The metabolic contribution of SKN-1/Nrf2 to the lifespan of Caenorhabditis elegans.

BACKGROUND AND AIMS: SKN-1, a C. elegans transcription factor analogous to the mammalian NF-E2-related factor (Nrf2), has been known to promote oxidative stress resistance aiding nematodes' longevity. Although SKN-1's functions suggest its implication in lifespan modulation through cellular metabolism, the actual mechanism of how metabolic rearrangements contribute to SKN-1's lifespan modulation has yet to be well characterized. Therefore, we performed the metabolomic profiling of the short-lived skn-1-knockdown C. elegans. METHODS: We analyzed the metabolic profile of the skn-1-knockdown worms with nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography-tandem mass spectrometry (LC-MS/MS) and obtained distinctive metabolomic profiles compared to WT worms. We further extended our study with gene expression analysis to examine the expression level of genes encoding all metabolic enzymes. RESULTS: A significant increase in the phosphocholine and AMP/ATP ratio, potential biomarkers of aging, was observed, accompanied by a decrease in the transsulfuration metabolites, NADPH/NADP+ ratio, and total glutathione (GSHt), which are known to be involved in oxidative stress defense. skn-1-RNAi worms also exhibited an impairment in the phase II detoxification system, confirmed by the lower conversion rate of paracetamol to paracetamol-glutathione. By further examining the transcriptomic profile, we found a decrease in the expression of cbl-1, gpx, T25B9.9, ugt, and gst, which are involved in GSHt and NADPH synthesis as well as in the phase II detoxification system. CONCLUSION: Our multi-omics results consistently revealed that the cytoprotective mechanisms, including cellular redox reactions and xenobiotic detoxification system, contribute to the roles of SKN-1/Nrf2 in the lifespan of worms.

Real-Time Monitoring of Host-Gut Microbial Interspecies Interaction in Anticancer Drug Metabolism.

Gut microbiome can affect drug metabolism considerably, leading to modified drug response. However, quantitative estimation of host vs. microbial contributions in a living host-gut microbiome system has been challenging. Using the interspecies system of Caenorhabditis elegans and gut bacteria, we developed a real-time approach for monitoring their metabolic interaction in vivo during anticancer drug 5-fluorouracil (5-FU) metabolism. The fluorine NMR-based approach yielded the quantitative contributions to the host 5-FU metabolism made by human gut-microbial species of variable genetic backgrounds. It also experimentally confirmed a bacterial gene-metabolism relationship. Differential 5-FU catabolism among bacterial substrains and the contributions to the host metabolism, unobservable by conventional 16S rRNA metagenomic sequencing, were also found. The metabolic contributions could be correlated with phenotypic developmental toxicity of 5-FU to the host fed with different substrains. Our convenient platform should help to reveal heterogeneity in host-gut microbiome interactions for many drugs in a living symbiotic system.

Real-Time In-Organism NMR Metabolomics Reveals Different Roles of AMP-Activated Protein Kinase Catalytic Subunits.

AMP-activated protein kinase (AMPK in human and AAK in C. elegans) is a master regulator of metabolism. It has many isotypes, but its isotype-dependent functions are largely unknown. By developing real-time in-organism NMR metabolomics for C. elegans, we were able to study different roles of the isotypic catalytic subunits of AAK/AMPK, AAK-1, and AAK-2 in live worms at the whole organism level. The aak-1 knockout animals exhibited enhanced glucose production under starvation, strikingly opposite to aak-2 knockout animals. Unusually high compensatory expression of the reciprocal isotypes in each KO strain and the results for the double KO animals suggested an unconventional phenotype-genotype relationship and the dominance of aak-2 in glucose production. The gene expression patterns showed that the differential phenotypes of aak-1 KO strain are due to reduced TCA and glycolysis and enhanced gluconeogenesis compared to the aak-2 KO strain. Subsequent 13C-isotope incorporation experiment showed that the glucose production in aak-1 KO occurs through the activation of fatty acid oxidation and glyoxylate shunt. Revealing differential roles of the isotypes of AAK/AMPK, our convenient approach is readily applicable to many C. elegans models for human metabolic diseases.

GPX8 regulates clear cell renal cell carcinoma tumorigenesis through promoting lipogenesis by NNMT.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC), with its hallmark phenotype of high cytosolic lipid content, is considered a metabolic cancer. Despite the implication of this lipid-rich phenotype in ccRCC tumorigenesis, the roles and regulators of de novo lipid synthesis (DNL) in ccRCC remain largely unexplained. METHODS: Our bioinformatic screening focused on ccRCC-lipid phenotypes identified glutathione peroxidase 8 (GPX8), as a clinically relevant upstream regulator of DNL. GPX8 genetic silencing was performed with CRISPR-Cas9 or shRNA in ccRCC cell lines to dissect its roles. Untargeted metabolomics, RNA-seq analyses, and other biochemical assays (e.g., lipid droplets staining, fatty acid uptake, cell proliferation, xenograft, etc.) were carried out to investigate the GPX8's involvement in lipid metabolism and tumorigenesis in ccRCC. The lipid metabolic function of GPX8 and its downstream were also measured by isotope-tracing-based DNL flux measurement. RESULTS: GPX8 knockout or downregulation substantially reduced lipid droplet levels (independent of lipid uptake), fatty acid de novo synthesis, triglyceride esterification in vitro, and tumor growth in vivo. The downstream regulator was identified as nicotinamide N-methyltransferase (NNMT): its knockdown phenocopied, and its expression rescued, GPX8 silencing both in vitro and in vivo. Mechanically, GPX8 regulated NNMT via IL6-STAT3 signaling, and blocking this axis suppressed ccRCC survival by activating AMPK. Notably, neither the GPX8-NNMT axis nor the DNL flux was affected by the von Hippel Lindau (VHL) status, the conventional regulator of ccRCC high lipid content. CONCLUSIONS: Taken together, our findings unravel the roles of the VHL-independent GPX8-NNMT axis in ccRCC lipid metabolism as related to the phenotypes and growth of ccRCC, which may be targeted for therapeutic purposes.

Snail augments fatty acid oxidation by suppression of mitochondrial ACC2 during cancer progression.

Despite the importance of mitochondrial fatty acid oxidation (FAO) in cancer metabolism, the biological mechanisms responsible for the FAO in cancer and therapeutic intervention based on catabolic metabolism are not well defined. In this study, we observe that Snail (SNAI1), a key transcriptional repressor of epithelial-mesenchymal transition, enhances catabolic FAO, allowing pro-survival of breast cancer cells in a starved environment. Mechanistically, Snail suppresses mitochondrial ACC2 (ACACB) by binding to a series of E-boxes located in its proximal promoter, resulting in decreased malonyl-CoA level. Malonyl-CoA being a well-known endogenous inhibitor of fatty acid transporter carnitine palmitoyltransferase 1 (CPT1), the suppression of ACC2 by Snail activates CPT1-dependent FAO, generating ATP and decreasing NADPH consumption. Importantly, combinatorial pharmacologic inhibition of pentose phosphate pathway and FAO with clinically available drugs efficiently reverts Snail-mediated metabolic reprogramming and suppresses in vivo metastatic progression of breast cancer cells. Our observations provide not only a mechanistic link between epithelial-mesenchymal transition and catabolic rewiring but also a novel catabolism-based therapeutic approach for inhibition of cancer progression.