Brown fat ATP-citrate lyase links carbohydrate availability to thermogenesis and guards against metabolic stress.

Brown adipose tissue (BAT) engages futile fatty acid synthesis-oxidation cycling, the purpose of which has remained elusive. Here, we show that ATP-citrate lyase (ACLY), which generates acetyl-CoA for fatty acid synthesis, promotes thermogenesis by mitigating metabolic stress. Without ACLY, BAT overloads the tricarboxylic acid cycle, activates the integrated stress response (ISR) and suppresses thermogenesis. ACLY's role in preventing BAT stress becomes critical when mice are weaned onto a carbohydrate-plentiful diet, while removing dietary carbohydrates prevents stress induction in ACLY-deficient BAT. ACLY loss also upregulates fatty acid synthase (Fasn); yet while ISR activation is not caused by impaired fatty acid synthesis per se, deleting Fasn and Acly unlocks an alternative metabolic programme that overcomes tricarboxylic acid cycle overload, prevents ISR activation and rescues thermogenesis. Overall, we uncover a previously unappreciated role for ACLY in mitigating mitochondrial stress that links dietary carbohydrates to uncoupling protein 1-dependent thermogenesis and provides fundamental insight into the fatty acid synthesis-oxidation paradox in BAT.

Modeling compound lipid homeostasis using stable isotope tracing.

Lipids represent the most diverse pool of metabolites found in cells, facilitating compartmentation, signaling, and other functions. Dysregulation of lipid metabolism is linked to disease states such as cancer and neurodegeneration. However, limited tools are available for quantifying metabolic fluxes across the lipidome. To directly measure reaction fluxes encompassing compound lipid homeostasis, we applied stable isotope tracing, high-resolution mass spectrometry, and network-based isotopologue modeling to non-small cell lung cancer (NSCLC) models. Compound lipid metabolic flux analysis (CL-MFA) enables the concurrent quantitation of fatty acid synthesis, elongation, headgroup assembly, and salvage reactions within virtually any biological system. Here, we resolve liver kinase B1 (LKB1)-mediated regulation of sphingolipid recycling in NSCLC cells and precision-cut lung slice cultures. We also demonstrate that widely used tissue culture conditions drive cells to upregulate fatty acid synthase flux to supraphysiological levels. Finally, we identify previously uncharacterized isozyme specificity of ceramide synthase inhibitors, highlighting the molecular detail revealed by CL-MFA.

Bempedoic acid suppresses diet-induced hepatic steatosis independently of ATP-citrate lyase.

ATP citrate lyase (ACLY) synthesizes acetyl-CoA for de novo lipogenesis (DNL), which is elevated in metabolic dysfunction-associated steatotic liver disease. Hepatic ACLY is inhibited by the LDL-cholesterol-lowering drug bempedoic acid (BPA), which also improves steatosis in mice. While BPA potently suppresses hepatic DNL and increases fat catabolism, it is unclear if ACLY is its primary molecular target in reducing liver triglyceride. We show that on a Western diet, loss of hepatic ACLY alone or together with the acetyl-CoA synthetase ACSS2 unexpectedly exacerbates steatosis, linked to reduced PPARα target gene expression and fatty acid oxidation. Importantly, BPA treatment ameliorates Western diet-mediated triacylglyceride accumulation in both WT and liver ACLY knockout mice, indicating that its primary effects on hepatic steatosis are ACLY independent. Together, these data indicate that hepatic ACLY plays an unexpected role in restraining diet-dependent lipid accumulation and that BPA exerts substantial effects on hepatic lipid metabolism independently of ACLY.

Rhythmic IL-17 production by γδ T cells maintains adipose de novo lipogenesis.

The circadian rhythm of the immune system helps to protect against pathogens1-3; however, the role of circadian rhythms in immune homeostasis is less well understood. Innate T cells are tissue-resident lymphocytes with key roles in tissue homeostasis4-7. Here we use single-cell RNA sequencing, a molecular-clock reporter and genetic manipulations to show that innate IL-17-producing T cells-including γδ T cells, invariant natural killer T cells and mucosal-associated invariant T cells-are enriched for molecular-clock genes compared with their IFNγ-producing counterparts. We reveal that IL-17-producing γδ (γδ17) T cells, in particular, rely on the molecular clock to maintain adipose tissue homeostasis, and exhibit a robust circadian rhythm for RORγt and IL-17A across adipose depots, which peaks at night. In mice, loss of the molecular clock in the CD45 compartment (Bmal1∆Vav1) affects the production of IL-17 by adipose γδ17 T cells, but not cytokine production by αß or IFNγ-producing γδ (γδIFNγ) T cells. Circadian IL-17 is essential for de novo lipogenesis in adipose tissue, and mice with an adipocyte-specific deficiency in IL-17 receptor C (IL-17RC) have defects in de novo lipogenesis. Whole-body metabolic analysis in vivo shows that Il17a-/-Il17f-/- mice (which lack expression of IL-17A and IL-17F) have defects in their circadian rhythm for de novo lipogenesis, which results in disruptions to their whole-body metabolic rhythm and core-body-temperature rhythm. This study identifies a crucial role for IL-17 in whole-body metabolic homeostasis and shows that de novo lipogenesis is a major target of IL-17.

Serine and glycine physiology reversibly modulate retinal and peripheral nerve function.

Metabolic homeostasis is maintained by redundant pathways to ensure adequate nutrient supply during fasting and other stresses. These pathways are regulated locally in tissues and systemically via the liver, kidney, and circulation. Here, we characterize how serine, glycine, and one-carbon (SGOC) metabolism fluxes across the eye, liver, and kidney sustain retinal amino acid levels and function. Individuals with macular telangiectasia (MacTel), an age-related retinal disease with reduced circulating serine and glycine, carrying deleterious alleles in SGOC metabolic enzymes exhibit an exaggerated reduction in circulating serine. A Phgdh+/- mouse model of this haploinsufficiency experiences accelerated retinal defects upon dietary serine/glycine restriction, highlighting how otherwise silent haploinsufficiencies can impact retinal health. We demonstrate that serine-associated retinopathy and peripheral neuropathy are reversible, as both are restored in mice upon serine supplementation. These data provide molecular insights into the genetic and metabolic drivers of neuro-retinal dysfunction while highlighting therapeutic opportunities to ameliorate this pathogenesis.

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.

Protocol to generate human liver spheroids to study liver fibrosis induced by metabolic stress.

Currently, there is no effective treatment for obesity and alcohol-associated liver diseases, partially due to the lack of translational human models. Here, we present a protocol to generate 3D human liver spheroids that contain all the liver cell types and mimic "livers in a dish." We describe strategies to induce metabolic and alcohol-associated hepatic steatosis, inflammation, and fibrosis. We outline potential applications, including using human liver spheroids for experimental and translational research and drug screening to identify potential anti-fibrotic therapies.

Drp1 controls complex II assembly and skeletal muscle metabolism by Sdhaf2 action on mitochondria.

The dynamin-related guanosine triphosphatase, Drp1 (encoded by Dnm1l), plays a central role in mitochondrial fission and is requisite for numerous cellular processes; however, its role in muscle metabolism remains unclear. Here, we show that, among human tissues, the highest number of gene correlations with DNM1L is in skeletal muscle. Knockdown of Drp1 (Drp1-KD) promoted mitochondrial hyperfusion in the muscle of male mice. Reduced fatty acid oxidation and impaired insulin action along with increased muscle succinate was observed in Drp1-KD muscle. Muscle Drp1-KD reduced complex II assembly and activity as a consequence of diminished mitochondrial translocation of succinate dehydrogenase assembly factor 2 (Sdhaf2). Restoration of Sdhaf2 normalized complex II activity, lipid oxidation, and insulin action in Drp1-KD myocytes. Drp1 is critical in maintaining mitochondrial complex II assembly, lipid oxidation, and insulin sensitivity, suggesting a mechanistic link between mitochondrial morphology and skeletal muscle metabolism, which is clinically relevant in combatting metabolic-related diseases.

Tracing the Diverse Paths of One-Carbon Metabolism in Cancer and Beyond.

One-carbon (1C) metabolism is a network of biochemical reactions distributed across organelles that delivers folate-activated 1C units to support macromolecule synthesis, methylation, and reductive homeostasis. Fluxes through these pathways are up-regulated in highly proliferative cancer cells, and anti-folates, which target enzymes within the 1C pathway, have long been used in the treatment of cancer. In this work, we review fundamental aspects of 1C metabolism and place it in context with other biosynthetic and redox pathways, such that 1C metabolism acts to bridge pathways across compartments. We further discuss the importance of stable-isotope-tracing techniques combined with mass spectrometry analysis to study 1C metabolism and conclude by highlighting therapeutic approaches that could exploit cancer cells' dependency on 1C metabolism.

Metabolic Deficiencies Underlie Plasmacytoid Dendritic Cell Exhaustion After Viral Infection.

Type I Interferons (IFN-I) are central to host protection against viral infections 1 . While any cell can produce IFN-I, Plasmacytoid Dendritic Cells (pDCs) make greater quantities and more varieties of these cytokines than any other cell type 2 . However, following an initial burst of IFN- I, pDCs lose their exceptional IFN-I production capacity and become "exhausted", a phenotype that associates with enhanced susceptibility to secondary infections 3-5 . Despite this apparent cost for the host, pDC exhaustion is conserved across multiple species and viral infections, but the underlying mechanisms and the potential evolutionary advantages are not well understood. Here we characterize pDC exhaustion and demonstrate that it is associated with a reduced capacity of pDCs to engage both oxidative and glycolytic metabolism. Mechanistically, we identify lactate dehydrogenase B (LDHB) as a novel positive regulator of pDC IFN-I production in mice and humans, show that LDHB deficiency is associated with suppressed IFN-I production, pDC metabolic capacity, and viral control following a viral infection, and demonstrate that preservation of LDHB expression is sufficient to partially restore exhausted pDC function in vitro and in vivo . Furthermore, restoring LDHB in vivo in exhausted pDCs increased IFNAR dependent infection- associated pathology. Therefore, our work identifies a novel and conserved mechanism for balancing immunity and pathology during viral infections, while also providing insight into the highly preserved but previously unexplained phenomenon of pDC exhaustion.

EGFR-driven lung adenocarcinomas coopt alveolar macrophage metabolism and function to support EGFR signaling and growth.

The limited efficacy of currently approved immunotherapies in EGFR-driven lung adenocarcinoma (LUAD) underscores the need to better understand alternative mechanisms governing local immunosuppression to fuel novel therapies. Elevated surfactant and GM-CSF secretion from the transformed epithelium induces tumor-associated alveolar macrophage (TA-AM) proliferation which supports tumor growth by rewiring inflammatory functions and lipid metabolism. TA-AM properties are driven by increased GM-CSF-PPARγ signaling and inhibition of airway GM-CSF or PPARγ in TA-AMs suppresses cholesterol efflux to tumor cells, which impairs EGFR phosphorylation and restrains LUAD progression. In the absence of TA-AM metabolic support, LUAD cells compensate by increasing cholesterol synthesis, and blocking PPARγ in TA-AMs simultaneous with statin therapy further suppresses tumor progression and increases proinflammatory immune responses. These results reveal new therapeutic combinations for immunotherapy resistant EGFR-mutant LUADs and demonstrate how cancer cells can metabolically co-opt TA-AMs through GM-CSF-PPARγ signaling to provide nutrients that promote oncogenic signaling and growth.

Long chain monomethyl branched-chain fatty acid levels in human milk vary with gestational weight gain.

Breastfeeding is an important determinant of infant health and there is immense interest in understanding its metabolite composition so that key beneficial components can be identified. The aim of this research was to measure the fatty acid composition of human milk in an Irish cohort where we examined changes depending on lactation stage and gestational weight gain trajectory. Utilizing a chromatography approach optimal for isomer separation, we identified 44 individual fatty acid species via GCMS and showed that monomethyl branched-chain fatty acids(mmBCFA's), C15:0 and C16:1 are lower in women with excess gestational weight gain versus low gestational weight gain. To further explore the potential contribution of the activity of endogenous metabolic pathways to levels of these fatty acids in milk, we administered D2O to C57BL/6J dams fed a purified lard based high fat diet (HFD) or low-fat diet during gestation and quantified the total and de novo synthesized levels of fatty acids in their milk. We found that de novo synthesis over three days can account for between 10 and 50 % of mmBCFAs in milk from dams on the low-fat diet dependent on the branched-chain fatty acid species. However, HFD fed mice had significantly decreased de novo synthesized fatty acids in milk resulting in lower total mmBCFAs and medium chain fatty acid levels. Overall, our findings highlight the diverse fatty acid composition of human milk and that human milk mmBCFA levels differ between gestational weight gain phenotypes. In addition, our data indicates that de novo synthesis contributes to mmBCFA levels in mice milk and thus may also be a contributory factor to mmBCFA levels in human milk. Given emerging data indicating mmBCFAs may be beneficial components of milk, this study contributes to our knowledge around the phenotypic factors that may impact their levels.

EGFR-Driven Lung Adenocarcinomas Co-opt Alveolar Macrophage Metabolism and Function to Support EGFR Signaling and Growth.

The limited efficacy of currently approved immunotherapies in EGFR-driven lung adenocarcinoma (LUAD) underscores the need to better understand alternative mechanisms governing local immunosuppression to fuel novel therapies. Elevated surfactant and GM-CSF secretion from the transformed epithelium induces tumor-associated alveolar macrophage (TA-AM) proliferation, which supports tumor growth by rewiring inflammatory functions and lipid metabolism. TA-AM properties are driven by increased GM-CSF-PPARγ signaling and inhibition of airway GM-CSF or PPARγ in TA-AMs suppresses cholesterol efflux to tumor cells, which impairs EGFR phosphorylation and restrains LUAD progression. In the absence of TA-AM metabolic support, LUAD cells compensate by increasing cholesterol synthesis, and blocking PPARγ in TA-AMs simultaneous with statin therapy further suppresses tumor progression and increases proinflammatory immune responses. These results reveal new therapeutic combinations for immunotherapy-resistant EGFR-mutant LUADs and demonstrate how cancer cells can metabolically co-opt TA-AMs through GM-CSF-PPARγ signaling to provide nutrients that promote oncogenic signaling and growth. SIGNIFICANCE: Alternate strategies harnessing anticancer innate immunity are required for lung cancers with poor response rates to T cell-based immunotherapies. This study identifies a targetable, mutually supportive, metabolic relationship between macrophages and transformed epithelium, which is exploited by tumors to obtain metabolic and immunologic support to sustain proliferation and oncogenic signaling.

Enhanced SREBP2-driven cholesterol biosynthesis by PKCλ/ι deficiency in intestinal epithelial cells promotes aggressive serrated tumorigenesis.

The metabolic and signaling pathways regulating aggressive mesenchymal colorectal cancer (CRC) initiation and progression through the serrated route are largely unknown. Although relatively well characterized as BRAF mutant cancers, their poor response to current targeted therapy, difficult preneoplastic detection, and challenging endoscopic resection make the identification of their metabolic requirements a priority. Here, we demonstrate that the phosphorylation of SCAP by the atypical PKC (aPKC), PKCλ/ι promotes its degradation and inhibits the processing and activation of SREBP2, the master regulator of cholesterol biosynthesis. We show that the upregulation of SREBP2 and cholesterol by reduced aPKC levels is essential for controlling metaplasia and generating the most aggressive cell subpopulation in serrated tumors in mice and humans. Since these alterations are also detected prior to neoplastic transformation, together with the sensitivity of these tumors to cholesterol metabolism inhibitors, our data indicate that targeting cholesterol biosynthesis is a potential mechanism for serrated chemoprevention.

Stable Isotope-Assisted Untargeted Metabolomics Identifies ALDH1A1-Driven Erythronate Accumulation in Lung Cancer Cells.

Using an untargeted stable isotope-assisted metabolomics approach, we identify erythronate as a metabolite that accumulates in several human cancer cell lines. Erythronate has been reported to be a detoxification product derived from off-target glycolytic metabolism. We use chemical inhibitors and genetic silencing to define the pentose phosphate pathway intermediate erythrose 4-phosphate (E4P) as the starting substrate for erythronate production. However, following enzyme assay-coupled protein fractionation and subsequent proteomics analysis, we identify aldehyde dehydrogenase 1A1 (ALDH1A1) as the predominant contributor to erythrose oxidation to erythronate in cell extracts. Through modulating ALDH1A1 expression in cancer cell lines, we provide additional support. We hence describe a possible alternative route to erythronate production involving the dephosphorylation of E4P to form erythrose, followed by its oxidation by ALDH1A1. Finally, we measure increased erythronate concentrations in tumors relative to adjacent normal tissues from lung cancer patients. These findings suggest the accumulation of erythronate to be an example of metabolic reprogramming in cancer cells, raising the possibility that elevated levels of erythronate may serve as a biomarker of certain types of cancer.

Sources and Sinks of Serine in Nutrition, Health, and Disease.

Amino acid dysregulation has emerged as an important driver of disease progression in various contexts. l-Serine lies at a central node of metabolism, linking carbohydrate metabolism, transamination, glycine, and folate-mediated one-carbon metabolism to protein synthesis and various downstream bioenergetic and biosynthetic pathways. l-Serine is produced locally in the brain but is sourced predominantly from glycine and one-carbon metabolism in peripheral tissues via liver and kidney metabolism. Compromised regulation or activity of l-serine synthesis and disposal occurs in the context of genetic diseases as well as chronic disease states, leading to low circulating l-serine levels and pathogenesis in the nervous system, retina, heart, and aging muscle. Dietary interventions in preclinical models modulate sensory neuropathy, retinopathy, tumor growth, and muscle regeneration. A serine tolerance test may provide a quantitative readout of l-serine homeostasis that identifies patients who may be susceptible to neuropathy or responsive to therapy.

EGFR + lung adenocarcinomas coopt alveolar macrophage metabolism and function to support EGFR signaling and growth.

The limited efficacy of currently approved immunotherapies in EGFR-mutant lung adenocarcinoma (LUAD) underscores the need to better understand mechanisms governing local immunosuppression. Elevated surfactant and GM-CSF secretion from the transformed epithelium induces tumor-associated alveolar macrophages (TA-AM) to proliferate and support tumor growth by rewiring inflammatory functions and lipid metabolism. TA-AM properties are driven by increased GM-CSF-PPARγ signaling and inhibition of airway GM-CSF or PPARγ in TA-AMs suppresses cholesterol efflux to tumor cells, which impairs EGFR phosphorylation and restrains LUAD progression. In the absence of TA-AM metabolic support, LUAD cells compensate by increasing cholesterol synthesis, and blocking PPARγ in TA-AMs simultaneous with statin therapy further suppresses tumor progression and increases T cell effector functions. These results reveal new therapeutic combinations for immunotherapy resistant EGFR-mutant LUADs and demonstrate how such cancer cells can metabolically co-opt TA-AMs through GM-CSF-PPARγ signaling to provide nutrients that promote oncogenic signaling and growth.

Inter-organelle cross-talk supports acetyl-coenzyme A homeostasis and lipogenesis under metabolic stress.

Proliferating cells rely on acetyl-CoA to support membrane biogenesis and acetylation. Several organelle-specific pathways are available for provision of acetyl-CoA as nutrient availability fluctuates, so understanding how cells maintain acetyl-CoA homeostasis under such stresses is critically important. To this end, we applied 13C isotope tracing cell lines deficient in these mitochondrial [ATP-citrate lyase (ACLY)]-, cytosolic [acetyl-CoA synthetase (ACSS2)]-, and peroxisomal [peroxisomal biogenesis factor 5 (PEX5)]-dependent pathways. ACLY knockout in multiple cell lines reduced fatty acid synthesis and increased reliance on extracellular lipids or acetate. Knockout of both ACLY and ACSS2 (DKO) severely stunted but did not entirely block proliferation, suggesting that alternate pathways can support acetyl-CoA homeostasis. Metabolic tracing and PEX5 knockout studies link peroxisomal oxidation of exogenous lipids as a major source of acetyl-CoA for lipogenesis and histone acetylation in cells lacking ACLY, highlighting a role for inter-organelle cross-talk in supporting cell survival in response to nutrient fluctuations.

iPSC-derived retinal pigmented epithelial cells from patients with macular telangiectasia show decreased mitochondrial function.

Patient-derived induced pluripotent stem cells (iPSCs) provide a powerful tool for identifying cellular and molecular mechanisms of disease. Macular telangiectasia type 2 (MacTel) is a rare, late-onset degenerative retinal disease with an extremely heterogeneous genetic architecture, lending itself to the use of iPSCs. Whole-exome sequencing screens and pedigree analyses have identified rare causative mutations that account for less than 5% of cases. Metabolomic surveys of patient populations and GWAS have linked MacTel to decreased circulating levels of serine and elevated levels of neurotoxic 1-deoxysphingolipids (1-dSLs). However, retina-specific, disease-contributing factors have yet to be identified. Here, we used iPSC-differentiated retinal pigmented epithelial (iRPE) cells derived from donors with or without MacTel to screen for novel cell-intrinsic pathological mechanisms. We show that MacTel iRPE cells mimicked the low serine levels observed in serum from patients with MacTel. Through RNA-Seq and gene set enrichment pathway analysis, we determined that MacTel iRPE cells are enriched in cellular stress pathways and dysregulation of central carbon metabolism. Using respirometry and mitochondrial stress testing, we functionally validated that MacTel iRPE cells had a reduction in mitochondrial function that was independent of defects in serine biosynthesis and 1-dSL accumulation. Thus, we identified phenotypes that may constitute alternative disease mechanisms beyond the known serine/sphingolipid pathway.