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Integrated metabolomic and transcriptomic analysis reveals metabolic adaptation of rats to Abcg2/Abcb1a knockout

ABSTRACT: Breast Cancer Resistance Protein (Bcrp) and P-glycoprotein (Pgp) are xenobiotic efflux transporters that limit permeability of their substrates in the gastrointestinal system and brain, and increase drug clearance in liver and kidney. Bcrp and Pgp are also known to transport different endogenous metabolites such as riboflavin, urate, sulfated steroids and glucocorticoids. However, a systemic approach to understand the endogenous substrates and function of these transporters is still lacking. We hypothesized that Bcrp and Pgp have important endogenous functions and play a crucial role in maintaining metabolomic homeostasis in the biological system. To understand the impact of Bcrp and Pgp on endogenous metabolome, we performed untargeted metabolomics of CSF and plasma of male Sprague Dawley (SD) rats (WT) and SD rats lacking both Bcrp and Pgp (double knock-out, dKO rats) (n= 8 per genotype). In addition, we performed genome-wide microarray analysis of brain frontal cortex, kidney and liver from the same animals to analyze changes in gene expression (n=5 per genotype). Our results indicate distinct differences in metabolites and gene expression between WT and dKO rats. Rank order based on both fold change (>1.5 or <0.67) and significance (p <0.05) identified 29 metabolites in CSF and 47 metabolites in plasma as most significantly altered between WT and dKO rats, which includes, among other biochemicals, known substrates of Bcrp e.g., riboflavin, urate, daidzein and its metabolites. Significantly altered metabolites in plasma as well as CSF showed signatures of altered fatty acid metabolism and oxidative stress, which was further supported by the pathway analysis of significantly altered genes. These findings may lead to new insights in understanding the endogenous function of Bcrp and Pgp transporters and identification of functional biomarkers of these transporters

ORGANISM(S): Rattus norvegicus

PROVIDER: GSE159058 | GEO | 2021/07/01

REPOSITORIES: GEO

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Project description:Mdr1a-, Bcrp-, and Mrp2-knockout rats are a more practical species for ADME studies than murine models and previously demonstrated expected alterations in pharmacokinetics of various probe substrates. At present, gene expression and pathology changes were systematically studied in small intestine, liver, kidney, and brain tissue from male SAGE Mdr1a-, Bcrp-, and Mrp2-knockout rats versus wild-type Sprague Dawley controls. Gene expression data supported the relevant knockout genotype. As expected, Mrp2-knockout rats were hyperbilirubinemic and exhibited upregulation of hepatic Mrp3. Overall, few alterations were observed within 137 ADME-relevant genes. The two most consequential changes were upregulation of intestinal carboxylesterase in Mdr1a-knockouts and catechol-O-methyltransferase in all tissues of Bcrp-knockout rats. Previously reported upregulation of hepatic Mdr1b P-glycoprotein in proprietary Wistar Mdr1a-knockout rats was not observed in the SAGE counterpart investigated herein. Relative liver and kidney weights were 22-53% higher in all three knockouts, with microscopic increases in hepatocyte size in Mdr1a- and Mrp2-knockout rats, and glomerular size in Bcrp- and Mrp2-knockouts. Increased relative weight of clearing organs is quantitatively consistent with reported increases in clearance of drugs that are not substrates of the knocked-out transporter. Overall, SAGE knockout rats demonstrated modest compensatory changes, which do not preclude their general application to study transporter-mediated pharmacokinetics. However until future studies elucidate the magnitude of functional change, caution is warranted in rare instances of extensive metabolism by catechol-O-methyltransferase in Bcrp-knockouts and intestinal carboxylesterase in Mdr1a-knockout rats, specifically for molecules with free catechol groups and esters subject to gut wall hydrolysis. 3 groups of knockout animals compared to wild-type male Sprague Dawley control rats. 4 animals per group. Assayed the following tissues: brain, duodenum, ileum, jejunum, kidney (cortex-anterior pole), kidney (medulla), liver. Hybridized to Affymetrix Rat Genome 230 v2 [Rat230_2] arrays.

Project description:Genomic analysis of the model lignocellulosic biomass degrading bacteria C. phytofermentans indicates that it can degrade, transport, and utilize a wide-range of carbohydrates as possible growth substrates. Previous experiments characterized the expression of the degradation and transport machinery using custom whole genome oligonucleotide microarrays. The results indicate that C. phytofermentans utilizes ATP-binding cassette (ABC) transporters for carbohydrate uptake and does not use the sole phosphoenolpyruvate-phosphotransferase system (PTS) for any of the tested substrates. While some ABC transporters are specific for a single carbohydrate, the expression profiles indicate that others may be capable of transporting multiple substrates. Distinct sets of Carbohydrate Active Enzymes (CAZy) genes were also up-regulated on specific substrates indicative of C. phytofermentans ability to selectively degrade plant biomass. We also identified a highly expressed cluster of genes which includes seven extracellular glycoside hydrolases and two ABC transporters with unknown specificity. These results lead to the hypothesis that when grown on plant biomass, C. phytofermentans is capable of degrading and transporting all major carbohydrate components of the plant cell. To test this, C. phytofermentans was grown on cornstover and switchgrass. Results from this expression data and HPLC analysis indicates that C. phytofermentans is utilizing multiple substrates. with multiple sugar ABC transporter clusters and glycoside hydrolases being expressed. Interestingly all of the transporters were initially identified on disaccharides or oligio-saccharides, and none of the transporters identified as monosaccharide specific transporters were expressed. This could be an indication that C. phytofermentans prefers to transport oligiosacchrides over monosaccharides. The results presented here corroborate the genomic data which indicates the breath of the carbohydrate degradation, transport, and utilization machinery of C. phytofermentans. C. phytofermentans was cultured anaerobically on switchgrass and corn stover to determine specific expression patterns. The data in this series consists three independent RNA preparations from replicate cultures.

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Integrated metabolomic and transcriptomic analysis reveals metabolic adaptation of rats to Abcg2/Abcb1a knockout

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