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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。
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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。
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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。
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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。
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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。
表 1 相关引物序列
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。
The increasement of blood METRNL protein by insulin sensitizer rosiglitazone
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摘要:
目的 探讨胰岛素增敏剂罗格列酮对血液中镍纹样蛋白(METRNL)水平的影响。 方法 高脂饮食3个月诱导胰岛素抵抗的肥胖小鼠,给予罗格列酮治疗1个月,葡萄糖耐量实验检测罗格列酮对小鼠糖耐量的作用,酶联免疫吸附实验检测血清中METRNL水平,实时定量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,以及棕色脂肪中线粒体蛋白的表达。 结果 罗格列酮治疗改善了高脂饮食动物的糖耐量,同时血液中METRNL浓度也显著增高;罗格列酮治疗增加了棕色脂肪和肾脏组织中METRNL的表达,对肌肉、肝脏、白色脂肪、脑、脾脏的METRNL表达没有影响;罗格列酮治疗增加了棕色脂肪线粒体相关蛋白的表达。 结论 胰岛素增敏剂罗格利酮可能通过提高棕色脂肪和肾组织的METRNL表达升高血清METRNL水平,提示METRNL可能参与了罗格列酮对糖尿病的治疗作用。 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. -
Key words:
- insulin sensitizer /
- rosiglitazone /
- METRNL /
- insulin resistance
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表 1 相关引物序列
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 -
[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