Central Carbon Metabolites Research Articles

Understanding metabolic plasticity at single cell resolution.

It is increasingly clear that cellular metabolic function varies not just between cells of different tissues, but also within tissues and cell types. In this essay, we envision how differences in central carbon metabolism arise from multiple sources, including the cell cycle, circadian rhythms, intrinsic metabolic cycles, and others. We also discuss and compare methods that enable such variation to be detected, including single-cell metabolomics and RNA-sequencing. We pay particular attention to biosensors for AMPK and central carbon metabolites, which when used in combination with metabolic perturbations, provide clear evidence of cellular variance in metabolic function.

Exploring fermentative metabolic response to varying exogenous supplies of redox cofactor precursors in selected wine yeast species.

The use of non-Saccharomyces yeasts in winemaking is gaining traction due to their specific phenotypes of technological interest, including their unique profile of central carbon metabolites and volatile compounds. However, the lack of knowledge about their physiology hinders their industrial exploitation. The intracellular redox status, involving NAD/NADH and NADP/NADPH cofactors, is a key driver of yeast activity during fermentation, notably directing the formation of metabolites that contribute to the wine bouquet. The biosynthesis of these cofactors can be modulated by the availability of their precursors, nicotinic acid and tryptophan, and their ratio by that of thiamine. In this study, a multifactorial experiment was designed to assess the effects of these three nutrients and their interactions on the metabolic response of various wine yeast species. The data indicated that limiting concentrations of nicotinic acid led to a species-dependent decrease in intracellular NAD(H) concentrations, resulting in variations of fermentation performance and production of metabolic sinks. Thiamine limitation did not directly affect redox cofactor concentrations or balance, but influenced redox management and subsequently the production of metabolites. Overall, this study identified nicotinic acid and thiamine as key factors to consider for species-specific modulation of the metabolic footprint of wine yeasts.

Characteristics of Myocardial Structure and Central Carbon Metabolism during the Early and Compensatory Stages of Cardiac Hypertrophy.

Cardiac hypertrophy is a classical forerunner of heart failure and myocardial structural and metabolic remodeling are closely associated with cardiac hypertrophy. We aim to investigate the characteristics of myocardial structure and central carbon metabolism of cardiac hypertrophy at different stages. Using echocardiography and pathological staining, early and compensatory cardiac hypertrophy were respectively defined as within 7 days and from 7 to 14 days after transverse aortic constriction (TAC) in mice. Among mass-spectrometry-based metabolomics, we identified 45 central carbon metabolites. Differential metabolite analysis showed that six metabolites, including citrate, cis-aconitate and so on, decreased significantly on day 1 after TAC. Ten metabolites, including l-lactate, (S)-2-hydroxyglutarate and so on, were obviously changed on days 10 and 14. Pathway analysis showed that these metabolites were involved in seven metabolic pathways, including carbohydrates, amino acids and so on. Western blot showed the expression of ATP-citrate lyase, malate dehydrogenase 1 and lactate dehydrogenase A in myocardium changed markedly on day 3, while the phosphorylation level of AMP-activated protein kinase did not show significantly difference. We hope our research will promote deeper understanding and early diagnosis of cardiac hypertrophy in clinical practice. All raw data were deposited in MetaboLights (MTBLS10555).

In vivo biosensing of subcellular pyruvate pools reveals photosynthesis-dependent metabolite dynamics in Nicotiana benthamiana

Abstract Pyruvate is central to metabolism across biology. It acts as a metabolic hub linking major pathways including glycolysis, the Krebs cycle, fermentation, and synthesis of amino acids, fatty acids, isoprenoids, and nucleotides. Even though the central role of pyruvate is well established biochemically, there is a remarkable gap in our understanding of how pyruvate levels behave within cells, where pyruvate is distributed across different compartments. Moreover, differential changes in pyruvate pools may occur rapidly upon changes in metabolic fluxes. Recently, this problem has been addressed by the development of a genetically encoded pyruvate biosensor to provide first insights into the pyruvate dynamics in animal cells. Here, we established in vivo biosensing of pyruvate in plants. We provided advanced characterization of the biosensor properties and demonstrated the functionality of the sensor in the cytosol, the mitochondria, and the chloroplasts of Nicotiana benthamiana epidermal cells. Finally, we harnessed the tool to investigate the impact of photosynthesis on pyruvate with unprecedented spatial and temporal resolution, revealing pronounced changes in subcellular pyruvate pools. While highlighting the current limitations of the biosensor, this study provides proof-of-concept for how the dynamics and regulation of central carbon metabolites can be revealed in living plant tissues.

Identification of Succinate Dehydrogenase Gene Variant Carriers by Blood Biomarkers.

Carriers of germline pathogenic variants (PVs) in succinate dehydrogenase genes (SDHx) are at risk of developing tumors, including paragangliomas, gastrointestinal stromal tumors, and renal cell carcinomas. Early tumor detection is paramount for improved clinical outcome. Blood-based biomarkers could aid in identifying individuals with PVs early and provide functional evidence in patients with variants of unknown significance. Blood plasma, urine, peripheral blood mononuclear cells, and erythrocytes from patients with and without SDHx PVs were investigated for central carbon metabolites. These were measured by liquid chromatography-tandem mass spectrometry and nuclear magnetic resonance spectroscopy and included among others, succinate, fumarate, α-ketoglutarate, and lactate. Plasma succinate to fumarate ratios effectively distinguished tumor-bearing and asymptomatic patients with and without SDHx PV with promising diagnostic performance (areas under the receiver operating characteristic curve 0.86-0.95), although higher levels were noted in individuals with SDHB PV. Metabolites in urine and in peripheral blood mononuclear cell extracts were largely similar between groups. Erythrocytes showed strong metabolic alterations in patients with SDHx PV compared to controls, with 8 of 13 low-molecular organic acids being significantly different (P < .05). The lactate-α-ketoglutarate-ratio of erythrocytes identified individuals with SDHx PV equally well as plasma, with a sensitivity and specificity of 92% (AUC 0.97). Blood biomarkers have been underutilized for identifying carriers of SDHx PV or to validate variants of unknown significance. Our findings advocate for further investigation into a combined approach involving plasma and erythrocytes for future diagnostic strategies.

A perfusion host-microbe bioreactor (HMB) system that captures dynamic interactions of secreted metabolites between epithelial cells cocultured with a human gut anaerobe.

The human microbiota impacts a variety of diseases and responses to therapeutics. Due to a lack of robust in vitro models, detailed mechanistic explanations of host-microbiota interactions cannot often be recapitulated. We describe the design and development of a novel, versatile and modular in vitro system that enables indirect coculture of human epithelial cells with anaerobic bacteria for the characterization of host-microbe secreted metabolite interactions. This system was designed to compartmentalize anaerobes and human cells in separate chambers conducive to each organism's requisite cell growth conditions. Using perfusion, fluidic mixing, and automated sample collection, the cells continuously received fresh media, while in contact with their corresponding compartments conditioned supernatant. Supernatants from each chamber were collected in a cell-free time-resolved fashion. The system sustained low oxygen conditions in the anaerobic chamber, while also supporting the growth of a representative anaerobe (Bacteroides thetaiotaomicron) and a human colonic epithelial cell line (Caco-2) in the aerobic chamber. Caco-2 global gene expression changes in response to coculture with B. thetaiotaomicron was characterized using RNA sequencing. Extensive, targeted metabolomics analysis of over 150 central carbon metabolites was performed on the serially collected supernatants. We observed broad metabolite changes in host-microbe coculture, compared to respective mono-culture controls. These effects were dependent both on sampling time and the compartment probed (apical vs. basolateral). Coculturing resulted in the depletion of several important metabolites, including guanine, uridine 5'-monophosphate, asparagine, and thiamine. Additionally, while Caco-2 cells cultured alone predominantly affected the basolateral metabolite milieu, increased abundance of 2,3-dihydroxyisovalerate and thymine on the basolateral side, occurred when the cells were cocultured with B. thetaiotaomicron. Thus, our system can capture the dynamic, competitive and cooperative processes between host cells and gut microbes.

Moxibustion for declined cardiorespiratory fitness of apparently healthy older adults: A study protocol for a randomized controlled trial.

Aging and age-related declines lead to varying degrees of decreased cardiorespiratory fitness (CRF) in apparently healthy older adults. Exercise training, the primary approach for enhancing CRF, encounters several constraints when used with elderly individuals. Existing evidence implies that moxibustion might enhance the CRF of older adults. However, clinical research in this area still needs to be improved. This study will employ a randomized, assessor-blinded, controlled trial design involving 126 eligible participants. These participants will be stratified and randomly assigned to one moxibustion group, one sham moxibustion group, and one blank control group. Acupoints of bilateral Zusanli (ST36), Shenque (CV8), and Guanyuan (CV4) are selected for both real and sham moxibustion groups. The treatment will last 60 min per session, 5 sessions a week for 12 weeks. The blank control group will not receive any intervention for CRF improvement. Primary outcomes will be the mean change in peak oxygen uptake (VO2peak), anaerobic threshold (AT), and serum central carbon metabolites (CCB) from the baseline to observation points. Secondary outcome measures involve the six-minute walk distance (6MWD), the Short Form 36 Health Survey (SF-36), and the Qi and Blood Status Questionnaire (QBSQ). Outcome assessments will be conducted at weeks 4, 8, 12, and 24 as part of the follow-up. Adverse events will be assessed at each visit. This trial can potentially ascertain moxibustion's effectiveness and safety in enhancing CRF among apparently healthy older adults. ChiCTR, ChiCTR2300070303. Registered on April 08, 2023.

Abstract 4452: The macropinosome: Uncovering the molecular anatomy of an oncogene-driven organelle

Abstract As an integral aspect of the metabolic reprogramming that occurs in cancer, oncogenic KRAS mutations drive the stimulation of macropinocytosis, a type of endocytosis that mediates nonselective fluid-phase uptake. Using KRAS-driven models of pancreatic ductal adenocarcinoma (PDAC), we were the first to demonstrate that macropinocytosis functions in tumor cells as a nutrient acquisition pathway. Macropinocytosis triggers the internalization of extracellular proteins via discrete endocytic vesicles called macropinosomes. The incoming protein cargo is targeted for lysosome-dependent degradation, causing the intracellular release of amino acids. These protein-derived amino acids support metabolic fitness by contributing to the intracellular amino acid pools, as well as to the biosynthesis of central carbon metabolites. In this way, macropinocytosis represents a novel amino acid supply route that tumor cells use to survive the nutrient-poor conditions of the tumor microenvironment. While the tenets of the signal transduction events that drive macropinocytosis in cancer have emerged, a detailed picture of the macropinosome itself is not yet available. With the aid of nanotechnology, we have developed a methodology to isolate a pure fraction of macropinosomes from PDAC cells. Using a large-scale proteomic approach, we have been able to generate a list of proteins that reside in macropinosomes. The specificity of this approach was confirmed through the identification and validation of proteins known to specifically localize to these organelles in different contexts, such as Rab5 or SDC1. Thanks to this innovative method, we have demonstrated the involvement of two calcium channels, ATP2B1 and ATP2B4, in the formation of macropinosomes. By elucidating the molecular anatomy of KRAS-driven macropinosomes, we will gain further insight into the regulation and function of macropinocytosis in cancer. Moreover, having a clearer picture of the macropinosome will make us better positioned to exploit this pathway therapeutically and deliver breakthroughs to patients. Citation Format: Ambroise Manceau, Koen M. Galenkamp, Cheska M. Galapate, Cosimo Commisso. The macropinosome: Uncovering the molecular anatomy of an oncogene-driven organelle [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4452.

Insights into intraspecific diversity of central carbon metabolites in Saccharomyces cerevisiae during wine fermentation

Saccharomyces cerevisiae is a major actor in winemaking that converts sugars from the grape must into ethanol and CO2 with outstanding efficiency. Primary metabolites produced during fermentation have a great importance in wine. While ethanol content contributes to the overall profile, other metabolites like glycerol, succinate, acetate or lactate also have significant impacts, even when present in lower concentrations. S. cerevisiae is known for its great genetic diversity that is related to its natural or technological environment. However, the variation range of metabolic diversity which can be exploited to enhance wine quality depends on the pathway considered. Our experiment assessed the diversity of primary metabolites production in a set of 51 S. cerevisiae strains from various genetic backgrounds. Results pointed out great yield differences depending on the metabolite considered, with ethanol having the lowest variation. A negative correlation between ethanol and glycerol was observed, confirming glycerol synthesis as a suitable lever to reduce ethanol yield. Genetic groups were linked to specific yields, such as the wine group and high α-ketoglutarate and low acetate yields. This research highlights the potential of using natural yeast diversity in winemaking. It also provides a detailed data set on production of well known (ethanol, glycerol, acetate) or little-known (lactate) primary metabolites.

Differences in Fatty Acid and Central Carbon Metabolite Distribution among Different Tissues of Alfalfa–Rhizobia Symbiotic System

Fatty acid and central carbon metabolism are crucial energy metabolism reactions. However, to date, few studies have examined their distribution characteristics within the alfalfa–rhizobia symbiotic system. To clarify the distributional differences and accumulation rates of fatty acids and central carbon with this system, we measured the plant phenotype, nodule formation, nitrogen fixation capacity, and key nitrogen metabolism enzyme activities of Medicago sativa ‘Gannong No. 9’ 35 days post-inoculation (dpi) with Sinorhizobia meliloti LL11. Additionally, we employed targeted metabolomics to analyze central carbon and fatty acid metabolites in various tissue samples of symbiotic and control (C.K.) plants, as well as in S. meliloti LL11. We found that plant height; root length; aboveground fresh and dry weights; underground fresh and dry weights; and nitrate reductase, nitrogen reductase, glutamine synthetase, and glutamate synthase activities were significantly higher in the leaves and roots of symbiotic plants than in those of C.K. plants. Compared to symbiotic plants, C.K. plants exhibited higher total central carbon and fatty acid metabolite content, accounting for 38.61% and 48.17% of C.K. plants, respectively. We detected 32 central carbon and 40 fatty acid metabolites in S. meliloti LL11, with succinate (343,180.8603 ng·mL−1) and hexadecanoic acid (4889.7783 ng·mL−1) being the most. In both symbiotic and C.K. plants, central carbon metabolite was considerably higher than the fatty acid metabolite central. Moreover, the carbon metabolites found in symbiotic plants were primarily distributed in pink nodule roots (PNRs), with malate exhibiting the highest content (4,800,612.3450 ng·g−1), accounting for 53.09% of total central carbon metabolite content. Fatty acid metabolites were mainly found in pink root nodules (P.N.s), which are sites of nitrogen fixation. Trans-10-nonadecenoic acid and hexadecanoic acid exhibited the highest contents, comprising &gt;15% of the total fatty acid metabolite content. We found that petroselaidic acid is only present in P.N., which seems to be closely related to the nitrogen fixation reaction in P.N. In general, symbiotic plants transfer central carbon metabolites to nodules via PNRs to drive nitrogen fixation. However, in P.N.s, these metabolites are limited, leading to accumulation in PNRs. Fatty acid metabolites, crucial for nitrogen fixation, are prevalent in P.N.s. Conversely, C.K. plants without nitrogen fixation distribute these metabolites primarily to the stems, emphasizing growth. This study provides new insights into the energy metabolism of symbiotic nitrogen fixation.

Abstract B088: The macropinosome: Uncovering the molecular anatomy of an oncogene-driven organelle

Abstract As an integral aspect of the metabolic reprogramming that occurs in cancer, oncogenic KRAS mutations drive the stimulation of macropinocytosis, a type of endocytosis that mediates nonselective fluid-phase uptake. Using KRAS-driven models of pancreatic ductal adenocarcinoma (PDAC), we were the first to demonstrate that macropinocytosis functions in tumor cells as a nutrient acquisition pathway. Macropinocytosis triggers the internalization of extracellular proteins via discrete endocytic vesicles called macropinosomes. The incoming protein cargo is targeted for lysosome-dependent degradation, causing the intracellular release of amino acids. These protein-derived amino acids support metabolic fitness by contributing to the intracellular amino acid pools, as well as to the biosynthesis of central carbon metabolites. In this way, macropinocytosis represents a novel amino acid supply route that tumor cells use to survive the nutrient-poor conditions of the tumor microenvironment. While the tenets of the signal transduction events that drive macropinocytosis in cancer have emerged, a detailed picture of the macropinosome itself is not yet available. With the aid of nanotechnology, we have developed a methodology to isolate a pure fraction of macropinosomes from PDAC cells. Using a large-scale proteomic approach, we have been able to generate a list of proteins that reside in macropinosomes. The specificity of this approach was confirmed through the identification and validation of proteins known to specifically localize to these organelles in different contexts, such as Rab5 or SDC1. Thanks to this innovative method, we have demonstrated the involvement of two calcium channels, ATP2B1 and ATP2B4, in the formation of macropinosomes. By elucidating the molecular anatomy of KRAS-driven macropinosomes, we will gain further insight into the regulation and function of macropinocytosis in cancer. Moreover, having a clearer picture of the macropinosome will make us better positioned to exploit this pathway therapeutically and deliver breakthroughs to patients. Citation Format: Ambroise Manceau, Koen M.O. Galenkamp, Cheska M. Galapate, Cosimo Commisso. The macropinosome: Uncovering the molecular anatomy of an oncogene-driven organelle [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B088.

Targeted metabolomics reveals the association between central carbon metabolism and pulmonary nodules.

With the widespread application of low-dose computed tomography (LDCT) technology, pulmonary nodules have aroused more attention. Significant alteration in plasma metabolite levels, mainly amino acid and lipid, have been observed in patients of PNs. However, evidence on the association between central carbon metabolism and PNs are largely unknown. The aim of this study was to investigate the underlying association of PNs and plasma central carbon metabolites. We measured the levels of 16 plasma central carbon metabolites in 1954 participants who gained LDCT screening in MALSC cohort. The inverse probability weighting (IPW) technique was used to control for bias due to self-selection for LDCT in the assessed high-risk population. The least absolute shrinkage and selection operator (LASSO) penalized regression was used to deal with the problem of multicollinearity among metabolites and the combined association of central carbon metabolites with PNs was estimated by using quantile g-computation (QgC) models. A quartile increase in 3-hydroxybutyric acid, gluconic acid, succinic acid and hippuric acid was positively associated with the PNs risk, whereas a quartile increase in 2-oxadipic acid and fumaric acid was negatively associated with the risk of PNs in multiple-metabolite models. A positive but insignificant joint associations of the mixture of 16 metabolites with PNs was observed by using QgC models analyses. Further studies are warranted to clarify the association between circulating metabolites and PNs and the biological mechanisms.

1618-P: Fasting Decreases Pyruvate Carboxylase Flux in the Perfused Exocrine Pancreas

The exocrine pancreas displays structural and functional abnormalities in type 1 diabetes, such as acinar atrophy and fibrosis. Metabolic dysfunction may occur alongside these changes. We established a method to investigate exocrine metabolism using nuclear magnetic resonance (NMR) and hyperpolarized (HP) substrates. This approach conserves the native environment while permitting analysis of metabolic flux in the living pancreas. Here, we compared exocrine pancreas metabolism in fed (n=4) and fasted (n=5) male C57BL/6J mice. Each pancreas was perfused for 30 minutes with oxygenated Krebs-Heinsleit buffer in a 14 T NMR instrument. NMR spectra were collected after injection of 4 mM HP [1-13C] pyruvate into the common bile duct. 13C signals from several central carbon metabolites were detected, such as alanine, malate, and fumarate. Pancreata from fasted mice produced less (p&amp;lt;0.05) fumarate and aspartate compared to those from the fed mice. After perfusion, all pancreata were analyzed by gas chromatography - mass spectrometry for metabolite quantification. Malate was significantly enriched (p&amp;lt;0.05) by 13C in the fed pancreas with similar total malate between groups. Overall, these results suggest that the exocrine pancreas displays higher pyruvate carboxylase (PC) flux in the fed state compared to the fasted state. The methodology presented here provides a unique avenue to assess pancreatic metabolism in real-time. Disclosure A.Rushin: None. M.Mcleod: None. M.Glanz: None. D.S.Graham: None. M.Merritt: None. Funding National Institutes of Health (T32DK10876); National Science Foundation (DMR-1644779)

S-lactoyl modification of KEAP1 by a reactive glycolytic metabolite activates NRF2 signaling.

KEAP1 (Kelch-like ECH-associated protein), a cytoplasmic repressor of the oxidative stress responsive transcription factor Nuclear factor erythroid 2-related factor 2 (NRF2), senses the presence of electrophilic agents by modification of its sensor cysteine residues. In addition to xenobiotics, several reactive metabolites have been shown to covalently modify key cysteines on KEAP1, although the full repertoire of these molecules and their respective modifications remain undefined. Here, we report the discovery of sAKZ692, a small molecule identified by high-throughput screening that stimulates NRF2 transcriptional activity in cells by inhibiting the glycolytic enzyme pyruvate kinase. sAKZ692 treatment promotes the buildup of glyceraldehyde 3-phosphate, a metabolite which leads to S-lactate modification of cysteine sensor residues of KEAP1, resulting in NRF2-dependent transcription. This work identifies a posttranslational modification of cysteine derived from a reactive central carbon metabolite and helps further define the complex relationship between metabolism and the oxidative stress-sensing machinery of the cell.

How Central Carbon Metabolites of Mexican Mint (Plectranthus amboinicus) Plants Are Impacted under Different Watering Regimes.

Plants are sessile, and their ability to reprogram their metabolism to adapt to fluctuations in soil water level is crucial but not clearly understood. A study was performed to determine alterations in intermediate metabolites involved in central carbon metabolism (CCM) following exposure of Mexican mint (Plectranthus amboinicus) to varying watering regimes. The water treatments were regular watering (RW), drought (DR), flooding (FL), and resumption of regular watering after flooding (DHFL) or after drought (RH). Leaf cluster formation and leaf greening were swift following the resumption of regular watering. A total of 68 key metabolites from the CCM routes were found to be significantly (p < 0.01) impacted by water stress. Calvin cycle metabolites in FL plants, glycolytic metabolites in DR plants, total tricarboxylic acid (TCA) cycle metabolites in DR and DHFL plants, and nucleotide biosynthetic molecules in FL and RH plants were significantly (p < 0.05) increased. Pentose phosphate pathway (PPP) metabolites were equally high in all the plants except DR plants. Total Calvin cycle metabolites had a significantly (p < 0.001) strong positive association with TCA cycle (r = 0.81) and PPP (r = 0.75) metabolites. Total PPP metabolites had a moderately positive association with total TCA cycle metabolites (r = 0.68; p < 0.01) and a negative correlation with total glycolytic metabolites (r = -0.70; p < 0.005). In conclusion, the metabolic alterations of Mexican mint plants under different watering regimes were revealed. Future studies will use transcriptomic and proteomic approaches to identify genes and proteins that regulate the CCM route.

Lipidome, central carbon metabolites, and sleep rhythm in coronary heart disease with nontraditional risks: An exploratory pilot study

AimsAltered lipid, energy metabolism and sleep disorders had been linked with coronary heart disease (CHD), however, the metabolic signatures and sleep rhythm in non-obstructive coronary atherosclerosis-CHD remain unclear. This pilot study aims to investigate the lipidome and central carbon metabolites profiles and associated sleep characteristics among CHD patients without traditional risk factors. MethodsFrom January to July 2021, 15 CHD patients and 15 healthy controls were randomly selected from the cardiology unit of Zhongshan Hospital, Shanghai. A total of 464 lipids and 45 central carbon metabolites (CCM) were quantified in blood plasma. Metabolic signatures were selected through orthogonal projections to latent structures discriminant analysis (OPLS-DA) and principal component analysis (PCA) was conducted to link the profiles of identified metabolites with CHD risk, sleep patterns, cardiometabolic traits and cardiac electrophysiologic parameters. ResultsUsing OPLS-DA, we identified 40 metabolites (variable influence on projection >1) that were altered in CHD patients, with 38 lipids, including 25 triacylglycerols (TAGs), 8 diacylglycerols (DAGs), being elevated and two CCM metabolites (i.e., succinic acid and glycolic acid) being reduced. Using PCA, four principal components (PCs) were identified and associated with increased risk of CHD. Specifically, one standard unit increasement in the PC that was characterized by high levels of DAG (18:1) and low succinic acid and the PC that was characterized by high levels of two sphingomyelins [SM (26:0) and SM (24:0)] was associated with 21% [odds ratio (OR) = 1.21, 95% CI: 1.02,1.43] and 14% (OR = 1.14,1.02,1.29) increased risk of CHD, respectively. Further regression analyses confirmed that the identified metabolites and the four PCs were positively associated with TG and ALT. Interestingly, glycolic acid was negatively associated with sleep quality and PSQI. Participants with night sleep mode tended to have a high level of the identified lipids, especially FFA (20:4). ConclusionIn the present pilot study, our findings provide clues on alterations of lipid and energy metabolism in CHD patients without traditional risk factors, with multiple triacylglycerols and diacylglycerols metabolites seemingly elevated and certain nonlipids metabolites (e.g., succinic acid and glycolic acid) decreased in cases. Considering the limit sample size, further studies are warranted to confirm our results.

Central carbon metabolite profiling reveals vector-associated differences in the recombinant protein production host Escherichia coli BL21

The Gram-negative bacterium Escherichia coli is the most widely used host for recombinant protein production, both as an industrial expression platform and as a model system at laboratory scale. The recombinant protein production industry generates proteins with direct applications as biopharmaceuticals and in technological processes central to a plethora of fields. Despite the increasing economic significance of recombinant protein production, and the importance of E. coli as an expression platform and model organism, only few studies have focused on the central carbon metabolic landscape of E. coli during high-level recombinant protein production. In the present work, we applied four targeted CapIC- and LC-MS/MS methods, covering over 60 metabolites, to perform an in-depth metabolite profiling of the effects of high-level recombinant protein production in strains derived from E. coli BL21, carrying XylS/Pm vectors with different characteristics. The mass-spectrometric central carbon metabolite profiling was complemented with the study of growth kinetics and protein production in batch bioreactors. Our work shows the robustness in E. coli central carbon metabolism when introducing increased plasmid copy number, as well as the greater importance of induction of recombinant protein production as a metabolic challenge, especially when strong promoters are used.

Plasma central carbon metabolite changes associated with KRAS mutation and circulating tumor DNA (ctDNA) status in colorectal cancer (CRC).

181 Background: KRAS mutations have been widely characterized as markers of poor prognosis in CRC. In stage IV CRC, KRAS mutations are predictive of benefit to anti-EGFR therapy. ctDNA has increasingly been recognized as a prognostic biomarker in CRC as well. We evaluated the association between plasma metabolites and KRAS mutation or ctDNA status in a longitudinal, observational cohort of patients with stage I-IV CRC. Methods: This was a retrospective analysis of prospectively collected blood samples from a single-institute cohort of patients with stage I-IV CRC. All blood samples were collected at pre-chemotherapy baseline. A modified Epi proColon 2.0 CE (Epigenomics AG) assay was used for plasma ctDNA testing on the methylated SEPTIN9 gene (mSEPT9). ctDNA positivity was defined as a mSEPT9 percentage of methylation reference (PMR) value greater than zero. Up to 150 metabolites of central carbon metabolism were analyzed by mass spectrometry and high-performance liquid chromatography. Analytes were compared by relative area under the curve (AUC) and differences evaluated by ANOVA. The mean AUC was used in patients with metabolites measured from &gt; 1 timepoint of collection. Patients were stratified by ctDNA status (positive or negative) and KRAS mutant (MT) or wildtype (WT) status. Results: A total of 32 patients were included with median age 65 years (range 20-90). The majority were female (53%) and had stage IV disease (78%). Of 25 patients with stage IV CRC, 88% had pre-chemotherapy blood samples collected in the first-line setting. Most patients were KRAS MT (44%) compared to KRAS WT (37%) or unknown KRAS status (19%). The most common KRAS MT subtypes were G12D (29%), G12V (29%), G13D (21%), and G12C (14%). The mean overall survival in this cohort was 27.4 months while the mean mSEPT9 PMR value was 2553.6. When stratified by ctDNA status, ctDNA positivity was associated with decreased levels of essential amino acids (lysine, methionine, threonine) and the non-essential amino acid arginine (all p &lt; 0.05). Compared to KRAS WT tumors, KRAS MT tumors were associated with increased levels of proline, phenylalanine, and intermediates of glycolysis (lactate), MTA cycle (SAM, 5-Methioadenosine), and O-GlcNAcylation (GlcNac, all p &lt; 0.05). Conclusions: We are the first to demonstrate the feasibility of associating central carbon metabolites with ctDNA and KRAS mutation status. As ctDNA positivity and KRAS MT status have evolving prognostic potential in CRC, associated metabolic signatures may identify metabolic pathways for novel biomarker development. Our findings also show that KRAS MT CRC appears to be enriched in intermediates of glycolytic, methyl donor, and O-GlcNAcylation pathways.

Serum Starvation Accelerates Intracellular Metabolism in Endothelial Cells.

Periods of low energy supply are challenging conditions for organisms and cells during fasting or famine. Although changes in nutrient levels in the blood are first sensed by endothelial cells, studies on their metabolic adaptations to diminished energy supply are lacking. We analyzed the dynamic metabolic activity of human umbilical vein endothelial cells (HUVECs) in basal conditions and after serum starvation. Metabolites of glycolysis, the tricarboxylic acid (TCA) cycle, and the glycerol pathway showed lower levels after serum starvation, whereas amino acids had increased levels. A metabolic flux analysis with 13C-glucose or 13C-glutamine labeling for different time points reached a plateau phase of incorporation after 30 h for 13C-glucose and after 8 h for 13C-glutamine under both experimental conditions. Notably, we observed a faster label incorporation for both 13C-glucose and 13C-glutamine after serum starvation. In the linear range of label incorporation after 3 h, we found a significantly faster incorporation of central carbon metabolites after serum starvation compared to the basal state. These findings may indicate that endothelial cells develop increased metabolic activity to cope with energy deficiency. Physiologically, it can be a prerequisite for endothelial cells to form new blood vessels under unfavorable conditions during the process of angiogenesis in vivo.

Lactate metabolism is essential in early-onset mitochondrial myopathy.

Myopathies secondary to mitochondrial electron transport chain (ETC) dysfunction can result in devastating disease. While the consequences of ETC defects have been extensively studied in culture, little in vivo data are available. Using a mouse model of severe, early-onset mitochondrial myopathy, we characterized the proteomic, transcriptomic, and metabolic characteristics of disease progression. Unexpectedly, ETC dysfunction in muscle results in reduced expression of glycolytic enzymes in our animal model and patient muscle biopsies. The decrease in glycolysis was mediated by loss of constitutive Hif1α signaling, down-regulation of the purine nucleotide cycle enzyme AMPD1, and activation of AMPK. In vivo isotope tracing experiments indicated that myopathic muscle relies on lactate import to supply central carbon metabolites. Inhibition of lactate import reduced steady-state levels of tricarboxylic acid cycle intermediates and compromised the life span of myopathic mice. These data indicate an unexpected mode of metabolic reprogramming in severe mitochondrial myopathy that regulates disease progression.