Research Article
Green tea polyphenols boost gut-microbiota-dependent mitochondrial TCA and urea cycles in Sprague–Dawley rats

https://doi.org/10.1016/j.jnutbio.2020.108395Get rights and content

Abstract

Green tea polyphenols (GTPs) were found to boost mammal energy conversion by modulating gut-microbial community structure, gene orthologs and metabolic pathways. Here we examined the metabolites present in the gut-microbiota-dependent mitochondrial tricarboxylic acid (TCA) cycle and urea cycle using hydrophilic interaction liquid chromatography (HILIC)-heated electrospray ionization (HESI)-tandem liquid chromatogram mass spectrometry (LC–MS). Six groups (n=12) of Sprague–Dawley rats (6-mo, ~250 g) were administered with water containing 0%, 0.5%, and 1.5% GTPs (wt/vol or g/dL). Gut-content samples were collected at 3- and 6-mo. Untargeted metabolomics detected 2177 features, with 91 features demonstrating significant dose- and time-dependencies on the GTPs treatment. Targeted metabolomics analysis revealed remarkable changes of 39 metabolites in the mitochondrial TCA cycle and urea cycle, including argininosuccunic acid (0.9-fold vs control), dihydrouracil (1.14-fold vs control), fumaric acid (1.19-fold vs control), malic acid (2.17-fold vs control), citrulline (1.86-fold vs control), and succinic acid (0.4-fold vs control). The untargeted metabolomics data were mined using bioinformatics approaches, such as analysis of variance-simultaneous component analysis (ASCA), enrichment pathway analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway mapping analysis. The results of 16S rRNA survey, metagenomics analysis, and metabolomics analysis were extrapolated and integrated using databases of Integrated Microbial Genomes and Microbiomes (IMG/M) and KEGG. Our analysis demonstrates that GTPs enhance energy conversion by boosting mitochondrial TCA cycle and urea cycle of gut-microbiota in rats. This metabolic modulation is achieved by enriching many gene orthologs, following the increase of beneficial microbials in families C. Ruminococcaceae, C. Lachnospiraceae and B. Bacteroidaceae.

Introduction

Green tea has been considered a recreational health-promoting beverage since ancient times [1]. The major functional compounds of green tea leaves (Camellia sinensis) are flavonoids, amino acids and polysaccharides [2]. As the primary flavonoids contained in tea leaves, green tea polyphenols (GTPs) constitute up to 30% dry weight of tea leaves [2,3]. In recent in vitro and in vivo studies, GTPs have demonstrated positive effects that are similar with the health benefits associated with drinking green tea, e.g. antioxidant activity, cancer protection, alleviation of high blood pressure, enhancement of bone quality, and the reductions of body fat, cholesterol level and blood sugar [[4], [5], [6], [7], [8], [9]]. Accordingly, GTPs have received remarkable attention from both industry and academia, and significant effort has been made to explore its novel clinical use and underlying mechanisms [10].

We have previously noticed that GTPs can effectively modify gut-microbiota community structure and its dependent metabolism in Sprague–Dawley rats [[11], [12], [13]]. From the gut-content samples collected from GTPs-treated rats, we observed significant enrichment of Clostridia Ruminococcaceae, C. Lachnospiraceae and B. Bacteroidaceae. Metagenomics analysis showed that GTPs elevated a number of critical gene orthologs of gut-microbiota that are associated with mitochondrial tricaboxylic acid (TCA) cycle and ATP synthesis [11]. Through normal phase gas chromatogram mass spectrometry (GC–MS) metabolomics and high-performance liquid chromatogram (HPLC) profiling analysis, we found that GTPs reduced calorific carbohydrates, such as glucose, fructose and trehalose in the gut-content of the experimental rats. The gut-microbiota dependent metabolism of bile acids, fatty acids, and amino acids were also elevated. The data collected from these aspects were highly consistent. On the other hand, it has been reported that the major part of cellular metabolites generated by gut-microbiota are more hydrophilic compounds, which, compared with more hydrophobic metabolites, can better reflect and represent the metabolic changes of gut-microbiota [14]. However, the water-soluble composition of the gut-microbiota dependent metabolome was not well characterized due to the undesirable separation of more hydrophilic metabolites in C18-column based reverse phase chromatography [[12], [13], [14]].

Hydrophilic interaction liquid chromatography (HILIC) silica column has been widely employed to analyze the more hydrophilic metabolites in complex bio-samples [15]. In practice, HILIC-column is usually coupled with LC-triple quadrupole (TsQ)-MS, which is featured by “selected reaction monitoring (SRM)” analysis. This line-up setting provides convenient targeted metabolomics analysis for the more hydrophilic partition of a global metabolome when reference MS/MS library is available[20]. In the current study, we performed HILIC technique-based untargeted and targeted metabolomics analyses to observe the GTPs-induced responses of more hydrophilic metabolites derived by gut-microbiota in the GTPs-treated rats. Bioinformatics analysis was performed to bridge the datasets of 16S rRNA survey, gas chromatogram (GC) MS metabolomics, and metagenomics analysis into a whole paradigm. The overall aim of the current work was to complete our consecutive studies, and provide a systematic mechanism on how GTPs enhance gut-microbiota dependent energy conversion in a rat model.

Section snippets

Chemicals and reagents

LC–MS grade acetonitrile, formic acid and water were purchased from Honeywell (Morris Plains, NJ, USA). High-purity GTPs decaffeinated powder was ordered from Zhejiang Yixin Pharmaceutical Co., Ltd. (Zhejiang, China). The mixture contains 65.37% of epigallocatechin gallate (EGCG), 19.08% of epicatechin gallate (ECG), 9.87% of epicatechin (EC), 4.14% of epigallocatechin (EGC), and 1.54% of catechin. Qiagen Fast DNA Stool Mini Kit was purchased from QIAGEN (Valencia, CA). KAPA LTP Library

HILIC-LC–MS metabolomic analysis

A total of 2177 features were aligned from the data of 60 samples analyzed by MS. ASCA analysis showed that there were 35 features with significant dose-dependency (Fig. 1 A), 54 features showing significant time-dependency (Fig. 1 B), and 2 features with significant dependency on the interaction effect of treatment (Fig. 1 C, D and E). The majority of the more hydrophilic metabolites were the dietary components and complex lipids with moderate polarity (Supplemental Table 1). A set of 15

Discussion

The results of HILIC-LC–MS based metabolomics analysis revealed how GTPs could modify gut-microbiota dependent energy conversion in the rat model. GTPs treatment up to 1.5% in water induced significant time- and dose-dependent alterations of the more hydrophilic metabolites and nutrients in the gut of Sprague Dawley rats (Table 1, Table 2, Fig. 1). The altered metabolic pathways include TCA cycle, bile acid metabolism and the metabolisms of purine, pyrimidine, and various amino acids (Table 3).

Acknowledgements and statement of authors' contributions to manuscript

This work was supported by USAID via Peanut and Mycotoxin Innovation Laboratory at University of Georgia [grant number ECG-A-00-13-00001-00]; National Center for Complementary and Integrative Health [grant number U01AT006691]. Special thanks are due to Interdisciplinary Toxicology Program at the University of Georgia for stipend supports. Shen CL, Wang JS, Zhou J and Tang L designed research; Zhou J conducted research; Zhou J analyzed data; Zhou J and Wang JS wrote the paper; Shen CL and Tang L

Author statement

Shen CL, Wang JS, Zhou J and Tang L designed research; Zhou J conducted research; Zhou J analyzed data; Zhou J and Wang JS wrote the paper; Shen CL and Tang L provided some of the reagents used in this study. Wang JS and Zhou J had primary responsibility for final content. All authors read and approved the final manuscript.

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