Metabolic heterogeneity in humans.

Recent advancements in technology, especially the emergence of single-cell technologies, genomic sequencing, metabolomics, and artificial intelligence, have enabled us to understand the distinct metabolic changes in different cell types, tissues, genders, disease states, ages, and populations. Six scientists whose work intersects with metabolism in various capacities tell us about their vision for human metabolic heterogeneity.

Systematic identification and characterization of genes in the regulation and biogenesis of photosynthetic machinery.

Photosynthesis is central to food production and the Earth's biogeochemistry, yet the molecular basis for its regulation remains poorly understood. Here, using high-throughput genetics in the model eukaryotic alga Chlamydomonas reinhardtii, we identify with high confidence (false discovery rate [FDR] < 0.11) 70 poorly characterized genes required for photosynthesis. We then enable the functional characterization of these genes by providing a resource of proteomes of mutant strains, each lacking one of these genes. The data allow assignment of 34 genes to the biogenesis or regulation of one or more specific photosynthetic complexes. Further analysis uncovers biogenesis/regulatory roles for at least seven proteins, including five photosystem I mRNA maturation factors, the chloroplast translation factor MTF1, and the master regulator PMR1, which regulates chloroplast genes via nuclear-expressed factors. Our work provides a rich resource identifying regulatory and functional genes and placing them into pathways, thereby opening the door to a system-level understanding of photosynthesis.

Gut bacterial nutrient preferences quantified in vivo.

Great progress has been made in understanding gut microbiomes' products and their effects on health and disease. Less attention, however, has been given to the inputs that gut bacteria consume. Here, we quantitatively examine inputs and outputs of the mouse gut microbiome, using isotope tracing. The main input to microbial carbohydrate fermentation is dietary fiber and to branched-chain fatty acids and aromatic metabolites is dietary protein. In addition, circulating host lactate, 3-hydroxybutyrate, and urea (but not glucose or amino acids) feed the gut microbiome. To determine the nutrient preferences across bacteria, we traced into genus-specific bacterial protein sequences. We found systematic differences in nutrient use: most genera in the phylum Firmicutes prefer dietary protein, Bacteroides dietary fiber, and Akkermansia circulating host lactate. Such preferences correlate with microbiome composition changes in response to dietary modifications. Thus, diet shapes the microbiome by promoting the growth of bacteria that preferentially use the ingested nutrients.

A Dual-Mechanism Antibiotic Kills Gram-Negative Bacteria and Avoids Drug Resistance.

The rise of antibiotic resistance and declining discovery of new antibiotics has created a global health crisis. Of particular concern, no new antibiotic classes have been approved for treating Gram-negative pathogens in decades. Here, we characterize a compound, SCH-79797, that kills both Gram-negative and Gram-positive bacteria through a unique dual-targeting mechanism of action (MoA) with undetectably low resistance frequencies. To characterize its MoA, we combined quantitative imaging, proteomic, genetic, metabolomic, and cell-based assays. This pipeline demonstrates that SCH-79797 has two independent cellular targets, folate metabolism and bacterial membrane integrity, and outperforms combination treatments in killing methicillin-resistant Staphylococcus aureus (MRSA) persisters. Building on the molecular core of SCH-79797, we developed a derivative, Irresistin-16, with increased potency and showed its efficacy against Neisseria gonorrhoeae in a mouse vaginal infection model. This promising antibiotic lead suggests that combining multiple MoAs onto a single chemical scaffold may be an underappreciated approach to targeting challenging bacterial pathogens.

Obesity Shapes Metabolism in the Tumor Microenvironment to Suppress Anti-Tumor Immunity.

Obesity is a major cancer risk factor, but how differences in systemic metabolism change the tumor microenvironment (TME) and impact anti-tumor immunity is not understood. Here, we demonstrate that high-fat diet (HFD)-induced obesity impairs CD8+ T cell function in the murine TME, accelerating tumor growth. We generate a single-cell resolution atlas of cellular metabolism in the TME, detailing how it changes with diet-induced obesity. We find that tumor and CD8+ T cells display distinct metabolic adaptations to obesity. Tumor cells increase fat uptake with HFD, whereas tumor-infiltrating CD8+ T cells do not. These differential adaptations lead to altered fatty acid partitioning in HFD tumors, impairing CD8+ T cell infiltration and function. Blocking metabolic reprogramming by tumor cells in obese mice improves anti-tumor immunity. Analysis of human cancers reveals similar transcriptional changes in CD8+ T cell markers, suggesting interventions that exploit metabolism to improve cancer immunotherapy.

Metabolomics and Isotope Tracing.

Great strides have been made over the past decade toward comprehensive study of metabolism. Mass spectrometry (MS) has played a central role by enabling measurement of many metabolites simultaneously. Tracking metabolite labeling from stable isotope tracers can in addition reveal pathway activities. Here, we describe the basics of metabolite measurement by MS, including sample preparation, metabolomic analysis, and data interpretation. In addition, drawing on examples of successful experiments, we highlight the ways in which metabolomics and isotope tracing can illuminate biology.

Diet-Induced Circadian Enhancer Remodeling Synchronizes Opposing Hepatic Lipid Metabolic Processes.

Overnutrition disrupts circadian metabolic rhythms by mechanisms that are not well understood. Here, we show that diet-induced obesity (DIO) causes massive remodeling of circadian enhancer activity in mouse liver, triggering synchronous high-amplitude circadian rhythms of both fatty acid (FA) synthesis and oxidation. SREBP expression was rhythmically induced by DIO, leading to circadian FA synthesis and, surprisingly, FA oxidation (FAO). DIO similarly caused a high-amplitude circadian rhythm of PPARα, which was also required for FAO. Provision of a pharmacological activator of PPARα abrogated the requirement of SREBP for FAO (but not FA synthesis), suggesting that SREBP indirectly controls FAO via production of endogenous PPARα ligands. The high-amplitude rhythm of PPARα imparted time-of-day-dependent responsiveness to lipid-lowering drugs. Thus, acquisition of rhythmicity for non-core clock components PPARα and SREBP1 remodels metabolic gene transcription in response to overnutrition and enables a chronopharmacological approach to metabolic disorders.

Metabolite Measurement: Pitfalls to Avoid and Practices to Follow.

Metabolites are the small biological molecules involved in energy conversion and biosynthesis. Studying metabolism is inherently challenging due to metabolites' reactivity, structural diversity, and broad concentration range. Herein, we review the common pitfalls encountered in metabolomics and provide concrete guidelines for obtaining accurate metabolite measurements, focusing on water-soluble primary metabolites. We show how seemingly straightforward sample preparation methods can introduce systematic errors (e.g., owing to interconversion among metabolites) and how proper selection of quenching solvent (e.g., acidic acetonitrile:methanol:water) can mitigate such problems. We discuss the specific strengths, pitfalls, and best practices for each common analytical platform: liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), nuclear magnetic resonance (NMR), and enzyme assays. Together this information provides a pragmatic knowledge base for carrying out biologically informative metabolite measurements.

Post-transcriptional Regulation of De Novo Lipogenesis by mTORC1-S6K1-SRPK2 Signaling.

mTORC1 is a signal integrator and master regulator of cellular anabolic processes linked to cell growth and survival. Here, we demonstrate that mTORC1 promotes lipid biogenesis via SRPK2, a key regulator of RNA-binding SR proteins. mTORC1-activated S6K1 phosphorylates SRPK2 at Ser494, which primes Ser497 phosphorylation by CK1. These phosphorylation events promote SRPK2 nuclear translocation and phosphorylation of SR proteins. Genome-wide transcriptome analysis reveals that lipid biosynthetic enzymes are among the downstream targets of mTORC1-SRPK2 signaling. Mechanistically, SRPK2 promotes SR protein binding to U1-70K to induce splicing of lipogenic pre-mRNAs. Inhibition of this signaling pathway leads to intron retention of lipogenic genes, which triggers nonsense-mediated mRNA decay. Genetic or pharmacological inhibition of SRPK2 blunts de novo lipid synthesis, thereby suppressing cell growth. These results thus reveal a novel role of mTORC1-SRPK2 signaling in post-transcriptional regulation of lipid metabolism and demonstrate that SRPK2 is a potential therapeutic target for mTORC1-driven metabolic disorders.

Macrophage de novo NAD+ synthesis specifies immune function in aging and inflammation.

Recent advances highlight a pivotal role for cellular metabolism in programming immune responses. Here, we demonstrate that cell-autonomous generation of nicotinamide adenine dinucleotide (NAD+) via the kynurenine pathway (KP) regulates macrophage immune function in aging and inflammation. Isotope tracer studies revealed that macrophage NAD+ derives substantially from KP metabolism of tryptophan. Genetic or pharmacological blockade of de novo NAD+ synthesis depleted NAD+, suppressed mitochondrial NAD+-dependent signaling and respiration, and impaired phagocytosis and resolution of inflammation. Innate immune challenge triggered upstream KP activation but paradoxically suppressed cell-autonomous NAD+ synthesis by limiting the conversion of downstream quinolinate to NAD+, a profile recapitulated in aging macrophages. Increasing de novo NAD+ generation in immune-challenged or aged macrophages restored oxidative phosphorylation and homeostatic immune responses. Thus, KP-derived NAD+ operates as a metabolic switch to specify macrophage effector responses. Breakdown of de novo NAD+ synthesis may underlie declining NAD+ levels and rising innate immune dysfunction in aging and age-associated diseases.

Peripheral TREM1 responses to brain and intestinal immunogens amplify stroke severity.

Stroke is a multiphasic process in which initial cerebral ischemia is followed by secondary injury from immune responses to ischemic brain components. Here we demonstrate that peripheral CD11b+CD45+ myeloid cells magnify stroke injury via activation of triggering receptor expressed on myeloid cells 1 (TREM1), an amplifier of proinflammatory innate immune responses. TREM1 was induced within hours after stroke peripherally in CD11b+CD45+ cells trafficking to ischemic brain. TREM1 inhibition genetically or pharmacologically improved outcome via protective antioxidant and anti-inflammatory mechanisms. Positron electron tomography imaging using radiolabeled antibody recognizing TREM1 revealed elevated TREM1 expression in spleen and, unexpectedly, in intestine. In the lamina propria, noradrenergic-dependent increases in gut permeability induced TREM1 on inflammatory Ly6C+MHCII+ macrophages, further increasing epithelial permeability and facilitating bacterial translocation across the gut barrier. Thus, following stroke, peripheral TREM1 induction amplifies proinflammatory responses to both brain-derived and intestinal-derived immunogenic components. Critically, targeting this specific innate immune pathway reduces cerebral injury.

MTHFD2 is a metabolic checkpoint controlling effector and regulatory T cell fate and function.

Antigenic stimulation promotes T cell metabolic reprogramming to meet increased biosynthetic, bioenergetic, and signaling demands. We show that the one-carbon (1C) metabolism enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) regulates de novo purine synthesis and signaling in activated T cells to promote proliferation and inflammatory cytokine production. In pathogenic T helper-17 (Th17) cells, MTHFD2 prevented aberrant upregulation of the transcription factor FoxP3 along with inappropriate gain of suppressive capacity. MTHFD2 deficiency also promoted regulatory T (Treg) cell differentiation. Mechanistically, MTHFD2 inhibition led to depletion of purine pools, accumulation of purine biosynthetic intermediates, and decreased nutrient sensor mTORC1 signaling. MTHFD2 was also critical to regulate DNA and histone methylation in Th17 cells. Importantly, MTHFD2 deficiency reduced disease severity in multiple in vivo inflammatory disease models. MTHFD2 is thus a metabolic checkpoint to integrate purine metabolism with pathogenic effector cell signaling and is a potential therapeutic target within 1C metabolism pathways.

Slow TCA flux and ATP production in primary solid tumours but not metastases.

Tissues derive ATP from two pathways-glycolysis and the tricarboxylic acid (TCA) cycle coupled to the electron transport chain. Most energy in mammals is produced via TCA metabolism1. In tumours, however, the absolute rates of these pathways remain unclear. Here we optimize tracer infusion approaches to measure the rates of glycolysis and the TCA cycle in healthy mouse tissues, Kras-mutant solid tumours, metastases and leukaemia. Then, given the rates of these two pathways, we calculate total ATP synthesis rates. We find that TCA cycle flux is suppressed in all five primary solid tumour models examined and is increased in lung metastases of breast cancer relative to primary orthotopic tumours. As expected, glycolysis flux is increased in tumours compared with healthy tissues (the Warburg effect2,3), but this increase is insufficient to compensate for low TCA flux in terms of ATP production. Thus, instead of being hypermetabolic, as commonly assumed, solid tumours generally produce ATP at a slower than normal rate. In mouse pancreatic cancer, this is accommodated by the downregulation of protein synthesis, one of this tissue's major energy costs. We propose that, as solid tumours develop, cancer cells shed energetically expensive tissue-specific functions, enabling uncontrolled growth despite a limited ability to produce ATP.

mTORC1 promotes cell growth via m6A-dependent mRNA degradation.

Dysregulated mTORC1 signaling alters a wide range of cellular processes, contributing to metabolic disorders and cancer. Defining the molecular details of downstream effectors is thus critical for uncovering selective therapeutic targets. We report that mTORC1 and its downstream kinase S6K enhance eIF4A/4B-mediated translation of Wilms' tumor 1-associated protein (WTAP), an adaptor for the N6-methyladenosine (m6A) RNA methyltransferase complex. This regulation is mediated by 5' UTR of WTAP mRNA that is targeted by eIF4A/4B. Single-nucleotide-resolution m6A mapping revealed that MAX dimerization protein 2 (MXD2) mRNA contains m6A, and increased m6A modification enhances its degradation. WTAP induces cMyc-MAX association by suppressing MXD2 expression, which promotes cMyc transcriptional activity and proliferation of mTORC1-activated cancer cells. These results elucidate a mechanism whereby mTORC1 stimulates oncogenic signaling via m6A RNA modification and illuminates the WTAP-MXD2-cMyc axis as a potential therapeutic target for mTORC1-driven cancers.

Restoring metabolism of myeloid cells reverses cognitive decline in ageing.

Ageing is characterized by the development of persistent pro-inflammatory responses that contribute to atherosclerosis, metabolic syndrome, cancer and frailty1-3. The ageing brain is also vulnerable to inflammation, as demonstrated by the high prevalence of age-associated cognitive decline and Alzheimer's disease4-6. Systemically, circulating pro-inflammatory factors can promote cognitive decline7,8, and in the brain, microglia lose the ability to clear misfolded proteins that are associated with neurodegeneration9,10. However, the underlying mechanisms that initiate and sustain maladaptive inflammation with ageing are not well defined. Here we show that in ageing mice myeloid cell bioenergetics are suppressed in response to increased signalling by the lipid messenger prostaglandin E2 (PGE2), a major modulator of inflammation11. In ageing macrophages and microglia, PGE2 signalling through its EP2 receptor promotes the sequestration of glucose into glycogen, reducing glucose flux and mitochondrial respiration. This energy-deficient state, which drives maladaptive pro-inflammatory responses, is further augmented by a dependence of aged myeloid cells on glucose as a principal fuel source. In aged mice, inhibition of myeloid EP2 signalling rejuvenates cellular bioenergetics, systemic and brain inflammatory states, hippocampal synaptic plasticity and spatial memory. Moreover, blockade of peripheral myeloid EP2 signalling is sufficient to restore cognition in aged mice. Our study suggests that cognitive ageing is not a static or irrevocable condition but can be reversed by reprogramming myeloid glucose metabolism to restore youthful immune functions.

Mitochondrial ATP generation is more proteome efficient than glycolysis.

Metabolic efficiency profoundly influences organismal fitness. Nonphotosynthetic organisms, from yeast to mammals, derive usable energy primarily through glycolysis and respiration. Although respiration is more energy efficient, some cells favor glycolysis even when oxygen is available (aerobic glycolysis, Warburg effect). A leading explanation is that glycolysis is more efficient in terms of ATP production per unit mass of protein (that is, faster). Through quantitative flux analysis and proteomics, we find, however, that mitochondrial respiration is actually more proteome efficient than aerobic glycolysis. This is shown across yeast strains, T cells, cancer cells, and tissues and tumors in vivo. Instead of aerobic glycolysis being valuable for fast ATP production, it correlates with high glycolytic protein expression, which promotes hypoxic growth. Aerobic glycolytic yeasts do not excel at aerobic growth but outgrow respiratory cells during oxygen limitation. We accordingly propose that aerobic glycolysis emerges from cells maintaining a proteome conducive to both aerobic and hypoxic growth.

Riboneogenesis in yeast.

Glucose is catabolized in yeast via two fundamental routes, glycolysis and the oxidative pentose phosphate pathway, which produces NADPH and the essential nucleotide component ribose-5-phosphate. Here, we describe riboneogenesis, a thermodynamically driven pathway that converts glycolytic intermediates into ribose-5-phosphate without production of NADPH. Riboneogenesis begins with synthesis, by the combined action of transketolase and aldolase, of the seven-carbon bisphosphorylated sugar sedoheptulose-1,7-bisphosphate. In the pathway's committed step, sedoheptulose bisphosphate is hydrolyzed to sedoheptulose-7-phosphate by the enzyme sedoheptulose-1,7-bisphosphatase (SHB17), whose activity we identified based on metabolomic analysis of the corresponding knockout strain. The crystal structure of Shb17 in complex with sedoheptulose-1,7-bisphosphate reveals that the substrate binds in the closed furan form in the active site. Sedoheptulose-7-phosphate is ultimately converted by known enzymes of the nonoxidative pentose phosphate pathway to ribose-5-phosphate. Flux through SHB17 increases when ribose demand is high relative to demand for NADPH, including during ribosome biogenesis in metabolically synchronized yeast cells.

SLC25A51 is a mammalian mitochondrial NAD+ transporter.

Mitochondria require nicotinamide adenine dinucleotide (NAD+) to carry out the fundamental processes that fuel respiration and mediate cellular energy transduction. Mitochondrial NAD+ transporters have been identified in yeast and plants1,2, but their existence in mammals remains controversial3-5. Here we demonstrate that mammalian mitochondria can take up intact NAD+, and identify SLC25A51 (also known as MCART1)-an essential6,7 mitochondrial protein of previously unknown function-as a mammalian mitochondrial NAD+ transporter. Loss of SLC25A51 decreases mitochondrial-but not whole-cell-NAD+ content, impairs mitochondrial respiration, and blocks the uptake of NAD+ into isolated mitochondria. Conversely, overexpression of SLC25A51 or SLC25A52 (a nearly identical paralogue of SLC25A51) increases mitochondrial NAD+ levels and restores NAD+ uptake into yeast mitochondria lacking endogenous NAD+ transporters. Together, these findings identify SLC25A51 as a mammalian transporter capable of importing NAD+ into mitochondria.

Dietary fructose feeds hepatic lipogenesis via microbiota-derived acetate.

Consumption of fructose has risen markedly in recent decades owing to the use of sucrose and high-fructose corn syrup in beverages and processed foods1, and this has contributed to increasing rates of obesity and non-alcoholic fatty liver disease2-4. Fructose intake triggers de novo lipogenesis in the liver4-6, in which carbon precursors of acetyl-CoA are converted into fatty acids. The ATP citrate lyase (ACLY) enzyme cleaves cytosolic citrate to generate acetyl-CoA, and is upregulated after consumption of carbohydrates7. Clinical trials are currently pursuing the inhibition of ACLY as a treatment for metabolic diseases8. However, the route from dietary fructose to hepatic acetyl-CoA and lipids remains unknown. Here, using in vivo isotope tracing, we show that liver-specific deletion of Acly in mice is unable to suppress fructose-induced lipogenesis. Dietary fructose is converted to acetate by the gut microbiota9, and this supplies lipogenic acetyl-CoA independently of ACLY10. Depletion of the microbiota or silencing of hepatic ACSS2, which generates acetyl-CoA from acetate, potently suppresses the conversion of bolus fructose into hepatic acetyl-CoA and fatty acids. When fructose is consumed more gradually to facilitate its absorption in the small intestine, both citrate cleavage in hepatocytes and microorganism-derived acetate contribute to lipogenesis. By contrast, the lipogenic transcriptional program is activated in response to fructose in a manner that is independent of acetyl-CoA metabolism. These data reveal a two-pronged mechanism that regulates hepatic lipogenesis, in which fructolysis within hepatocytes provides a signal to promote the expression of lipogenic genes, and the generation of microbial acetate feeds lipogenic pools of acetyl-CoA.

Impact of acute stress on murine metabolomics and metabolic flux.

Plasma metabolite concentrations and labeling enrichments are common measures of organismal metabolism. In mice, blood is often collected by tail snip sampling. Here, we systematically examined the effect of such sampling, relative to gold-standard sampling from an in-dwelling arterial catheter, on plasma metabolomics and stable isotope tracing. We find marked differences between the arterial and tail circulating metabolome, which arise from two major factors: handling stress and sampling site, whose effects were deconvoluted by taking a second arterial sample immediately after tail snip. Pyruvate and lactate were the most stress-sensitive plasma metabolites, rising ~14 and ~5-fold. Both acute handling stress and adrenergic agonists induce extensive, immediate production of lactate, and modest production of many other circulating metabolites, and we provide a reference set of mouse circulatory turnover fluxes with noninvasive arterial sampling to avoid such artifacts. Even in the absence of stress, lactate remains the highest flux circulating metabolite on a molar basis, and most glucose flux into the TCA cycle in fasted mice flows through circulating lactate. Thus, lactate is both a central player in unstressed mammalian metabolism and strongly produced in response to acute stress.