Thomas R F, Liggett S B
Department of Medicine (Pulmonary), University of Cincinnati Medical Center, Ohio 45267.
Mol Pharmacol. 1993 Mar;43(3):343-8.
The beta 3-adrenergic receptor (beta 3AR) has been purported to play important roles in a number of metabolic functions, suggesting that beta 3AR agonists might be useful as antidiabetic and antiobesity therapeutic agents. However, these assertions are based entirely on extensive metabolic studies with such agonists in rodents. To clarify the role that the beta 3AR might have in humans, we sought to define the tissue distribution of the beta 3AR in adult human tissue by the use of a highly specific and sensitive approach. Northern blots of selected tissues failed to reveal any beta 3AR mRNA, suggesting little or no expression. To detect minute amounts of transcripts, we developed a reverse transcriptase-polymerase chain reaction (RT-PCR) method that uses primers to amplify a region of the beta 3AR that has little homology with the closely related beta 1- and beta 2-AR genes, and we verified the specificity of this approach using plasmids containing the cloned human beta 1-, beta 2-, and beta 3AR genes. RT-PCR performed on as little as 20 ng of total RNA from 3T3-F442A cells, which expressed beta 3AR at very low levels (approximately 20 fmol/mg of protein), provided an easily detectable signal by ethidium bromide staining and Southern blotting of electrophoresed products. RT-PCR was performed on RNA obtained from 23 different human tissues, using primers for the beta 3AR, the beta 2AR, and beta-actin, which acted as a control. Whereas beta-actin and the beta 2AR were detected in virtually all tissues, RT-PCR using beta 3AR primers gave products from 13 tissues, including skeletal muscle, lung, adipose tissue, kidney, small intestine, pancreas, spleen, and adrenal gland. An end-labeled 50-nucleotide probe identical to an internal region of the expected beta 3AR product hybridized under low stringency conditions to seven of these products. However, sequencing of these products, which were somewhat smaller in molecular size than expected, did not reveal beta 3AR DNA sequence. Given the specificity and sensitivity of our approach, we conclude that the beta 3AR is not expressed to any significant degree in the adult human tissues studied, including adipose tissue and other metabolic sites.
β3 - 肾上腺素能受体（β3AR）据称在多种代谢功能中发挥重要作用，这表明β3AR激动剂可能作为抗糖尿病和抗肥胖治疗药物有用。然而，这些论断完全基于在啮齿动物中使用此类激动剂进行的广泛代谢研究。为了阐明β3AR在人类中可能具有的作用，我们试图通过使用高度特异和灵敏的方法来确定β3AR在成人组织中的组织分布。对选定组织进行的Northern印迹未能检测到任何β3AR mRNA，表明其表达极少或无表达。为了检测微量转录本，我们开发了一种逆转录 - 聚合酶链反应（RT - PCR）方法，该方法使用引物扩增β3AR的一个区域，该区域与密切相关的β1 - 和β2 - AR基因几乎没有同源性，并且我们使用含有克隆的人β1 - 、β2 - 和β3AR基因的质粒验证了该方法的特异性。对来自3T3 - F442A细胞的低至20 ng总RNA进行RT - PCR，该细胞β3AR表达水平极低（约20 fmol/mg蛋白质），通过溴化乙锭染色和电泳产物的Southern印迹提供了易于检测的信号。使用β3AR、β2AR引物以及作为对照的β - 肌动蛋白引物，对从23种不同人类组织获得的RNA进行RT - PCR。虽然几乎在所有组织中都检测到了β - 肌动蛋白和β2AR，但使用β3AR引物进行的RT - PCR在13种组织中得到了产物，包括骨骼肌、肺、脂肪组织、肾脏、小肠、胰腺、脾脏和肾上腺。一个与预期β3AR产物内部区域相同的末端标记50核苷酸探针在低严格条件下与其中7种产物杂交。然而，对这些分子大小略小于预期的产物进行测序，未揭示β3AR DNA序列。鉴于我们方法的特异性和灵敏性，我们得出结论，在所研究的成人组织中，包括脂肪组织和其他代谢部位，β3AR没有显著表达。
