Microbial provision of essential amino acids: protein conservation in hibernation

项目2 -微生物释放的尿素氮和宿主必需氨基酸。 老年人特别容易患肌肉减少性肥胖症（SAO），由此瘦体重的损失（例如， 肌肉）伴随着脂肪的增加，并且难以通过生活方式的改变来治疗（例如，饮食）设计 针对肥胖症。虽然许多研究已经解决了饮食和肠道微生物作为因素的作用， 疾病，如肥胖症，肠道微生物在预防瘦体重损失方面的潜在有益作用， 诸如SAO之类的疾病受到了极大的关注。尿素氮回收（UNS）是指 由此尿素被肠道微生物降解，释放氮， 氨基酸，或被宿主重吸收和使用。UNS可能在氮代谢中起重要作用 在人类中，有证据表明UNS和肠道产生必需氨基酸（EAA） 微生物可能对人类健康产生重要影响。然而，尽管有微生物释放的 尿素氮（MLUN）用于肠道中EAA的合成，这两种微生物组分是宿主氮 代谢的研究主要是独立的，很少关注的结构和功能的 肠道微生物群落该项目的目标是确定使用MLUN的肠道微生物群潜力 在EAA的合成中，并证明了微生物合成的EAA对宿主蛋白质的提供 合成.我们使用北极地松鼠（AGS）作为研究物种，因为AGS能够保持瘦体重 在它们漫长的冬眠季节。我们假设AGS肠道中的MLUN被肠道微生物利用 用于合成在冬眠期间并入宿主组织的EAA。我们的方法依赖于 同时分析了肠道微生物群落的结构和功能，微生物的生产， 使用MLUN的EAA，以及在不同条件下微生物来源的EAA在宿主组织中的掺入。 宿主膳食蛋白质利用率和生理状态。在具体目标1和2中，我们利用同位素标记的 尿素（13 C和15 N），当腹腔注射时，尿素扩散到肠道中， 尿素降解菌在给冬眠的松鼠注射标记的尿素后，我们将测量 呼吸（以确认尿素分解活性），使用下一个测定肠道微生物群的EAA合成潜力 代测序技术（寻找EAA生物合成的宏基因组学和元转录组学 基因）和代谢组学（15 N-NMR以搜索肠内容物中标记的EAA），并确定AGS是否 将微生物来源的EAA掺入宿主组织中（15 N-NMR）。在具体目标3中，我们将隔离和 表征来自AGS胃肠道的细菌，并确定生长所需的培养条件， 在未来进行模拟肠道社区的实验。我们的方法有望增加知识 肠道微生物群落在宿主氮代谢和蛋白质保存中的作用，有助于 我们对肠道微生物群落与人类健康之间关系的理解。

Project Summary Project 2 – Microbially-liberated urea nitrogen and host essential amino acids. The elderly are particularly susceptible to sarcopenic obesity (SAO), whereby a loss of lean mass (e.g., muscle) accompanies an increase in fat, and which is difficult to treat via life style changes (e.g., diet) designed to target obesity. Although numerous studies have addressed the role of diet and gut microbes as factors in diseases such as obesity, the potential beneficial role of gut microbes in preventing loss of lean mass in disorders such as SAO has received comparably less attention. Urea-nitrogen salvage (UNS) is the process whereby urea is degraded by gut microbes releasing nitrogen that can be utilized by gut microbes to produce amino acids, or be reabsorbed and used by the host. UNS likely plays an important role in nitrogen metabolism in humans, and evidence suggests both UNS and the production of essential amino acids (EAAs) by gut microbes may have important impacts on human health. Yet, despite the availability of microbially-liberated urea-nitrogen (MLUN) for EAA synthesis in the gut, these two microbial components of host nitrogen metabolism have been studied primarily independently and with little focus on the structure and function of the gut microbial community. The goal of this project is to determine the gut microbiota potential for use of MLUN in synthesis of EAAs, and demonstrate the provision of microbially-synthesized EAAs for host protein synthesis. We use the arctic ground squirrel (AGS) as a study species, as AGS are able to preserve lean mass during their long hibernation season. Our hypothesis is that MLUN in the gut of AGS is utilized by gut microbes for synthesis of EAAs which are incorporated into host tissues during hibernation. Our approach relies upon simultaneous analyses of the structure and function of the gut microbial community, microbial production of EAAs using MLUN, and incorporation of microbial-derived EAAs in host tissues under varying conditions of host dietary protein availability and physiological state. In Specific Aims 1 and 2 we utilize isotopically labeled urea (13C and 15N), which when injected intra-peritoneally diffuses into the gut and becomes available for urea degrading microbes. After injecting hibernating squirrels with labeled urea, we will measure 13CO2 in breath (to confirm ureolytic activity), determine the EAA synthesis potential of the gut microbiota using next generation sequencing techniques (metagenomics and metatranscriptomics to look for EAA biosynthesis genes) and metabolomics (15N-NMR to search for labeled EAAs in gut contents), and determine if AGS incorporate microbially-derived EAAs into host tissues (15N-NMR). In Specific Aim 3, we will isolate and characterize bacteria from the GI tract of AGS and determine culture conditions necessary for growth to experiment with mock gut communities in the future. Our approach promises to yield an increased knowledge of the role of the gut microbial community in host nitrogen metabolism and protein conservation, contributing to our understanding of the relationship between the gut microbial community and human health.