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Despite the known metabolic benefits of exercise, an integrated metabolic understanding of exercise is lacking. Here, we use in vivo steady-state isotope-labeled infusions to quantify fuel flux and oxidation during exercise in fasted, fed, and exhausted female mice, revealing several novel findings. Exercise strongly promoted glucose fluxes from liver glycogen, lactate, and glycerol, distinct from humans. Several organs spared glucose, a process that broke down in exhausted mice despite concomitant hypoglycemia. Proteolysis increased markedly, also divergent from humans. Fatty acid oxidation dominated during fasted exercise. Ketone production and oxidation rose rapidly, seemingly driven by a hepatic bottleneck caused by gluconeogenesis-induced cataplerotic stress. Altered fuel consumption was observed in organs not directly involved in muscle contraction, including the pancreas and brown fat. Several futile cycles surprisingly persisted during exercise, despite their energy cost. In sum, we provide a comprehensive, integrated, holistic, and quantitative accounting of metabolism during exercise in an intact organism., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The activation of branched chain amino acid (BCAA) catabolism has garnered interest as a potential therapeutic approach to improve insulin sensitivity, enhance recovery from heart failure, and blunt tumor growth. Evidence for this interest relies in part on BT2, a small molecule that promotes BCAA oxidation and is protective in mouse models of these pathologies. BT2 and other analogs allosterically inhibit branched chain ketoacid dehydrogenase kinase (BCKDK) to promote BCAA oxidation, which is presumed to underlie the salutary effects of BT2. Potential "off-target" effects of BT2 have not been considered, however. We therefore tested for metabolic off-target effects of BT2 in Bckdk -/- animals. As expected, BT2 failed to activate BCAA oxidation in these animals. Surprisingly, however, BT2 strongly reduced plasma tryptophan levels and promoted catabolism of tryptophan to kynurenine in both control and Bckdk -/- mice. Mechanistic studies revealed that none of the principal tryptophan catabolic or kynurenine-producing/consuming enzymes (TDO, IDO1, IDO2, or KATs) were required for BT2-mediated lowering of plasma tryptophan. Instead, using equilibrium dialysis assays and mice lacking albumin, we show that BT2 avidly binds plasma albumin and displaces tryptophan, releasing it for catabolism. These data confirm that BT2 activates BCAA oxidation via inhibition of BCKDK but also reveal a robust off-target effect on tryptophan metabolism via displacement from serum albumin. The data highlight a potential confounding effect for pharmaceutical compounds that compete for binding with albumin-bound tryptophan.

Cold-induced thermogenesis (CIT) is widely studied as a potential avenue to treat obesity, but a thorough understanding of the metabolic changes driving CIT is lacking. Here, we present a comprehensive and quantitative analysis of the metabolic response to acute cold exposure, leveraging metabolomic profiling and minimally perturbative isotope tracing studies in unanesthetized mice. During cold exposure, brown adipose tissue (BAT) primarily fueled the tricarboxylic acid (TCA) cycle with fat in fasted mice and glucose in fed mice, underscoring BAT's metabolic flexibility. BAT minimally used branched-chain amino acids or ketones, which were instead avidly consumed by muscle during cold exposure. Surprisingly, isotopic labeling analyses revealed that BAT uses glucose largely for TCA anaplerosis via pyruvate carboxylation. Finally, we find that cold-induced hepatic gluconeogenesis is critical for CIT during fasting, demonstrating a key functional role for glucose metabolism. Together, these findings provide a detailed map of the metabolic rewiring driving acute CIT., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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.

Elevated levels of plasma branched-chain amino acids (BCAAs) have been associated with insulin resistance and type 2 diabetes since the 1960s. Pharmacological activation of branched-chain α-ketoacid dehydrogenase (BCKDH), the rate-limiting enzyme of BCAA oxidation, lowers plasma BCAAs and improves insulin sensitivity. Here we show that modulation of BCKDH in skeletal muscle, but not liver, affects fasting plasma BCAAs in male mice. However, despite lowering BCAAs, increased BCAA oxidation in skeletal muscle does not improve insulin sensitivity. Our data indicate that skeletal muscle controls plasma BCAAs, that lowering fasting plasma BCAAs is insufficient to improve insulin sensitivity and that neither skeletal muscle nor liver account for the improved insulin sensitivity seen with pharmacological activation of BCKDH. These findings suggest potential concerted contributions of multiple tissues in the modulation of BCAA metabolism to alter insulin sensitivity., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Pharmacologic activation of branched-chain amino acid (BCAA) catabolism is protective in models of heart failure (HF). How protection occurs remains unclear, although a causative block in cardiac BCAA oxidation is widely assumed. Here, we use in vivo isotope infusions to show that cardiac BCAA oxidation in fact increases, rather than decreases, in HF. Moreover, cardiac-specific activation of BCAA oxidation does not protect from HF even though systemic activation does. Lowering plasma and cardiac BCAAs also fails to confer significant protection, suggesting alternative mechanisms of protection. Surprisingly, activation of BCAA catabolism lowers blood pressure (BP), a known cardioprotective mechanism. BP lowering occurred independently of nitric oxide and reflected vascular resistance to adrenergic constriction. Mendelian randomization studies revealed that elevated plasma BCAAs portend higher BP in humans. Together, these data indicate that BCAA oxidation lowers vascular resistance, perhaps in part explaining cardioprotection in HF that is not mediated directly in cardiomyocytes., Competing Interests: Declaration of interests Z.A. received consulting fees from Pfizer. J.W.E. received consulting fees, unassociated with this work, from Jannsen Pharmaceuticals and Mitobridge, Inc. J.D.R. is an advisor and stockholder in Colorado Research Partners, Empress Therapeutics, L.E.A.F. Pharmaceuticals, Bantam Pharmaceuticals, Barer Institute, and Rafael Holdings; a founder, director, and stockholder of Farber Partners, Serien Therapeutics, and Sofro Pharmaceuticals; a founder and stockholder in Toran Therapeutics; inventor of patents held by Princeton University; and a director of the Princeton University-PKU Shenzhen collaboration., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of interests J.D.R. is a member of the Rutgers Cancer Institute of New Jersey and the University of Pennsylvania Diabetes Research Center; a co-founder and stockholder in Empress Therapeutics and Serien Therapeutics; and an advisor and stockholder in Agios Pharmaceuticals, Bantam Pharmaceuticals, Colorado Research Partners, Rafael Pharmaceuticals, Barer Institute, and L.E.A.F. Pharmaceuticals. M.S.D. is a member of the scientific advisory boards of DeepBiome Therapeutics and VastBiome., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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 13 C-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., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Insulin stimulates adipose tissue to extract fatty acids from circulation and sequester them inside adipose cells. How fatty acids are transported across the capillary endothelial barrier, and how this process is regulated, remains unclear. We modeled the relationship of adipocytes and endothelial cells in vitro to test the role of insulin in fatty acid transport. Treatment of endothelial cells with insulin did not affect endothelial fatty acid uptake, but endothelial cells took up more fatty acids when exposed to medium conditioned by adipocytes treated with insulin. Manipulations of this conditioned medium indicated that the secreted factor is a small, hydrophilic, non-proteinaceous metabolite. Factor activity was correlated with lactate concentration, and inhibition of lactate production in adipocytes abolished the activity. Finally, lactate alone was sufficient to increase endothelial uptake of both free fatty acids and lipids liberated from chylomicrons, and to promote transendothelial transport, at physiologically relevant concentrations. Taken together, these data suggest that insulin drives adipocytes to secrete lactate, which then acts in a paracrine fashion to promote fatty acid uptake and transport across the neighboring endothelial barrier., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Oxidation of branched-chain amino acids (BCAAs) is tightly regulated in mammals. We review here the distribution and regulation of whole-body BCAA oxidation. Phosphorylation and dephosphorylation of the rate-limiting enzyme, branched-chain α-ketoacid dehydrogenase complex directly regulates BCAA oxidation, and various other indirect mechanisms of regulation also exist. Most tissues throughout the body are capable of BCAA oxidation, and the flux of oxidative BCAA disposal in each tissue is influenced by three key factors: 1. tissue-specific preference for BCAA oxidation relative to other fuels, 2. the overall oxidative activity of mitochondria within a tissue, and 3. total tissue mass. Perturbations in BCAA oxidation have been implicated in many disease contexts, underscoring the importance of BCAA homeostasis in overall health., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Elevations in branched-chain amino acids (BCAAs) associate with numerous systemic diseases, including cancer, diabetes, and heart failure. However, an integrated understanding of whole-body BCAA metabolism remains lacking. Here, we employ in vivo isotopic tracing to systemically quantify BCAA oxidation in healthy and insulin-resistant mice. We find that most tissues rapidly oxidize BCAAs into the tricarboxylic acid (TCA) cycle, with the greatest quantity occurring in muscle, brown fat, liver, kidneys, and heart. Notably, pancreas supplies 20% of its TCA carbons from BCAAs. Genetic and pharmacologic suppression of branched-chain alpha-ketoacid dehydrogenase kinase, a clinically targeted regulatory kinase, induces BCAA oxidation primarily in skeletal muscle of healthy mice. While insulin acutely increases BCAA oxidation in cardiac and skeletal muscle, chronically insulin-resistant mice show blunted BCAA oxidation in adipose tissues and liver, shifting BCAA oxidation toward muscle. Together, this work provides a quantitative framework for understanding systemic BCAA oxidation in health and insulin resistance., (Copyright © 2018 Elsevier Inc. All rights reserved.)

[This corrects the article DOI: 10.1371/journal.pgen.1005378.].

Genome-wide association studies (GWAS) have identified more than 100 genetic variants contributing to BMI, a measure of body size, or waist-to-hip ratio (adjusted for BMI, WHRadjBMI), a measure of body shape. Body size and shape change as people grow older and these changes differ substantially between men and women. To systematically screen for age- and/or sex-specific effects of genetic variants on BMI and WHRadjBMI, we performed meta-analyses of 114 studies (up to 320,485 individuals of European descent) with genome-wide chip and/or Metabochip data by the Genetic Investigation of Anthropometric Traits (GIANT) Consortium. Each study tested the association of up to ~2.8M SNPs with BMI and WHRadjBMI in four strata (men ≤50y, men >50y, women ≤50y, women >50y) and summary statistics were combined in stratum-specific meta-analyses. We then screened for variants that showed age-specific effects (G x AGE), sex-specific effects (G x SEX) or age-specific effects that differed between men and women (G x AGE x SEX). For BMI, we identified 15 loci (11 previously established for main effects, four novel) that showed significant (FDR<5%) age-specific effects, of which 11 had larger effects in younger (<50y) than in older adults (≥50y). No sex-dependent effects were identified for BMI. For WHRadjBMI, we identified 44 loci (27 previously established for main effects, 17 novel) with sex-specific effects, of which 28 showed larger effects in women than in men, five showed larger effects in men than in women, and 11 showed opposite effects between sexes. No age-dependent effects were identified for WHRadjBMI. This is the first genome-wide interaction meta-analysis to report convincing evidence of age-dependent genetic effects on BMI. In addition, we confirm the sex-specificity of genetic effects on WHRadjBMI. These results may provide further insights into the biology that underlies weight change with age or the sexually dimorphism of body shape.

Maternal nutrition has a profound long-term impact on infant health. Poor maternal nutrition influences placental development and fetal growth, resulting in low birth weight, which is strongly associated with the risk of developing chronic diseases, including heart disease, hypertension, asthma, and type 2 diabetes, later in life. Few studies have delineated the mechanisms by which maternal nutrition affects fetal lung development. Here, we report that maternal exposure to a diet high in fat (HFD) causes placental inflammation, resulting in placental insufficiency, fetal growth restriction (FGR), and inhibition of fetal lung development. Notably, pre- and postnatal exposure to maternal HFD also results in persistent alveolar simplification in the postnatal period. Our novel findings provide a strong association between maternal diet and fetal lung development., (Copyright © 2015 the American Physiological Society.)

Objective: Roux-en-Y gastric bypass (RYGB) is an effective method of weight loss and remediation of type-2 diabetes; however, the mechanisms leading to these improvements are unclear. Additionally, adipocytes within white adipose tissue (WAT) depots can manifest characteristics of brown adipocytes. These 'BRITE/beige' adipocytes express uncoupling protein 1 (UCP1) and are associated with improvements in glucose homeostasis and protection from obesity. Interestingly, atrial and B-type natriuretic peptides (NPs) promote BRITE/beige adipocyte enrichment of WAT depots, an effect known as "browning." Here, we investigate the effect of RYGB surgery on NP, NP receptors, and browning in the gonadal adipose tissues of female mice. We propose that such changes may lead to improvements in metabolic homeostasis commonly observed following RYGB., Methods: Wild type, female, C57/Bl6 mice were fed a 60% fat diet ad libitum for six months. Mice were divided into three groups: Sham operated (SO), Roux-en-Y gastric bypass (RYGB), and Weight matched, sham operated (WM-SO). Mice were sacrificed six weeks following surgery and evaluated for differences in body weight, glucose homeostasis, adipocyte morphology, and adipose tissue gene expression., Results: RYGB and calorie restriction induced similar weight loss and improved glucose metabolism without decreasing food intake. β3-adrenergic receptor expression increased in gonadal adipose tissue, in addition to Nppb (BNP), and NP receptors, Npr1, and Npr2. The ratio of Npr1:Npr3 and Npr2:Npr3 increased in RYGB, but not WM-SO groups. Ucp1 protein and mRNA, as well as additional markers of BRITE/beige adipose tissue and lipolytic genes increased in RYGB mice to a greater extent than calorie-restricted mice., Conclusions: Upregulation of Nppb, Npr1, Npr2, and β3-adrenergic receptors in gonadal adipose tissue following RYGB was associated with increased markers of browning. This browning of gonadal adipose tissue may underpin the positive effect of RYGB on metabolic parameters and may in part be mediated through upregulation of natriuretic peptides.

Hypoxia Inducible Factor 1 (HIF-1) promotes fibrosis and inflammation in adipose tissues, while estrogens and Estrogen Receptor α (ERα) have the opposite effect. Here we identify an Estrogen Response Element (ERE) in the promoter of Phd3, which is a negative regulatory enzyme of HIF-1, and we demonstrate HIF-1α is ubiquitinated following 17-β estradiol (E2)/ERα mediated Phd3 transcription. Manipulating ERα in vivo increases Phd3 transcription and reduces HIF-1 activity, while addition of PHD3 ameliorates adipose tissue fibrosis and inflammation. Our findings outline a novel regulatory relationship between E2/ERα, PHD3 and HIF-1 in adipose tissues, providing a mechanistic explanation for the protective effect of E2/ERα in adipose tissue.