Propionate Metabolism Research Articles

Proteomic profiling reveals the potential mechanisms and regulatory targets of sirtuin 4 in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinson's mouse model.

Parkinson's disease (PD), as a common neurodegenerative disease, currently has no effective therapeutic approaches to delay or stop its progression. There is an urgent need to further define its pathogenesis and develop new therapeutic targets. An increasing number of studies have shown that members of the sirtuin (SIRT) family are differentially involved in neurodegenerative diseases, indicating their potential to serve as targets in therapeutic strategies. Mitochondrial SIRT4 possesses multiple enzymatic activities, such as deacetylase, ADP ribosyltransferase, lipoamidase, and deacylase activities, and exhibits different enzymatic activities and target substrates in different tissues and cells; thus, mitochondrial SIRT4 plays an integral role in regulating metabolism. However, the role and mechanism of SIRT4 in PD are not fully understood. This study aimed to investigate the potential mechanism and possible regulatory targets of SIRT4 in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mice. The expression of the SIRT4 protein in the MPTP-induced PD mouse mice or key familial Parkinson disease protein 7 knockout (DJ-1 KO) rat was compared against the control group by western blot assay. Afterwards, quantitative proteomics and bioinformatics analyses were performed to identify altered proteins in the vitro model and reveal the possible functional role of SIRT4. The most promising molecular target of SIRT4 were screened and validated by viral transfection, western blot assay and reverse transcription quantitative PCR (RT-qPCR) assays. The expression of the SIRT4 protein was found to be altered both in the MPTP-induced PD mouse mice and DJ-1KO rats. Following the viral transfection of SIRT4, a quantitative proteomics analysis identified 5,094 altered proteins in the vitro model, including 213 significantly upregulated proteins and 222 significantly downregulated proteins. The results from bioinformatics analyses indicated that SIRT4 mainly affected the ribosomal pathway, propionate metabolism pathway, peroxisome proliferator-activated receptor (PPAR) signaling pathway and peroxisome pathway in cells, and we screened 25 potential molecular targets. Finally, only fatty acid binding protein 4 (FABP4) in the PPAR signaling pathway was regulated by SIRT4 among the 25 molecules. Importantly, the alterations in FABP4 and PPARγ were verified in the MPTP-induced PD mouse model. Our results indicated that FABP4 in the PPAR signaling pathway is the most promising molecular target of SIRT4 in an MPTP-induced mouse model and revealed the possible functional role of SIRT4. This study provides a reference for future drug development and mechanism research with SIRT4 as a target or biomarker.

Decoding the carbon and nitrogen metabolism mechanism in anammox system treating pharmaceutical wastewater with varying COD/N ratios through metagenomic analysis

In current study, the underlying mechanism of coupling dissimilatory nitrate reduction to ammonium (DNRA) pathway with anaerobic ammonium oxidation (anammox) process was explored through metagenomics sequencing analysis in an anammox granular system treating pharmaceutical wastewater under varying COD/N ratios. It was found that average NH4+-N and NO2--N concentration in effluent was stabled around 2.89 ± 2.53 and 14.23 ± 9.23 mg L-1 with nitrogen removal efficiency (NRE) increase to 85.3% when influent COD/N ratio was around 0.63. Besides, metagenomics sequencing analysis revealed that different COD/N ratios significantly influenced the abundances of functional nitrogen and carbon metabolic genes and microbial community succession. The low COD/N ratio strengthened DNRA pathway in Ca. Kuenenia_stuttgartiensis, as well as the abundance of functional gene nrf. The abundance of the genes encoding acetate degradation, for example, AMP-acs, ADP-acs and AckA increased, suggesting the acetate could stimulate the multiple carbon metabolic potentials of AnAOB. On the contrary, the genes encoding for propionate metabolism were enriched in denitrification bacteria. Our findings revealed the new insight into the mixotrophic characteristic of AnAOB which might help widen engineering application of anammox proocess in various industrial wastewater.

Methylmalonic acidemia caused by Homozygous Missense variant c.1837C>T in Exon 11 of the MMUT gene which is Variant of Uncertain Significance: A Case Report

Methylmalonic academia (MMA) is an autosomal recessive Inborn Error of Metabolism originating from a disorder of propionate metabolism. Although rare, it is one of the most frequent inborn errors of organic acid metabolism. The disease can manifest itself in the neonatal period or have late onset in childhood. The therapy is based on protein restriction and carnitine supplementation. The case report presents 10 months old male child who came with complaint of fever, loose stools and severe dehydration. On workup he was diagnosed as a case of isolated Methylmalonic acidemia. Clinical Exome sequencing based gene panel study showed a Homozygote Missense variant c.1837C>T in Exon 11 of the MMUT gene that resulted in the amino acid substitution p. Arg613Cys. Thus, the advanced genetic analysis along with clinical findings and typical imaging will help in appropriate diagnosis and management of the Inborn Error of Metabolism.

Altered gut microbiomes are associated with the symptomatic status of unruptured intracranial aneurysms.

Gut microbiome has recently been recognized as an important environmental factor affecting the occurrence and development of unruptured intracranial aneurysms (UIA). This study aimed to investigate the relationship between gut microbiome and symptomatic UIA, which is a predictor of instability and a high propensity to rupture. A total of 132 patients including 86 asymptomatic UIA and 46 symptomatic UIA were recruited in the study. The composition of gut bacterial communities was determined by 16S ribosomal RNA gene sequencing. In addition, Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) was used to predict the functional composition of the gut microbiome. There is no difference in the fecal microbial alpha diversity between symptomatic and asymptomatic UIA, but gut microbiome composition changed significantly. At the order level, the relative abundance of Clostridiales was significantly enriched in the symptomatic compared with asymptomatic UIA (p = 0.043). In addition, similar alterations were observed at the family levels of Ruminococcaceae. The Linear discriminant analysis (LEfSe) revealed Fournierella, Ruthenibacterium, and Anaerotruncus as discriminative features in the symptomatic group. Notably, functional differences in gut microbiome of patients with symptomatic UIA included decreased propionate metabolism pathway and enrichment of peptidoglycan biosynthesis pathways. The present study comprehensively characterizes gut microbiome in a large cohort of different risk statuses of UIA patients and demonstrates the potential biological function of gut microbiome involved in the development of UIA. It may provide additional benefits in guiding UIA management and improving patient outcomes.

Analysis of the Propionate Metabolism in Bacillus subtilis during 3-Indolacetic Production.

The genera Bacillus belongs to the group of microorganisms that are known as plant growth-promoting bacteria, their metabolism has evolved to produce molecules that benefit the growth of the plant, and the production of 3-indole acetic acid (IAA) is part of its secondary metabolism. In this work, Bacillus subtilis was cultivated in a bioreactor to produce IAA using propionate and glucose as carbon sources in an M9-modified media; in both cases, tryptophan was added as a co-substrate. The yield of IAA using propionate is 17% higher compared to glucose. After 48 h of cultivation, the final concentration was 310 mg IAA/L using propionate and 230 mg IAA/L using glucose, with a concentration of 500 mg Trp/L. To gain more insight into propionate metabolism and its advantages, the genome-scale metabolic model of B. subtilis (iBSU 1147) and computational analysis were used to calculate flux distribution and evaluate the metabolic capabilities to produce IAA using propionate. The metabolic fluxes demonstrate that propionate uptake favors the production of precursors needed for the synthesis of the hormone, and the sensitivity analysis shows that the control of a specific growth rate has a positive impact on the production of IAA.

233 (PB-057) Poster - Serum methylmalonic acid concentrations at breast cancer diagnosis are not associated with distant metastases

Background: Methylmalonic acid (MMA), a metabolite and by-product of propionate metabolism, promotes breast cancer (BC) progression in mice via the transforming growth factor beta (TGFβ) signaling pathway (Gomes et al, Nature 2020). It is currently unknown if this effect also exists in patients with BC. This study is to investigate the association between baseline serum MMA concentrations in patients at BC diagnosis and the development of distant metastases via a matched case-control study.

Altered gut microbiome diversity and function in patients with propionic acidemia.

Propionic acidemia (PA) is an inherited metabolic disorder of propionate metabolism, where the gut microbiota may play a role in pathophysiology and therefore, represent a relevant therapeutic target. Little is known about the gut microbiota composition and activity in patients with PA. Although clinical practice varies between metabolic treatment centers, management of PA requires combined dietary and pharmaceutical treatments, both known to affect the gut microbiota. This study aimed to characterize the gut microbiota and its metabolites in fecal samples of patients with PA compared with healthy controls from the same household. Eight patients (aged 3-14y) and 8 controls (4-31y) were recruited from Center 1 (UK) and 7 patients (11-33y) and 6 controls (15-54y) from Center 2 (Austria). Stool samples were collected 4 times over 3 months, alongside data on dietary intakes and medication usage. Several microbial taxa differed between patients with PA and controls, particularly for Center 1, e.g., Proteobacteria levels were increased, whereas butyrate-producing genera, such as Roseburia and Faecalibacterium, were decreased. Most measured microbial metabolites were lower in patients with PA, and butyrate was particularly depleted in patients from Center 1. Furthermore, microbiota profile of these patients showed the lowest compositional and functional diversity, and lowest stability over 3 months. As the first study to map the gut microbiota of patients with PA, this work represents an important step forward for developing new therapeutic strategies to further improve PA clinical status. New dietary strategies should consider microbial propionate production as well as butyrate production and microbiota stability.

473 Acetate and propionate metabolism by Pseudomonas aeruginosa contributes to significant sinus inflammation in a rabbit model of sinusitis

473 Acetate and propionate metabolism by Pseudomonas aeruginosa contributes to significant sinus inflammation in a rabbit model of sinusitis

Breastfeeding might partially contribute to gut microbiota construction and stabilization of propionate metabolism in cesarean-section infants.

This study was aimed to determine how delivery mode and feeding pattern influence the infant's gut microbiota construction and the variation of fecal microbial metabolites from a birth cohort. Fecal samples collected from 61 full-term born Chinese infants at four time points: day 0, day 7, month 1, and month 3. Based on delivery mode (vaginal delivery [V] or cesarean section [C]) and feeding pattern (breastfeeding [B] or mixed feeding [M]), infants were divided into four groups, namely VB, CB, VM, and CM groups. The gut microbiota composition and bacterial diversity were assessed using 16S rRNA sequencing. Short-chain fatty acid (SCFA) concentrations were determined via gas chromatography-mass spectrometry (GC-MS). The CM group had a significantly higher relative abundance of Firmicutes (day 0 and month 1), Enterococcaceae (month 3), and Enterococcus (month 3) than the VB group and a significantly higher abundance of Firmicutes (month 1) and Blautia (month 3) than the CB group. The VB and CB groups exhibited a stable SCFA variation and a significantly lower level of propionate compared with the VM and CM groups. All groups showed an intense transition of enterotypes within 1month and became stable at 3months. The correlation between SCFA and enterotypes showed a significant positive correlation between Bifidobacteriaceae and acetate in the CB group (day 7 and month 3) and a significant positive correlation between Clostridiaceae and butyrate in the CB and VB groups (day 7 and month 3), respectively. These results indicated that C-section was associated with higher abundance of the phylum Firmicutes and family Enterococcaceae, and intense fluctuation of SCFA, at least propionate. And breastfeeding might partially contribute to gut microbiota construction and stabilization propionate metabolism in cesarean-section infants.

Biotin, folic acid, and vitamin B12 supplementation given in early lactation to Holstein dairy cows: Their effects on whole-body propionate, glucose, and protein metabolism

Biotin (B8), folic acid (B9), and vitamin B12 (B12) play key roles in energy and protein metabolism. The aim of this study was to evaluate the effects of a B8 supplement, administered alone or combined with B9 and B12 during the transition period on glucose, propionate, and protein metabolism. Thirty-two Holstein multiparous cows were assigned to one of the 4 treatments according to an incomplete block design in a 2 × 2 factorial arrangement: 1) 2-mL of weekly intramuscular injections of saline 9 g/L NaCl (B8 −/B9B12 −); 2) 20 mg/d of dietary B8 (unprotected from ruminal degradation) and 2-mL weekly intramuscular injections of saline 9 g/L NaCl (B8 +/B9B12 −); 3) 2.6 g/d of dietary B9 (unprotected) and 2-mL weekly intramuscular injections of 10 mg of B12 (B8 −/B9B12 +); 4) 20 mg/d of dietary B8, 2.6 g/d of dietary B9 and weekly intramuscular injections of 10 mg of B12 (B8 +/B9B12 +). Treatments were given from − 26–45 days relative to calving date. At 43 (SD:3) days in milk (DIM), D-[6,6-2H2]-glucose (23.9 mmol/h; jugular vein) and [1-13C]-sodium propionate (22.7 mmol/h; ruminal vein) were simultaneously infused for 4 h; the following 2 d, respectively, [13C]-sodium bicarbonate (3.0 mmol/h) and L[1-13C]-Leu (3.7 mmol/h) were infused into a jugular vein for 4 h. Blood samples were collected every 15 min during the last 1.5 h of infusions at 43 DIM and every 20 min at 44 and 45 DIM during the last 2 h of infusions. During the infusion week, there were no treatment effects on milk yield and major milk components, on whole-body propionate (1354 ± 116 mmol/h) and glucose (811 ± 34 mmol/h) rates of appearance, and on the proportion of glucose synthesized from propionate (65.1 ± 4.0%). Nevertheless, plasma glucose concentration during the infusions at 43 DIM for cows receiving the B8 supplement was greater when given in combination with B9 and B12, whereas no effect was noted without B8 supplementation. Whole-body Leu rate of appearance, Leu oxidation, and Leu used for protein synthesis and exportation averaged 133 ± 5, 23 ± 2, 111 ± 4, and 53 ± 2 mmol/h, respectively, and were not different among treatments. The present results might indicate that at the time of infusions between 43 and 45 days in milk under the current conditions, supplies of B8, B9, and B12 were sufficient to meet cow requirements. This reinforces the need to better define under which conditions cows would likely benefit from a supplementation caused by an imbalance between supplies and needs.

Specific activation of hypoxia-inducible factor-2α by propionate metabolism via a β-oxidation-like pathway stimulates MUC2 production in intestinal goblet cells

Microbiota-derived short-chain fatty acids (SCFAs) are known to stimulate mucin expression in the intestine, which contributes to the gut mucosal immune responses, and the gut mucosal immune system extends to the brain and other organs through several axes. Hypoxia-inducible factors (HIFs), especially HIF-1α, are known to act as the master regulator of mucin expression, however, underlying mechanism of mucin expression during hypoxia by SCFAs remains unclear. In this study, we investigated the mechanism of MUC2 expression by propionate, an SCFA, in intestinal goblet cells. The real time oxygen consumption rate (OCR) and ATPase activity were measured to investigate the induction of hypoxia by propionate. Using 2-dimensional electrophoresis (2-DE), microarray analysis, and siRNA-induced gene silencing, we found that propionate is metabolized via a β-oxidation-like pathway instead of the vitamin B12-dependent carboxylation pathway (also known as the methylmalonyl pathway). We verified the results by analyzing several intermediates in the pathway using LC-MS and GC-MS. Propionate metabolism via the β-oxidation-like pathway leads to the depletion of oxygen and thereby induces hypoxia. Analysis of HIFs revealed that HIF-2α is the primary HIF whose activation is induced by propionate metabolism in a hypoxic environment and that HIF-2α regulates the expression of MUC2. Thus, hypoxia induced during propionate metabolism via a β-oxidation-like pathway specifically activates HIF-2α, stimulating MUC2 production in LS 174 T goblet cells. Our findings show that propionate-induced selective HIF-2α stimulation contributes to intestinal mucosal defense.

Long-term evaluation of the anaerobic co-digestion of food waste and landfill leachate to alleviate ammonia inhibition

To evaluate the feasibility of the co-digestion of food waste (FW) and landfill leachate (LL) to alleviate ammonia inhibition, a series of batch co-digestion experiments were conducted to explore the optimal substrate mixing ratio, and semi-continuous experiments were conducted to evaluate the long-tern effects of LL addition on methane generation and digestate characteristics of the anaerobic co-digestion system under ammonia stress. The optimum methane generation and digestate properties were observed when the concentration of LL in the dilution liquid in the co-digestion reactor was 40 % (v/v). Significant volatile fatty acid accumulation and lower methane yields were both observed during the long-term FW mono-digestion when the total ammonia nitrogen (TAN) concentration reached 3391.27 mg·L–1. However, stable methane generation was observed in the co-digestion system, even when the TAN was 3721.59 mg·L–1, confirming that the LL alleviated the ammonia inhibition of the FW-based anaerobic digestion (AD) system. Microbial analysis of the AD reactors demonstrated that the relative abundance of Pelotomaculum was higher in the co-digestion system than in the mono-digestion system. Analysis of the phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) confirmed the enrichment of genes involved in butyrate, propionate, and methane metabolism in the co-digestion system. In addition, analysis of the digestate characteristics (i.e., dewaterability, germination index, and heavy metals concentrations) indicated that there was no ecological risk for utilization of the digestate after long-term co-digestion operation. These results aim to provide guidance for using LL to promote the processing of FW in the future.

Integrated gut microbiota and metabolomic analysis reveals immunomodulatory effects of Echinacea extract and Astragalus polysaccharides.

Immunosuppression in different animals increases the susceptibility of various infections caused by pathogenic microorganisms leading to increase risks posed by antibiotics in different animal farming sectors. Therefore, investigation of the interactions between natural medicines and the intestinal environmental ecosystem is of vital importance and crucial. This study for the first time investigated the effects of Echinacea Extract (EE) and Astragalus polysaccharide (APS) on the gut using 16S rRNA and metabolomic analysis approaches in immunosuppressed broiler chickens. There were four groups divided into control (C), immunosuppression (IS), EE, and APS groups. Sequencing of gut microbes showed that immunosuppression decreased the relative abundance of Anaerofustis, Anaeroplasma, Anaerotroncus, and Lachnospira in the gut while increasing that of c_115 and Holdemania. However, EE and APS diminished the effects on the immunosuppression on the microbiota. The results revealed up-regulation of the relative abundance of Enterococcus in broiler chickens. In addition, EE reduced the relative abundance of Ruminococcus and Blautia. The results on metabolomic analysis revealed that immunosuppression mainly affects cyanuric acid metabolism, starch and sucrose metabolism while interconversion of pentose and glucuronide. EE and APS, on the other hand mainly impact butyrate metabolism, alanine, aspartate and glutamate metabolism while the interconversion of pentose and glucuronide, and D-glutamine and D-glutamate metabolism. Results regarding correlation analysis revealed significantly metabolic pathways including TCA cycle, butyrate metabolism, glyoxylate and dicarboxylate metabolism, propionate metabolism, alanine, aspartate and glutamate metabolism associated with Ruminococcus and Blautia. Both EE and APS can antagonize the effects of immunosuppression by modulating the disrupted gut microbiota. Nevertheless, EE might have a bidirectional regulatory functions on the intestinal health and further studies are needed to know the exact and relevant mechanisms of action regarding the effects of EE and APS.

Responses of Fermentation Characteristics and Microbial Communities to Vitamin B12 Supplementation in In Vitro Ruminal Cultures

Vitamin B12, an important cofactor involved in propionate formation, is synthesized exclusively by bacteria and archaebacteria. Humans need to intake vitamin B12 through food, and dairy products are generally the best source of vitamin B12. In the present study, the effects of vitamin B12 supplementation in diets on in vitro ruminal fermentation characteristics and microbial communities were investigated to provide a reference for increasing the vitamin B12 content in milk by dietary supplementation. A completely randomized design was carried out using the in vitro rumen culture technique, and 5 vitamin B12 dose levels (0, 0.5, 1.0, 2.0, and 4 mg/g of dry matter) were used. The results showed that vitamin B12 supplementation in diets decreased acetate: propionate ratio and butyrate concentration. The change in the acetate: propionate ratio can be attributed to the increased relative abundances of the Proteobacteria phylum and the Negativicutes class, both of which are involved in propionate metabolism. The decrease in butyrate concentration can likely be attributed to a reduction in relative abundance of species belonging to the Clostridia class, which are known as the predominant butyrate producers in the mammalian intestine. In addition, vitamin B12 supplementation in diets reduced the CH4 production by altering the species composition of the archaeal community. In conclusion, dietary supplementation of vitamin B12 resulted in rumen perturbation. In vivo studies should be conducted cautiously when evaluating the effects of vitamin B12 supplementation on the synthesis and absorption of it, as well as its content in milk.

Commonalities of Mycobacterium tuberculosis Transcriptomes in Response to Defined Persisting Macrophage Stresses.

As the goal of a bacterium is to become bacteria, evolution has imposed continued selections for gene expression. The intracellular pathogen Mycobacterium tuberculosis, the causative agent of tuberculosis, has adopted a fine-tuned response to survive its host’s methods to aggressively eradicate invaders. The development of microarrays and later RNA sequencing has led to a better understanding of biological processes controlling the relationship between host and pathogens. In this study, RNA-seq was performed to detail the transcriptomes of M. tuberculosis grown in various conditions related to stresses endured by M. tuberculosis during host infection and to delineate a general stress response incurring during persisting macrophage stresses. M. tuberculosis was subjected to long-term growth, nutrient starvation, hypoxic and acidic environments. The commonalities between these stresses point to M. tuberculosis maneuvering to exploit propionate metabolism for lipid synthesis or to withstand propionate toxicity whilst in the intracellular environment. While nearly all stresses led to a general shutdown of most biological processes, up-regulation of pathways involved in the synthesis of amino acids, cofactors, and lipids were observed only in hypoxic M. tuberculosis. This data reveals genes and gene cohorts that are specifically or exclusively induced during all of these persisting stresses. Such knowledge could be used to design novel drug targets or to define possible M. tuberculosis vulnerabilities for vaccine development. Furthermore, the disruption of specific functions from this gene set will enhance our understanding of the evolutionary forces that have caused the tubercle bacillus to be a highly successful pathogen.

Abstract 2378: Methylmalonic acid is elevated in non-small cell lung cancer and promotes drug resistance

Abstract Lung cancer is the leading cause of cancer-related deaths in the United States. Identifying new factors that promote drug resistance is critical to restoring drug sensitivity and improving patient outcomes. Methylmalonic acid (MMA) is a dicarboxylic acid by-product of propionate metabolism. MMA levels rise in the serum with age and are associated with cancer-related mortality (the higher the MMA, the worse the mortality). We recently asked whether this metabolite had a functional impact and showed that treating non-small cell lung cancer (NSCLC) cells with MMA in vitro promotes drug resistance through triggering epithelial-to-mesenchymal transition (EMT). However, whether MMA is elevated in lung cancer is not known, and how MMA levels are regulated is poorly understood. To determine if MMA is elevated in NSCLC, we performed targeted metabolomics on 20 resected lung adenocarcinomas and squamous cell carcinomas with matched normal lung tissue. We found that MMA was 2.5 fold elevated in tumors compared to controls (p<0.001, paired t-test). To our knowledge, this is the first report of MMA being elevated in any cancer. MMAB is one of the key MMA regulatory genes. When MMAB is inactivated or underexpressed, MMA cannot be metabolized and its intracellular levels increase. We hypothesized that loss of MMAB expression may explain elevated MMA levels in lung cancer. To test this, we analyzed publicly available mRNA expression data sets and found that MMAB is underexpressed in NSCLC (suggesting high levels of MMA). Moreover, MMAB expression correlates with survival. The lower the MMAB expression (suggesting higher MMA), the worse the survival (Hazard Ratio 0.36, p<0.001). We thus asked if suppressing MMAB expression had a functional impact on cell lines. Using shRNA constructs, we knocked down MMAB in the A549 and H1975 NSCLC cell lines. Targeted metabolomics confirmed that knockdown of MMAB increased MMA levels. Moreover, knockdown of MMAB decreased cellular proliferation and increased resistance to carboplatin, pemetrexed, and osimertinib. RNA-seq analysis showed that suppressing MMAB induced EMT and senescence expression signatures. Furthermore, we found that hypoxia and nutrient stress downregulate MMAB expression at the mRNA and protein level. Collectively, our data suggest that loss of MMAB expression through hypoxia or nutrient stress can increase MMA levels, triggering EMT and resistance to chemotherapy and targeted therapy in NSCLC. Our results indicate that dysregulation of MMA plays an important role in lung cancer. Citation Format: Bobak Parang, Zhongchi Li, Vivien Low, Jennifer Endress, Murtaza Malbari, Ashish Saxena, Nasser Altorki, John Blenis. Methylmalonic acid is elevated in non-small cell lung cancer and promotes drug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2378.

Abstract 3175: Methylmalonic acid in TME signaling

Abstract The importance of metabolic reprogramming in cancer has been long demonstrated by the association of systemic metabolic changes, such as aging, diet and exercise, with cancer outcomes. These systemic shifts, combined with local metabolic alterations within the tumor microenvironment (TME), can all cooperate to foster an environment conducive to cancer progression. In cells derived from primary and metastatic patient tumors, we found that mesenchymal-like cells displayed dysregulated propionate metabolism, leading to increased accumulation and secretion of methylmalonic acid (MMA), a novel aging-induced oncometabolite. This tumor cell-secreted MMA, in addition to increased MMA in the serum of elderly individuals, combine to form high local accumulation of MMA in the TME. We discovered that MMA acts on fibroblasts in the TME, activating them to cancer-associated fibroblasts (CAFs). MMA modifies the cargo of CAF-secreted extracellular vesicles (EVs), which function as a messenger to tumor cells, further promoting epithelial-to-mesenchymal transition, drug resistance, and increased metastasis in vivo. Here, we reveal a novel function of MMA in cancer, demonstrating for the first time that tumor-secreted MMA recruits the tumor microenvironment to drive cancer progression and metastatic reprogramming. Citation Format: Vivien Low, Zhongchi Li, Ashley Laughney, Wenbing Jin, Noah Dephoure, Valbona Luga, Ethan Earlie, Bobak Parang, Chunjun Guo, Lewis Cantley, John Blenis. Methylmalonic acid in TME signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3175.

Direct anabolic metabolism of three-carbon propionate to a six-carbon metabolite occurs in vivo across tissues and species

Anabolic metabolism of carbon in mammals is mediated via the one- and two-carbon carriers S-adenosyl methionine and acetyl-coenzyme A. In contrast, anabolic metabolism of three-carbon units via propionate has not been shown to extensively occur. Mammals are primarily thought to oxidize the three-carbon short chain fatty acid propionate by shunting propionyl-CoA to succinyl-CoA for entry into the TCA cycle. Here, we found that this may not be absolute as, in mammals, one nonoxidative fate of propionyl-CoA is to condense to two three-carbon units into a six-carbon trans-2-methyl-2-pentenoyl-CoA (2M2PE-CoA). We confirmed this reaction pathway using purified protein extracts provided limited substrates and verified the product via LC-MS using a synthetic standard. In whole-body in vivo stable isotope tracing following infusion of 13C-labeled valine at steady state, 2M2PE-CoA was found to form via propionyl-CoA in multiple murine tissues, including heart, kidney, and to a lesser degree, in brown adipose tissue, liver, and tibialis anterior muscle. Using ex vivo isotope tracing, we found that 2M2PE-CoA also formed in human myocardial tissue incubated with propionate to a limited extent. While the complete enzymology of this pathway remains to be elucidated, these results confirm the in vivo existence of at least one anabolic three- to six-carbon reaction conserved in humans and mice that utilizes propionate.

Measurement of changes in CO2 generated from propionate in Caenorhabditis elegans using novel 13CO2 gas analysis.

Caenorhabditis elegans contains two pathways for propionate metabolism, the vitamin B12-dependent and shunt pathways, which are similar to those in humans. In this study, we monitored the changes in propionate metabolism in the whole body of C. elegans using novel 13CO2 gas analysis. We measured the increase in 13CO2 excreted in the air after administering [1-13C]-labeled propionate ([13C]propionate) to the worms. The 13CO2 generated from [13C]propionate in C. elegans increased in a dose-dependent manner, reaching a maximum at 48 h. Enhanced expression of propionate metabolism-related genes was observed after administration of propionate. Knockdown of mmcm-1, which encodes the rate-limiting enzyme of the propionate metabolism, using RNAi reduced 13CO2 excretion. Thus, 13CO2 gas analysis could be confirmed 13CO2 excretion associated with changes in the metabolism of [13C]propionate in nematodes. This analysis can contribute to further understanding of the physiological effects of short-chain fatty acids.

Altered propionate metabolism contributes to tumour progression and aggressiveness.

The alteration of metabolic pathways is a critical strategy for cancer cells to attain the traits necessary for metastasis in disease progression. Here, we find that dysregulation of propionate metabolism produces a pro-aggressive signature in breast and lung cancer cells, increasing their metastatic potential. This occurs through the downregulation of methylmalonyl-CoA epimerase (MCEE), mediated by an ERK2-driven SP1/EGR1 transcriptional switch driven by metastatic signaling at its promoter level. The loss of MCEE results in reduced propionate-driven anaplerotic flux and the intracellular and intratumoral accumulation of methylmalonic acid (MMA), a byproduct of propionate metabolism that promotes cancer cell invasiveness. Altogether, we present a previously uncharacterized dysregulation of propionate metabolism as an important contributor to cancer and a valuable potential target in the therapeutic treatment of metastatic carcinomas.