitazone[J]. Journal of Pharmaceutical Practice and Service, 2024, 42(4): 165-168. doi: 10.12206/j.issn.2097-2024.202308053
Citation: XU Fei, CHEN Jin, LI Zhiyong. The increasement of blood METRNL protein by insulin sensitizer rosiglitazone[J]. Journal of Pharmaceutical Practice and Service, 2024, 42(4): 165-168. doi: 10.12206/j.issn.2097-2024.202308053

The increasement of blood METRNL protein by insulin sensitizer rosiglitazone

doi: 10.12206/j.issn.2097-2024.202308053
  • Received Date: 2023-08-25
  • Accepted Date: 2024-02-13
  • Rev Recd Date: 2024-02-13
  • Available Online: 2024-04-24
  • Publish Date: 2024-04-25
  •   Objective  To investigate the effect of insulin sensitizer rosiglitazone on blood METRNL levels.   Methods  After fed with high fat diet (HFD) for 3 months, obese mice were treated with rosiglitazone for 1 month. Glucose tolerance was tested with glucose tolerance test (GTT), and METRNL levels in blood were measured by ELISA. Real time fluorescence quantitative PCR was used to detect the expression of METRNL in various tissues such as muscle, liver, white fat, brown fat, brain, spleen and kidney, as well as the expression of mitochondrial proteins in brown adipose tissue.   Results  Glucose tolerance of animals fed a high-fat diet was improved in rosiglitazone group, and blood METRNL levels were also increased significantly in this group. Rosiglitazone treatment increased the expression of METRNL in brown fat and kidney tissue. There was no effect on METRNL expression in muscle, liver, white fat, brain and spleen. Rosiglitazone increased the expression of mitochondrial-associated proteins in brown adipose tissue.   Conclusion  The insulin sensitizer rosiglitazone might increase the serum METRNL level by increasing the METRNL expression in brown fat and kidney tissue, suggesting that METRNL may be involved in the therapeutic effect of rosiglitazone on diabetes.
  • [1] LI Z Y, ZHENG S L, WANG P, et al. Subfatin is a novel adipokine and unlike Meteorin in adipose and brain expression[J]. CNS Neurosci Ther, 2014, 20(4):344-354. doi:  10.1111/cns.12219
    [2] JØRGENSEN J R, FRANSSON A, FJORD-LARSEN L, et al. Cometin is a novel neurotrophic factor that promotes neurite outgrowth and neuroblast migration in vitro and supports survival of spiral ganglion neurons in vivo[J]. Exp Neurol, 2012, 233(1):172-181. doi:  10.1016/j.expneurol.2011.09.027
    [3] NISHINO J, YAMASHITA K, HASHIGUCHI H, et al. Meteorin: a secreted protein that regulates glial cell differentiation and promotes axonal extension[J]. EMBO J, 2004, 23(9):1998-2008. doi:  10.1038/sj.emboj.7600202
    [4] JØRGENSEN J R, THOMPSON L, FJORD-LARSEN L, et al. Characterization of Meteorin: an evolutionary conserved neurotrophic factor[J]. J Mol Neurosci, 2009, 39(1-2):104-116. doi:  10.1007/s12031-009-9189-4
    [5] LI Z Y, FAN M B, ZHANG S L, et al. Intestinal Metrnl released into the gut lumen acts as a local regulator for gut antimicrobial peptides[J]. Acta Pharmacol Sin, 2016, 37(11):1458-1466. doi:  10.1038/aps.2016.70
    [6] USHACH I, BURKHARDT A M, MARTINEZ C, et al. METEORIN-LIKE is a cytokine associated with barrier tissues and alternatively activated macrophages[J]. Clin Immunol, 2015, 156(2):119-127. doi:  10.1016/j.clim.2014.11.006
    [7] LI Z Y, SONG J, ZHENG S L, et al. Adipocyte metrnl antagonizes insulin resistance through PPARγ signaling[J]. Diabetes, 2015, 64(12):4011-4022. doi:  10.2337/db15-0274
    [8] RAO R R, LONG J Z, WHITE J P, et al. Meteorin-like is a hormone that regulates immune-adipose interactions to increase beige fat thermogenesis[J]. Cell, 2014, 157(6):1279-1291. doi:  10.1016/j.cell.2014.03.065
    [9] LÖFFLER D, LANDGRAF K, ROCKSTROH D, et al. METRNL decreases during adipogenesis and inhibits adipocyte differentiation leading to adipocyte hypertrophy in humans[J]. Int J Obes, 2017, 41(1):112-119. doi:  10.1038/ijo.2016.180
    [10] MIAO Z W, HU W J, LI Z Y, et al. Involvement of the secreted protein Metrnl in human diseases[J]. Acta Pharmacol Sin, 2020, 41(12):1525-1530. doi:  10.1038/s41401-020-00529-9
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The increasement of blood METRNL protein by insulin sensitizer rosiglitazone

doi: 10.12206/j.issn.2097-2024.202308053

Abstract:   Objective  To investigate the effect of insulin sensitizer rosiglitazone on blood METRNL levels.   Methods  After fed with high fat diet (HFD) for 3 months, obese mice were treated with rosiglitazone for 1 month. Glucose tolerance was tested with glucose tolerance test (GTT), and METRNL levels in blood were measured by ELISA. Real time fluorescence quantitative PCR was used to detect the expression of METRNL in various tissues such as muscle, liver, white fat, brown fat, brain, spleen and kidney, as well as the expression of mitochondrial proteins in brown adipose tissue.   Results  Glucose tolerance of animals fed a high-fat diet was improved in rosiglitazone group, and blood METRNL levels were also increased significantly in this group. Rosiglitazone treatment increased the expression of METRNL in brown fat and kidney tissue. There was no effect on METRNL expression in muscle, liver, white fat, brain and spleen. Rosiglitazone increased the expression of mitochondrial-associated proteins in brown adipose tissue.   Conclusion  The insulin sensitizer rosiglitazone might increase the serum METRNL level by increasing the METRNL expression in brown fat and kidney tissue, suggesting that METRNL may be involved in the therapeutic effect of rosiglitazone on diabetes.

XU Fei, CHEN Jin, LI Zhiyong. The increasement of blood METRNL protein by insulin sensitizer rosiglitazone[J]. Journal of Pharmaceutical Practice and Service, 2024, 42(4): 165-168. doi: 10.12206/j.issn.2097-2024.202308053
Citation: XU Fei, CHEN Jin, LI Zhiyong. The increasement of blood METRNL protein by insulin sensitizer rosiglitazone[J]. Journal of Pharmaceutical Practice and Service, 2024, 42(4): 165-168. doi: 10.12206/j.issn.2097-2024.202308053
  • METRNL(Metrn-like)蛋白是近年来发现和证实的新的分泌蛋白[1-2],其与METRN构成了一个两蛋白的新蛋白家族。虽然最初的研究表明,该家族蛋白均可促进神经细胞轴突的生长[2-4],但两者表达差异很大,METRN在中枢神经系统中高特异性表达,而METRNL则在全身较为广泛地表达,提示其可能具有更广泛的生理功能[1-2, 5-6]

    最近的研究发现,METRNL对代谢具有重要的调节作用。其在脂肪组织中表达较高,特别是皮下脂肪,被认为是一种新的脂肪因子[1]。研究发现,该蛋白可以调节脂肪细胞的分化,脂肪细胞中METRNL过表达可提高全身胰岛素敏感性,减少脂肪炎症扩大脂肪细胞的体积等[7]。也有研究发现,METRNL可以在运动后由肌肉组织增加分泌,促进脂肪组织棕色化,从而提高代谢率,减轻体重和改善胰岛素敏感性[8]。这些研究提示,METRNL可能与提高胰岛素敏感性相关。

    噻唑烷二酮类药物,如罗格列酮,可以通过激动PPARγ受体,提高胰岛素增敏性,被称为胰岛素增敏剂。但是这类药物与METRNL蛋白之间的关系,至今尚不清楚。我们前期的研究发现,白色脂肪组织中METRNL过表达可以提高PPARγ的表达,促进脂肪重构,降低白色脂肪炎症,但是激动PPARγ对METRNL表达的影响尚未有报道。

    本研究拟通过高脂饮食(HFD)诱导的胰岛素抵抗小鼠模型,检测血液METRNL的浓度变化;通过给予胰岛素增敏剂罗格列酮治疗,构建胰岛素增敏动物模型,检测血液中METRNL的水平变化,从而明确激动PPARγ对血液METRNL水平影响,通过实时定量PCR检测不同组织中METRNL的表达,明确PPARγ通过何种组织调控METRNL的表达与血液浓度。

    • 12周龄的雄性C57BL/6小鼠与小鼠饲料,均购自上海斯莱克实验动物有限公司。为检测胰岛素抵抗对METRNL表达的影响,分两组小鼠,每组8只,分别给予正常饮食(NCD)和HFD,均饲养4个月;为了检测胰岛素增敏对于METRNL表达的影响,分两组小鼠,每组8只,两组均先HFD饲养3个月,而后实验组的饲料中加入药物罗格列酮(胰岛素增敏组),剂量为10 mg/kg·d,治疗1个月,对照组继续HFD饲养1个月。

    • 小鼠禁食18 h,腹腔注射30%葡萄糖溶液(2 g/kg),分别在0、30、60、90、120 min取尾静脉血,采用强生血糖仪(OneTouch Ultra)检测小鼠血糖水平。

    • 戊巴比妥钠麻醉小鼠后(80 mg/kg),心脏取血收集血液,室温静置2 h,3000 r/min离心20 min,取上清液。采用小鼠METRNL ELISA试剂盒(购自美国R&D biosystem公司)检测血清中METRNL浓度。操作步骤参照试剂盒说明书。

    • 取小鼠附睾周围白色脂肪、肩胛骨间棕色脂肪、肝脏、腓肠肌、脑组织、肾脏、脾脏组织,采用TRIzol试剂(购自美国Invitrogen公司),按照说明书抽提组织总RNA,用RT-PCR逆转录试剂盒(购自中国TARARA公司)逆转录为cDNA。1 μg的cDNA用于检测METRNL的表达,GAPDH作为内参。采用2−ΔΔCt法与SYBR® Green PCR Master Mix(Applied Biosystems)试剂,反应条件为,95 ℃,5 min, 1个循环;95 ℃, 15 s,60 ℃,30 s,72 ℃,30 s,40个循环。相关引物序列见表1

      基因 上游序列(5'—3') 下游序列(5'—3')
      METRNL CTGGAGCAGGGAGGCTTATTT GGACAACAAAGTCACTGGTACA
      GAPDH GTATGACTCCACTCACGGCAAA GGTCTCGCTCCTGGAAGATG
      ERRα GCCG CGATGTCCTTTTGTG CTGTACTCGATGCTCCCTGC
      UCP-1 CACGGGGACCTACAATGCTT ACAGTAAATGGCAGGGGACG
      clec10a TGGTGTCTTGGTTTCCGTCC AGCTCCTAGCTCTCCTTGGC
      Mrc-1 CTCTGTTCAGCTATTGGACGC TGGCACTCCCAAACATAATTTGA
      Lipe GTTATGAGTGCGCTCCGAGA GAGCAAAGCTAGAGTCGGGG
      LPL GGTTGCGCGTAGAGAGGATG CTCACGCTCTGACATGCCTTC
      FABP4 AAGGTGAAGAGCATCATAACCCT TCACGCCTTTCATAACACATTCC
      CD36 ATGGGCTGTGATCGGAACTG TTTGCCACGTCATCTGGGTTT
    • 所有数据均用($ \bar x \pm s $)表示,用SPSS 10.0处理。两样本均数比较采用t检验。

    • 对HFD 3个月的小鼠采用罗格列酮治疗1个月后,葡萄糖耐量实验检测其糖耐量情况如图1所示,罗格列酮治疗组给予葡萄糖后30、60、90、120 min的血糖浓度均明显低于单纯HFD组(P<0.01),说明罗格列酮治疗明显改善了小鼠的糖耐量,提高了机体胰岛素敏感性。

    • 取正常对照组、HFD组、HFD罗格列酮治疗组小鼠的血清,ELISA检测METRNL的水平,结果如图2所示,罗格列酮组血清中METRNL的浓度为(6 632±358) pg/ml,是单纯HFD组(4 271±310) pg/ml的1.6倍(P<0.05)。

    • 实时荧光定量PCR检测肌肉、肝脏、白色脂肪、棕色脂肪、脑、脾脏、肾脏等组织中METRNL的表达,结果如图3所示,与单纯HFD组相比,罗格列酮治疗组棕色脂肪组织METRNL表达升高1.6倍,肾脏组织METRNL表达升高1.3倍。

    • 实时荧光定量PCR检测棕色脂肪组织代谢与棕色脂肪标记蛋白等mRNA表达情况,结果如图4所示,与单纯HFD组相比,罗格列酮治疗组ERRα、UCP-1、clec10a、Mrc-1、Lipe、LPL、FABP4、CD36、PNPLA2等因子表达显著升高。

    • 本研究通过HFD诱导胰岛素抵抗的肥胖小鼠,发现HFD可以导致METRNL血液水平升高。对肥胖小鼠不同组织METRNL表达的检测显示,脂肪组织METRNL表达显著升高。HFD诱导的胰岛素抵抗小鼠,给予胰岛素增敏剂罗格列酮治疗后,小鼠糖耐量改善,同时,血清METRNL的浓度也升高。这些结果说明,METRNL并非胰岛素敏感性的特异性指标,脂肪可能是使血液METRNL水平改变的主要组织之一。

      Li等研究发现,肥胖小鼠的脂肪组织METRNL表达增加[7]。本研究也表明,METRNL的血液浓度在高脂诱导肥胖后升高。Löffler等研究发现,METRNL与脂肪细胞的肥大相关,而脂肪细胞肥大被认为与PPARγ活性的降低相关,是胰岛素抵抗的重要标志之一,进而认为METRNL是机体胰岛素抵抗的标志[9]。然而,在本研究中,胰岛素增敏剂罗格列酮治疗后小鼠胰岛素敏感性提高,同时METRNL表达也显著升高,可见METRNL血液浓度的升高并不能代表胰岛素抵抗的增加。

      罗格列酮可以显著提高胰岛素的敏感性,故也称为胰岛素增敏剂。本研究表明,其可以显著提高METRNL的表达,而METRNL又具有促进白色脂肪棕色化和提高胰岛素敏感性的作用,所以METRNL可能参与介导了罗格列酮的胰岛素增敏作用。

      我们前期的研究表明,增加白色脂肪表达可以提高血液中METRNL的水平。本研究发现,罗格列酮未促进高脂条件下白色脂肪METRNL的表达,在检测的7种组织中,罗格列酮显著提高了棕色脂肪和肾脏METRNL的表达,但是对脾脏、肝脏、肌肉、白色脂肪、脑组织METRNL的表达没有影响,说明罗格列酮可能主要通过棕色脂肪和肾脏提高血液METRNL浓度。此外,进一步实验发现,罗格列酮促进了棕色脂肪中代谢和棕色脂肪标记蛋白的表达,这与以往的研究结果一致[10],既往研究表明,METRNL可促进白色脂肪棕色化,提示罗格列酮促进棕色脂肪代谢的作用可能有METRNL蛋白参与。

      本研究发现了胰岛素增敏剂罗格利酮治疗可能通过提高棕色脂肪和肾组织的METRNL表达来升高血清METRNL水平,提示METRNL可能参与了罗格列酮对糖尿病的治疗过程。

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