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Objectives To perform an exploratory study to identify a JDM serum metabolic profile that differs from healthy controls (HCs) and responds to immunosuppressive treatment. Methods Blood was collected from 9 HCs and 10 patients diagnosed with probable (n = 4) or definite (n = 6) JDM based on the criteria of Bohan and Peter for myositis, with 7 of the 10 providing longitudinal samples following initiation of treatment; these patients comprised the treatment-naïve cohort. Sera underwent mass spectroscopy–based measurements of targeted metabolic intermediates, including 15 amino acids, 45 acylcarnitines (ACs), 15 ceramides and 29 sphingomyelins. Principal components analysis reduced metabolites into smaller sets of factors each comprised of correlated metabolic intermediates. Factor scores and metabolite concentrations were compared with HCs using two-sample t-tests while treatment effects were evaluated using paired t-tests. Results Of eight principal components analysis–derived metabolite factors (one AC, two amino acids, three sphingosine and two ceramide), two were significantly associated with JDM: one AC factor containing mostly long-chain ACs (P = 0.049) and one ceramide factor (P Conclusion While additional validation is needed, these lipids have potential as JDM serum diagnostic and/or treatment biomarkers. Additionally, the significant association of long-chain ACs and ceramides with JDM offers insights regarding pathogenesis, implicating dysregulation of mitochondrial fatty acid β-oxidation.

Elevations in circulating levels of branched-chain amino acids (BCAAs) are associated with a variety of cardiometabolic diseases and conditions. Restriction of dietary BCAAs in rodent models of obesity lowers circulating BCAA levels and improves whole-animal and skeletal-muscle insulin sensitivity and lipid homeostasis, but the impact of BCAA supply on heart metabolism has not been studied. Here, we report that feeding a BCAA-restricted chow diet to Zucker fatty rats (ZFRs) causes a shift in cardiac fuel metabolism that favors fatty acid relative to glucose catabolism. This is illustrated by an increase in labeling of acetyl-CoA from [1-13C]palmitate and a decrease in labeling of acetyl-CoA and malonyl-CoA from [U-13C]glucose, accompanied by a decrease in cardiac hexokinase II and glucose transporter 4 protein levels. Metabolomic profiling of heart tissue supports these findings by demonstrating an increase in levels of a host of fatty-acid-derived metabolites in hearts from ZFRs and Zucker lean rats (ZLRs) fed the BCAA-restricted diet. In addition, the twofold increase in cardiac triglyceride stores in ZFRs compared with ZLRs fed on chow diet is eliminated in ZFRs fed on the BCAA-restricted diet. Finally, the enzymatic activity of branched-chain ketoacid dehydrogenase (BCKDH) is not influenced by BCAA restriction, and levels of BCAA in the heart instead reflect their levels in circulation. In summary, reducing BCAA supply in obesity improves cardiac metabolic health by a mechanism independent of alterations in BCKDH activity.

Summary: The pro-longevity enzyme SIRT6 regulates various metabolic pathways. Gene expression analyses in SIRT6 heterozygotic mice identify significant decreases in PPARα signaling, known to regulate multiple metabolic pathways. SIRT6 binds PPARα and its response element within promoter regions and activates gene transcription. Sirt6+/− results in significantly reduced PPARα-induced β-oxidation and its metabolites and reduced alanine and lactate levels, while inducing pyruvate oxidation. Reciprocally, starved SIRT6 transgenic mice show increased pyruvate, acetylcarnitine, and glycerol levels and significantly induce β-oxidation genes in a PPARα-dependent manner. Furthermore, SIRT6 mediates PPARα inhibition of SREBP-dependent cholesterol and triglyceride synthesis. Mechanistically, SIRT6 binds PPARα coactivator NCOA2 and decreases liver NCOA2 K780 acetylation, which stimulates its activation of PPARα in a SIRT6-dependent manner. These coordinated SIRT6 activities lead to regulation of whole-body respiratory exchange ratio and liver fat content, revealing the interactions whereby SIRT6 synchronizes various metabolic pathways, and suggest a mechanism by which SIRT6 maintains healthy liver. : How the pro-longevity enzyme SIRT6 coordinates between various metabolic pathways is still obscure. Here, Naiman et al. show that SIRT6 activates PPARα to promote fatty acid beta oxidation and inhibit pyruvate oxidation during fasting. This ultimately decides the energy source under nutrient-limited conditions, promoting fat usage over other energy sources. Keywords: SIRT6, PPARα, beta-oxidation, liver, deacetylase, fasting

Branched-chain amino acid (BCAA; valine, leucine and isoleucine) supplementation is often beneficial to energy expenditure; however, increased circulatinglevels of BCAA are linked to obesity and diabetes. The mechanisms of this paradox remain unclear. Here we report that, on cold exposure, brown adipose tissue (BAT) actively utilizes BCAA in the mitochondria for thermogenesis and promotes systemic BCAA clearance in mice and humans. In turn, a BAT-specific defect in BCAA catabolism attenuates systemic BCAA clearance, BAT fuel oxidation and thermogenesis, leading to diet-induced obesity and glucose intolerance. Mechanistically, active BCAA catabolism in BAT is mediated by SLC25A44, which transports BCAAs into mitochondria. Our results suggest that BAT serves as a key metabolic filter that controls BCAA clearance via SLC25A44, thereby contributing to the improvement of metabolic health.

The consequences of damage to the mitochondrial genome (mtDNA) are poorly understood, although mtDNA is more susceptible to damage resulting from some genotoxicants than nuclear DNA (nucDNA), and many environmental toxicants target the mitochondria. Reports from the toxicological literature suggest that exposure to early-life mitochondrial damage could lead to deleterious consequences later in life (the “Developmental Origins of Health and Disease” paradigm), but reports from other fields often report beneficial (“mitohormetic”) responses to such damage. Here, we tested the effects of low (causing no change in lifespan) levels of ultraviolet C (UVC)-induced, irreparable mtDNA damage during early development in Caenorhabditis elegans. This exposure led to life-long reductions in mtDNA copy number and steady-state ATP levels, accompanied by increased oxygen consumption and altered metabolite profiles, suggesting inefficient mitochondrial function. Exposed nematodes were also developmentally delayed, reached smaller adult size, and were rendered more susceptible to subsequent exposure to chemical mitotoxicants. Metabolomic and genetic analysis of key signaling and metabolic pathways supported redox and mitochondrial stress-response signaling during early development as a mechanism for establishing these persistent alterations. Our results highlight the importance of early-life exposures to environmental pollutants, especially in the context of exposure to chemicals that target mitochondria., Graphical abstract Image 1, Highlights • Early life mtDNA damage led to lifelong deficits in mitochondrial function. • C. elegans developed slowly and were sensitive to chemical exposures as adults. • Redox signaling is a mechanism that establishes these persistent alterations. • Data are consistent with the Developmental Origins of Health and Disease model.

Background: To provide energy for cardiopulmonary function and maintenance of blood glucose, acute aerobic exercise induces lipolysis, fatty acid oxidation (FAO), glycolysis, and glycogenolysis/gluconeogenesis. These adaptations are mediated by increases in cortisol, growth hormone (GH), and catecholamines and facilitated by a decline in insulin. Branched-chain amino acids (BCAA) also undergo catabolism during intense exercise. Here, we investigated the relationship between BCAA catabolism and metrics of cardiopulmonary function in healthy, well-developed, mature adolescent athletes undergoing an acute bout of maximal aerobic exercise.Hypothesis: We hypothesized: (a) acute maximal exercise in adolescents induces lipolysis, FAO, and BCAA catabolism associated with increases in GH and cortisol and a reduction in insulin; (b) increases in GH are associated with increases in ghrelin; and (c) metrics of cardiopulmonary function (aVO2, rVO2, aVO2/HRmax) following maximal exercise correlate with increases in GH secretion, FAO, and BCAA catabolism.Methods: Blood samples before and after maximal cardiopulmonary exercise in 11 adolescent athletes were analyzed by tandem-mass spectrometry. Paired, two-tailed student's t-tests identified significant changes following exercise. Linear regression determined if pre-exercise metabolite levels, or changes in metabolite levels, were associated with aVO2, rVO2, and aVO2/HRmax. Sex and school of origin were included as covariates in all regression analyses.Results: Following exercise there were increases in GH and cortisol, and decreases in ghrelin, but no changes in glucose or insulin concentrations. Suggesting increased lipolysis and FAO, the levels of glycerol, ketones, β-hydroxybutyrate, and acetylcarnitine concentrations increased. Pyruvate, lactate, alanine, and glutamate concentrations also increased. Plasma concentrations of valine (a BCAA) declined (p = 0.002) while valine degradation byproducts increased in association with decreases in urea cycle amino acids arginine and ornithine. Metrics of cardiopulmonary function were associated with increases in propionylcarnitine (C3, p = 0.013) and Ci4-DC/C4-DC (p < 0.01), byproducts of BCAA catabolism.Conclusions: Induction of lipolysis, FAO, gluconeogenesis, and glycogenolysis provides critical substrates for cardiopulmonary function during exercise. However, none of those pathways were significantly associated with metrics of cardiopulmonary function. The associations between rVO2, and aVO2/HRmax and C3 and Ci4-DC/C4-DC suggest that the cardiopulmonary response to maximal exercise in adolescents is linked to BCAA utilization and catabolism.

Significance Employing an intergenerational model of diet restriction (DR) that reduces weight gain, we identify differences in carbohydrate metabolic enzyme gene content of DR versus full-fed (FF) pig gut microbiomes as animals experienced a phased-feeding program administered to farm-raised pigs during their growth cycle. Gnotobiotic mice harboring DR or FF pig microbiomes and fed a corn/soy-dominated pig diet disclosed that the DR microbiome has reduced capacity to produce butyrate, a key diet-derived energy source, and alters hepatic energy metabolism. Combining studies of farm animal microbiome development with functional assays of their microbiomes in gnotobiotic mice should help generate husbandry recommendations for promoting healthy growth of animals during this time of increasing food insecurity and mandates to eliminate subtherapeutic antibiotics for growth-promotion., The concept that gut microbiome-expressed functions regulate ponderal growth has important implications for infant and child health, as well as animal health. Using an intergenerational pig model of diet restriction (DR) that produces reduced weight gain, we developed a feature-selection algorithm to identify representative characteristics distinguishing DR fecal microbiomes from those of full-fed (FF) pigs as both groups consumed a common sequence of diets during their growth cycle. Gnotobiotic mice were then colonized with DR and FF microbiomes and subjected to controlled feeding with a pig diet. DR microbiomes have reduced representation of genes that degrade dominant components of late growth-phase diets, exhibit reduced production of butyrate, a key host-accessible energy source, and are causally linked to reduced hepatic fatty acid metabolism (β-oxidation) and the selection of alternative energy substrates. The approach described could aid in the development of guidelines for microbiome stewardship in diverse species, including farm animals, in order to support their healthy growth.

Statins are a class of drug widely prescribed for the prevention of cardiovascular disease, with pleiotropic cellular effects. Statins inhibit HMG-CoA reductase (HMGCR), which converts the reactive HMG-CoA into mevalonate. Recent discoveries revealed HMG-CoA is a highly reactive metabolite that can non-enzymatically modify proteins and impact their activity. Therefore, we predicted that inhibition of HMGCR by statins would increase HMG-CoA levels and subsequent protein modifications. We found a strong increase in HMG-CoA levels, but only a single protein being modified. Fatty acid synthase (FAS) was modified on active site residues and, importantly, the modification is located on non-lysine residues. The dynamic modifications occur only on a subpool of FAS that is located near HMGCR and alters cellular signaling around the ER and Golgi. These results uncover communication between cholesterol and lipid biosynthesis by the substrate of one pathway inhibiting another in a rapid and reversible manner.

Introduction Insulin resistance in adolescents with obesity associates with a sex‐dependent metabolic ‘signature’ comprising branched‐chain amino acids (BCAAs), glutamate and C3/C5 acylcarnitines (C3/C5), implicating altered flux through BCAA catabolic pathways. Here, we investigated the effects of lifestyle intervention on BCAA catabolism and insulin sensitivity. We hypothesized (1) weight reduction and improved insulin sensitivity associate with enhanced BCAA catabolism; (2) baseline BCAAs and their metabolic by‐products predict changes in weight and insulin sensitivity during lifestyle intervention. Methods A 33 adolescents with obesity were studied before and after 6 months of lifestyle intervention. Principal component analysis and multiple linear regression models were used to correlate changes in metabolic factors with changes in weight and insulin sensitivity assessed by HOMA‐IR, adiponectin and ratio of triglyceride (TG) to HDL. Baseline metabolic factors were used as explanatory variables in prediction models. Results Weight reduction was associated with reductions in BCAA, glutamate, and C3/C5 (p = .002) and increases in urea cycle AA (p = .029), suggesting an increase in BCAA catabolism. Increases in urea cycle AA during weight reduction were associated with increases in adiponectin, a marker of insulin sensitivity. Markers of insulin resistance (high BCAA, glutamate, and C3/C5 and low urea cycle AA) at baseline predicted increases in metrics of insulin sensitivity (decreased TG/HDL and increased adiponectin) during lifestyle intervention. Conclusions Weight reduction in adolescents is associated with increases in BCAA catabolism and improvements in insulin sensitivity. Our study underscores the therapeutic potential of manipulating BCAA catabolism to treat obesity‐associated insulin resistance in adolescents and prevent progression to T2D., Insulin resistance in adolescents with obesity associates with a sex‐dependent metabolic ‘signature’ comprising branched‐chain amino acids (BCAAs), glutamate and C3/C5 acylcarnitines (C3/C5), implicating altered flux through BCAA catabolic pathways. Here, we investigated the effects of lifestyle intervention on BCAA catabolism and insulin sensitivity.

Branched-chain amino acids (BCAA) and their cognate α-ketoacids (BCKA) are elevated in an array of cardiometabolic diseases. Here we demonstrate that the major metabolic fate of uniformly-13C-labeled α-ketoisovalerate ([U-13C]KIV) in the heart is reamination to valine. Activation of cardiac branched-chain α-ketoacid dehydrogenase (BCKDH) by treatment with the BCKDH kinase inhibitor, BT2, does not impede the strong flux of [U-13C]KIV to valine. Sequestration of BCAA and BCKA away from mitochondrial oxidation is likely due to low levels of expression of the mitochondrial BCAA transporter SLC25A44 in the heart, as its overexpression significantly lowers accumulation of [13C]-labeled valine from [U-13C]KIV. Finally, exposure of perfused hearts to levels of BCKA found in obese rats increases phosphorylation of the translational repressor 4E-BP1 as well as multiple proteins in the MEK-ERK pathway, leading to a doubling of total protein synthesis. These data suggest that elevated BCKA levels found in obesity may contribute to pathologic cardiac hypertrophy via chronic activation of protein synthesis., Systemic modulation of branched-chain keto acid (BCKA) metabolism alters cardiac health. Here, the authors define the major fates of BCKA in the heart and demonstrate that acute exposure to BCKA levels found in obesity activates cardiac protein synthesis and markedly alters the heart phosphoproteome.

Skeletal muscle is a major determinant of systemic metabolic homeostasis that plays a critical role in glucose metabolism and insulin sensitivity. By contrast, despite being a major user of fatty acids, and evidence that muscular disorders can lead to abnormal lipid deposition (e.g., nonalcoholic fatty liver disease in myopathies), our understanding of skeletal muscle regulation of systemic lipid homeostasis is not well understood. Here we show that skeletal muscle Krüppel-like factor 15 (KLF15) coordinates pathways central to systemic lipid homeostasis under basal conditions and in response to nutrient overload. Mice with skeletal muscle-specific KLF15 deletion demonstrated (a) reduced expression of key targets involved in lipid uptake, mitochondrial transport, and utilization, (b) elevated circulating lipids, (c) insulin resistance/glucose intolerance, and (d) increased lipid deposition in white adipose tissue and liver. Strikingly, a diet rich in short-chain fatty acids bypassed these defects in lipid flux and ameliorated aspects of metabolic dysregulation. Together, these findings establish skeletal muscle control of lipid flux as critical to systemic lipid homeostasis and metabolic health.

There is marked heterogeneity in the response to weight loss interventions with regards to weight loss amount and metabolic improvement. We sought to identify biomarkers predictive of type 2 diabetes remission and amount of weight loss in individuals with severe obesity enrolled in the Longitudinal Assessment of Bariatric Surgery (LABS) and the Look AHEAD (Action for Health in Diabetes) studies. Targeted mass spectrometry-based profiling of 135 metabolites was performed in pre-intervention blood samples using a nested design for diabetes remission over five years (n = 93 LABS, n = 80 Look AHEAD; n = 87 remitters), and for extremes of weight loss at five years (n = 151 LABS; n = 75 with high weight loss). Principal components analysis (PCA) was used for dimensionality reduction, with PCA-derived metabolite factors tested for association with both diabetes remission and weight loss. Metabolic markers were tested for incremental improvement to clinical models, including the DiaRem score. Two metabolite factors were associated with diabetes remission: one primarily composed of branched chain amino acids (BCAA) and tyrosine (odds ratio (95% confidence interval) [OR (95% CI)] = 1.4 [1.0–1.9], p = 0.045), and one with betaine and choline (OR [95% CI] = 0.7 [0.5–0.9], p = 0.02).These results were not significant after adjustment for multiple tests. Inclusion of these two factors in clinical models yielded modest improvements in model fit and performance: in a constructed clinical model, the C-statistic improved from 0.87 to 0.90 (p = 0.02), while the net reclassification index showed improvement in prediction compared to the DiaRem score (NRI = 0.26, p = 0.0013). No metabolite factors associated with weight loss at five years. Baseline levels of metabolites in the BCAA and trimethylamine-N-oxide (TMAO)-microbiome-related pathways are independently and incrementally associated with sustained diabetes remission after weight loss interventions in individuals with severe obesity. These metabolites could serve as clinically useful biomarkers to identify individuals who will benefit the most from weight loss interventions.

Circulating branched chain amino acid (BCAA) levels are elevated in obese humans and genetically obese rodents. However, the relationship of BCAAs to insulin resistance in diet-induced obese mice, a commonly used model to study glucose homeostasis, is still ill-defined. Here we examined how high-fat high-sucrose (HFHS) or high-fat diet (HFD) feeding, with or without BCAA supplementation in water, alters the metabolome in serum/plasma and tissues in mice and whether raising circulating BCAA levels worsens insulin resistance and glucose intolerance. Neither HFHS nor HFD feeding raised circulating BCAA levels in insulin-resistant diet-induced obese mice. BCAA supplementation raised circulating BCAA and branched-chain α-keto acid levels and C5-OH/C3-DC acylcarnitines (AC) in muscle from mice fed an HFHS diet or HFD, but did not worsen insulin resistance. A set of short- and long-chain acyl CoAs were elevated by diet alone in muscle, liver, and white adipose tissue (WAT), but not increased further by BCAA supplementation. HFD feeding reduced valine and leucine oxidation in WAT but not in muscle. BCAA supplementation markedly increased valine oxidation in muscle from HFD-fed mice, while leucine oxidation was unaffected by diet or BCAA treatment. Here we establish an extensive metabolome database showing tissue-specific changes in mice on 2 different HFDs, with or without BCAA supplementation. We conclude that mildly elevating circulating BCAAs and a subset of ACs by BCAA supplementation does not worsen insulin resistance or glucose tolerance in mice. This work highlights major differences in the effects of BCAAs on glucose homeostasis in diet-induced obese mice versus data reported in obese rats and in humans.

Animal models of human diseases are classically fed purified diets that contain casein as the unique protein source. We show that provision of a mixed protein source mirroring that found in the western diet exacerbates diet-induced obesity and insulin resistance by potentiating hepatic mTORC1/S6K1 signaling as compared to casein alone. These effects involve alterations in gut microbiota as shown by fecal microbiota transplantation studies. The detrimental impact of the mixed protein source is also linked with early changes in microbial production of branched-chain fatty acids (BCFA) and elevated plasma and hepatic acylcarnitines, indicative of aberrant mitochondrial fatty acid oxidation. We further show that the BCFA, isobutyric and isovaleric acid, increase glucose production and activate mTORC1/S6K1 in hepatocytes. Our findings demonstrate that alteration of dietary protein source exerts a rapid and robust impact on gut microbiota and BCFA with significant consequences for the development of obesity and insulin resistance., Diet-induced changes in the microbiome have been associated with obesity. Here, using a mouse model, the authors show that a mixed protein source found in western diets exacerbates diet-induced obesity and insulin resistance by potentiating hepatic mTORC1/S6K1 signaling via microbial production of branched-chain fatty acids (BCFA).

The consequences of damage to the mitochondrial genome (mtDNA) are poorly understood, although mtDNA is more susceptible to damage than nuclear DNA (nucDNA), and many environmental toxicants target the mitochondria. Reports from the toxicological literature suggest that exposure to early-life mitochondrial damage could lead to deleterious consequences later in life (the “Developmental Origins of Health and Disease” paradigm) but reports from other fields often report beneficial (“mitohormetic”) responses to such damage. Here, we test the effects of low (causing no change in lifespan) levels of ultraviolet C (UVC)-induced, irreparable mtDNA damage during early development inCaenorhabditis elegans. This exposure led to life-long reductions in mtDNA copy number and steady-state ATP levels, accompanied by increased oxygen consumption and altered metabolite profiles, suggesting inefficient mitochondrial function. Exposed nematodes were also developmentally delayed, reached smaller adult size, and were rendered more susceptible to subsequent exposure to chemical mitotoxicants. Metabolomic and genetic analysis of key signaling and metabolic pathways supported redox signaling during early development as a mechanism for establishing these persistent alterations. Our results highlight the importance of early-life exposures to environmental pollutants, especially in the context of exposure to chemicals that target mitochondria.

Background Allogeneic hematopoietic stem cell transplantation (HCT) is an often curative intent treatment, however it is associated with significant gastrointestinal (GI) toxicity and treatment related mortality. Graft-versus-host disease is a significant contributor to transplant-related mortality. We performed a phase 2 trial of the somatostatin analog pasireotide to prevent gastrointestinal toxicity and GVHD after myeloablative allogeneic HCT. Methods Patients received 0.9mg pasireotide every 12 hours from the day prior to conditioning through day +4 after HCT (or a maximum of 14 days). The primary outcomes were grade 3–4 gastrointestinal toxicity through day 30 and acute GVHD. Secondary outcomes were chronic GVHD, overall survival and relapse free survival at one year. Stool and blood samples were collected from before and after HCT for analyses of stool microbiome, local inflammatory markers, and systemic inflammatory and metabolic markers. Results were compared with matched controls. Results Twenty-six patients received pasireotide and were compared to 52 matched contemporaneous controls using a 1–2 match. Grade 3–4 GI toxicity occurred in 21 (81%) patients who received pasireotide and 35 (67%) controls (p = 0.33). Acute GVHD occurred in 15 (58%) patients in the pasireotide group and 28 (54%) controls (p = 0.94). Chronic GVHD occurred in 16 patients in the pasireotide group (64%) versus 22 patients in the control group (42%) (p = 0.12). Overall survival at 1 year in the pasireotide group was 63% (95% CI: 47%,86%) versus 82% (95% CI: 72%, 93%) in controls (log-rank p = 0.006). Relapse-free survival rate at one year was 40% (95% CI: 25%, 65%) in the pasireotide group versus 78% (95% CI: 68%, 91%) in controls (log-rank p = 0.002). After controlling for the effect of relevant covariates, patients in the pasireotide group had attenuated post-HCT loss of microbial diversity. Analysis of systemic inflammatory markers and metabolomics demonstrated feasibility of such analyses in patients undergoing allogeneic HCT. Baseline level and pre-to-post transplant changes in several inflammatory markers (including MIP1a, MIP1b, TNFa, IL8Pro, and IL6) correlated with likelihood of survival. Conclusions Pasireotide did not prevent gastrointestinal toxicity or acute GVHD compared to contemporaneous controls. Pasireotide was associated with numerically higher chronic GVHD and significantly decreased OS and RFS compared to contemporaneous controls. Pasireotide may provide a locally protective effect in the stool microbiome and in local inflammation as measured by stool calprotectin, stool beta-defensin, and stool diversity index.

FIT2 is a protein important for ER lipid metabolism and lipid droplet formation, but its function has remained mysterious. Becuwe et al. show that FIT2 is an acyl coenzyme A diphosphatase, and this activity is crucial for ER homeostasis and cellular lipid storage., The endoplasmic reticulum is a cellular hub of lipid metabolism, coordinating lipid synthesis with continuous changes in metabolic flux. Maintaining ER lipid homeostasis despite these fluctuations is crucial to cell function and viability. Here, we identify a novel mechanism that is crucial for normal ER lipid metabolism and protects the ER from dysfunction. We identify the molecular function of the evolutionarily conserved ER protein FIT2 as a fatty acyl–coenzyme A (CoA) diphosphatase that hydrolyzes fatty acyl–CoA to yield acyl 4′-phosphopantetheine. This activity of FIT2, which is predicted to be active in the ER lumen, is required in yeast and mammalian cells for maintaining ER structure, protecting against ER stress, and enabling normal lipid storage in lipid droplets. Our findings thus solve the long-standing mystery of the molecular function of FIT2 and highlight the maintenance of optimal fatty acyl–CoA levels as key to ER homeostasis.

Rotenone, a mitochondrial complex I inhibitor, has been widely used to study the effects of mitochondrial dysfunction on dopaminergic neurons in the context of Parkinson’s disease. Although the deleterious effects of rotenone are well documented, we found that young adult Caenorhabditis elegans showed resistance to 24- and 48-hour rotenone exposures. To better understand the response to rotenone in C. elegans, we evaluated mitochondrial bioenergetic parameters after 24- and 48-hour exposures to 1μM or 5 μM rotenone. Results suggested upregulation of mitochondrial complexes II and V following rotenone exposure, without major changes in oxygen consumption or steady-state ATP levels after rotenone treatment at the tested concentrations. We found evidence that the glyoxylate pathway (an alternate pathway not present in higher metazoans) was induced by rotenone exposure; gene expression measurements showed increases in mRNA levels for two complex II subunits and for isocitrate lyase, the key glyoxylate pathway enzyme. Targeted metabolomics analyses showed alterations in the levels of organic acids, amino acids, and acylcarnitines, consistent with the metabolic restructuring of cellular bioenergetics pathways including activation of complex II, the glyoxylate pathway, glycolysis, and fatty acid oxidation. This expanded understanding of how C. elegans responds metabolically to complex I inhibition via multiple bioenergetic adaptations, including the glyoxylate pathway, will be useful in interrogating the effects of mitochondrial and bioenergetic stressors and toxicants.

A wide range of protein acyl modifications has been identified on enzymes across various metabolic processes; however, the impact of these modifications remains poorly understood. Protein glutarylation is a recently identified modification that can be non-enzymatically driven by glutaryl-CoA. In mammalian systems, this unique metabolite is only produced in the lysine and tryptophan oxidative pathways. To better understand the biology of protein glutarylation, we studied the relationship between enzymes within the lysine/tryptophan catabolic pathways, protein glutarylation, and regulation by the deglutarylating enzyme Sirtuin 5 (SIRT5). Here, we identify glutarylation on the lysine oxidation pathway enzyme glutaryl-CoA dehydrogenase (GCDH). We show increased GCDH glutarylation when glutaryl-CoA production is stimulated by lysine catabolism. Our data reveal glutarylation of GCDH impacts its function, ultimately decreasing lysine oxidation. We then demonstrate the ability of SIRT5 to deglutarylate GCDH, restoring its enzymatic activity. Finally, metabolomic and bioinformatic analyses indicate an expanded role for SIRT5 in regulating amino acid metabolism. Together, these data support a model whereby a feedback loop exists within the lysine/tryptophan oxidation pathway, in which glutaryl-CoA is produced, in turn inhibiting GCDH function via glutaryl modification of GCDH lysine residues, and can be relieved by SIRT5 deacylation activity.

Low potassium intake can affect cardiovascular disease (CVD) risk and cardiometabolic risk factors. We hypothesize that potassium chloride (KCl) supplementation can improve cardiovascular risk metabolomic profile. In this secondary analysis of a pilot randomized clinical trial (RCT) of 26 participants with prediabetes randomized to KCl or placebo, we performed targeted mass-spectrometry-based metabolomic profiling on baseline and 12-week (end-of-study) plasma samples. Principal component analysis (PCA) was used to reduce the many correlated metabolites into fewer, independent factors that retain most of the information in the original data. Those taking KCl had significant reductions (corresponding to lower cardiovascular risk) in the branched-chain amino acids (BCAA) factor (P = 0.004) and in valine levels (P = 0.02); and non-significant reductions in short-chain acylcarnitines (SCA) factor (P = 0.11). KCl supplementation may improve circulating BCAA levels, which may reflect improvements in overall cardiometabolic risk profile. Clinicaltrials.gov identifier: NCT02236598; https://clinicaltrials.gov/ct2/show/NCT02236598 .

ObjectiveTo establish a biorepository of clinical, metabolomic, and microbiome samples from adolescents with obesity as they undergo lifestyle modification.MethodsWe enrolled 223 adolescents aged 10-18 years with Body Mass Index ≥ 95th percentile, along with 71 healthy weight participants. We collected clinical data, fasting serum, and fecal samples at repeated intervals over 6 months. Here we present our study design, data collection methods, and an interim analysis, including targeted serum metabolite measurements and fecal 16S rRNA gene amplicon sequencing among adolescents with obesity (n=27) and healthy weight controls (n=27).ResultsAdolescents with obesity have higher serum alanine aminotransferase, C-reactive protein, and glycated hemoglobin, and lower high-density lipoprotein cholesterol when compared with healthy weight controls. Metabolomics revealed differences in branched chain amino acid related metabolites. We also observed differential abundance of specific microbial taxa and lower species diversity among adolescents with obesity when compared with the healthy weight group.ConclusionsThe Duke Pediatric Metabolism and Microbiome Study biorepository is available as a shared resource. Early findings suggest evidence of a metabolic signature of obesity unique to adolescents, along with confirmation of previously reported findings describing metabolic and microbiome markers of obesity.Clinical Trial RegistrationBiorepository: NCT02959034Observational Trial: NCT03139877What is already known about this subject?The intestinal microbiome plays an important role in adult obesity and regulation of metabolism. Although it is well-established that obesity has its roots in childhood, very little is known about the role of the microbiome in pediatric obesity and how it changes during adolescence.What are the new findings in your manuscript?This manuscript provides details of a new shared biorepository including clinical data, stool samples and plasma samples from a diverse cohort of 223 adolescents with obesity followed longitudinally over 6 months during a weight management intervention, as well as 71 adolescents with healthy weight as a comparison group.Interim analyses suggest that adolescents with obesity have microbiome signatures and metabolite profiles similar to adults, however key differences in microbial communities and metabolic by-products are identified.How might your results change the direction of research or focus of clinical practice?The POMMS biorepository will be available for investigators to use in future research, to elucidate the underlying mechanisms of obesity and related chronic health conditions.Preliminary data reveal metabolite profiles that suggest adolescence may be a window of metabolic plasticity and disease reversibilityMicrobiome and metabolomic signatures suggest potential biomarkers that may serve as prognostic or predictive factors in disease remission, or targets for future therapeutics.

Background: Urine tricarboxylic acid (TCA) cycle organic anions (OAs) are elevated in diabetes and may be biomarkers for diabetic kidney disease (DKD) progression. Methods: We conducted an ancillary study to the Simultaneous Risk Factor Control Using Telehealth to Slow Progression of Diabetic Kidney Disease (STOP-DKD) Trial - a randomized trial of pharmacist-led medication and behavior management in 281 patients with early to moderate DKD at Duke from 2014-2015. In this study, we used linear mixed models to assess associations of 10 urine TCA cycle OAs with estimated glomerular filtration rate (eGFR) and eGFR decline. TCA cycle OAs were measured in 24h urine samples by targeted MS and modeled as: (1) the average of z-scores for each OAs; and (2) factor scores derived by principal component analysis (PCA). We extended follow-up for eGFR to mid-2019 using electronic health records. Results: Among 132 participants (50% men; 58% Black; mean age 64 years [SD 9]; median eGFR 72 ml/min/1.73m2 [IQR 59-89] and urine albumin-to-creatinine [UACR] 25 mg/g [IQR 9-81]), PCA identified 3 OA metabolite factors (F). Fumarate, malate, α-ketoglutarate, citrate, lactate, and pyruvate loaded positively on F1; succinate, pyruvate, and lactate loaded negatively on F2; and methylsuccinate, ethylmalonate, and methylmalonate loaded positively on F3. Over a median follow-up of 1 year [IQR 1.3], higher average OA z score, F1, and F3 were each strongly associated with higher baseline eGFR after adjustment for age, sex, race, and log(UACR) at baseline (all p Conclusion: Lower urine TCA cycle OAs, most notably a factor comprising lower succinate, pyruvate and lactate, are potential biomarkers for faster DKD progression. Disclosure J. Lunyera: None. M. Nguyen: None. C.J. Diamantidis: None. H.B. Bosworth: Board Member; Self; Preventric Diagnostic. Consultant; Self; Abbott, Medicines Company, Novartis Pharmaceuticals Corporation. Research Support; Self; improved patient outcomes, Novo Nordisk Foundation, Otsuka Pharmaceutical Co., Ltd., Otsuka Pharmaceutical Co., Ltd., Pharma foundation, Proteus Digital Health, Proteus Digital Health, Sanofi. U.D. Patel: Board Member; Self; Goldfinch Bio. Employee; Self; Gilead Sciences, Inc. C.A. Davenport: None. J.R. Bain: None. M. Muehlbauer: None. O. Ilkayeva: None. S.H. Shah: Research Support; Self; AstraZeneca, Verily Life Sciences LLC. J. Scialla: Advisory Panel; Self; Tricida. Other Relationship; Self; GlaxoSmithKline plc., Sanofi. Funding National Institute of Diabetes and Digestive and Kidney Diseases (DK076169)

OBJECTIVES: Metabolomic profiling is used to identify biological pathways and biomarkers for cardiovascular disease (CVD) and diabetes. Thus, metabolomics could be used to identify metabolic effects and demonstrate short-term efficacy of interventions designed to reduce longer-term risk of these conditions. Low potassium (K) intake has been linked to high blood pressure and increased CVD and diabetes risk. The effects of increasing K intake on metabolomic measures related to CVD and diabetes risk are not known. In this ancillary study, we tested the hypothesis that potassium chloride (KCl) supplementation would be associated with improvements in metabolomic biomarkers, such as branched-chain amino acids (BCAA) which are associated with CVD and diabetes risk. METHODS: We performed targeted mass-spectrometry-based metabolomic profiling of 60 metabolites on baseline and 12-week (end-of-study) plasma samples from 26 African-American participants with prediabetes randomized to KCl supplements vs. placebo. Principal component analysis (PCA) was used for dimensionality reduction. Univariate and multivariable analyses were used to assess differences between the two intervention arms in the changes in metabolomic factor scores and individual metabolites. RESULTS: In univariate comparisons, compared to placebo, a PCA factor composed of long-chain acylcarnitines (LCA) increased in the KCl arm (P = 0.02). In multivariable models adjusted for baseline factor score, age, and sex, this association with the LCA factor was no longer significant; but those taking KCl had significant reductions in the BCAA factor (P = 0.004) and in valine levels (P = 0.02). CONCLUSIONS: These results suggest that KCl supplementation may be associated with improvements in BCAA metabolism. Further studies among a larger population and with longer-term follow-up are warranted to verify these results and to determine if KCl supplementation may be an effective intervention for CVD and diabetes prevention. FUNDING SOURCES: NIH/Duke CTSA.

Progesterone is primarily a pregnancy-related hormone, produced in substantial quantities after ovulation and during gestation. Traditionally known to function via nuclear receptors for transcriptional regulation, there is also evidence of nonnuclear action. A previously identified mitochondrial progesterone receptor (PR-M) increases cellular respiration in cell models. In these studies, we demonstrated that expression of PR-M in rat H9c2 cardiomyocytes resulted in a ligand-dependent increase in oxidative cellular respiration and beta-oxidation. Cardiac expression in a TET-On transgenic mouse resulted in gene expression of myofibril proteins for remodeling and proteins involved in oxidative phosphorylation and fatty acid metabolism. In a model of increased afterload from constant transverse aortic constriction, mice expressing PR-M showed a ligand-dependent preservation of cardiac function. From these observations, we propose that PR-M is responsible for progesterone-induced increases in cellular energy production and cardiac remodeling to meet the physiological demands of pregnancy.

The myocardium is metabolically flexible and can use fatty acids, glucose, lactate/pyruvate, ketones, or amino acids to fuel mechanical work. However, impaired metabolic flexibility is associated with cardiac dysfunction in conditions including diabetes and heart failure. The mitochondrial pyruvate carrier (MPC) is required for pyruvate metabolism and is composed of a hetero-oligomer of two proteins known as MPC1 and MPC2. Interestingly, MPC1 and MPC2 expression is downregulated in failing human hearts and in a mouse model of heart failure. Mice with cardiac-specific deletion of MPC2 (CS-MPC2-/-) exhibited loss of both MPC2 and MPC1 proteins and reduced pyruvate-stimulated mitochondrial respiration. CS-MPC2-/- mice exhibited normal cardiac size and function at 6-weeks old, but progressively developed cardiac dilation and contractile dysfunction thereafter. Feeding CS-MPC2-/- mice a ketogenic diet (KD) completely prevented or reversed the cardiac remodeling and dysfunction. Other diets with higher fat content and enough carbohydrate to limit ketosis also improved heart failure in CS-MPC2-/- mice, but direct ketone body provisioning provided only minor improvements in cardiac remodeling. Finally, KD was also able to prevent further remodeling in an ischemic, pressure-overload mouse model of heart failure. In conclusion, loss of mitochondrial pyruvate utilization leads to dilated cardiomyopathy that can be corrected by a ketogenic diet.

AIMS/HYPOTHESIS: We aimed to determine the association of maternal metabolites with newborn adiposity and hyperinsulinaemia in a multi-ethnic cohort of mother–newborn dyads. METHODS: Targeted and non-targeted metabolomics assays were performed on fasting and 1 h serum samples from a total of 1600 mothers in four ancestry groups (Northern European, Afro-Caribbean, Mexican American and Thai) who participated in the Hyperglycemia and Adverse Pregnancy Outcome (HAPO) study, underwent an OGTT at ~28 weeks gestation and whose newborns had anthropometric measurements at birth. RESULTS: In this observational study, meta-analyses demonstrated significant associations of maternal fasting and 1 h metabolites with birthweight, cord C-peptide and/or sum of skinfolds across ancestry groups. In particular, maternal fasting triacylglycerols were associated with newborn sum of skinfolds. At 1 h, several amino acids, fatty acids and lipid metabolites were associated with one or more newborn outcomes. Network analyses revealed clusters of fasting acylcarnitines, amino acids, lipids and fatty acid metabolites associated with cord C-peptide and sum of skinfolds, with the addition of branched-chain and aromatic amino acids at 1 h. CONCLUSIONS/INTERPRETATION: The maternal metabolome during pregnancy is associated with newborn outcomes. Maternal levels of amino acids, acylcarnitines, lipids and fatty acids and their metabolites during pregnancy relate to fetal growth, adiposity and cord C-peptide, independent of maternal BMI and blood glucose levels.

BACKGROUND: Gastric bypass surgery for weight reduction often corrects dysglycemia in diabetic patients, but a full understanding of the underlying biochemical pathways continues to be investigated. We explored the effects of weight loss by surgical and dietary interventions on plasma metabolites using both targeted and discovery-oriented metabolomics platforms. SETTING: An academic medical center in the United States. METHODS: Improvement in HOMA-IR, as an index of insulin resistance, was compared at six months in eleven patients that underwent Roux-en-Y gastric bypass (RYGB) against eleven patients that were matched for weight loss in the Weight Loss Maintenance (WLM) program. Metabolites in plasma were evaluated by non-targeted gas chromatography/mass spectrometry (GC/MS) for the potential detection of more than 1100 biochemical markers. RESULTS: Among multiple metabolites detected, 2-hydroxybutyric acid (2-HBA) declined most significantly after six months in comparing patients that underwent RYGB versus those in WLM (P < 0.001), corresponding with declines in HOMA-IR (P = 0.025). Baseline levels of 2-HBA for all patients were correlated with pre-intervention levels of HOMA-IR (R(2) = 0.565, P < 0.001). Moreover, the changes in 2-HBA after six months were correlated with changes in HOMA-IR (R(2) = 0.399, P = 0.0016). CONCLUSIONS: Correlation between insulin resistance and 2-hydroxybutyric acid suggests the utility of the latter as an excellent biomarker for tracking glycemic improvement, and offers further insight into the pathways that control diabetes. This is the first report of a decline in 2-HBA in response to bariatric surgery.

The mRNA modification N6-methyladenosine (m6A) is involved in many post-transcriptional regulatory processes including mRNA stability and translational efficiency. However, it is also imperative to correlate these processes with phenotypic outputs during cancer progression. Here we report that m6A levels are significantly decreased in genetically-defined immortalized and oncogenically-transformed human mammary epithelial cells (HMECs), as compared with their primary cell predecessor. Furthermore, the m6A methyltransferase (METTL3) is decreased and the demethylase (ALKBH5) is increased in the immortalized and transformed cell lines, providing a possible mechanism for this basal change in m6A levels. Although the immortalized and transformed cells showed lower m6A levels than their primary parental cell line, overexpression of METTL3 and METTL14, or ALKBH5 knockdown to increase m6A levels in transformed cells increased proliferation and migration. Remarkably, these treatments had little effect on the immortalized cells. Together, these results suggest that m6A modification may be downregulated in immortalized cells as a brake against malignant progression. Finally, we found that m6A levels in the immortalized and transformed cells increased in response to hypoxia without corresponding changes in METTL3, METTL14 or ALKBH5 expression, suggesting a novel pathway for regulation of m6A levels under stress.

SUMMARY SIRT3 is a nicotinamide adenine dinucleotide (NAD+)- dependent mitochondrial protein deacetylase purported to influence metabolism through post-translational modification of metabolic enzymes. Fuel-stimulated insulin secretion, which involves mitochondrial metabolism, could be susceptible to SIRT3-mediated effects. We used CRISPR/Cas9 technology to manipulate SIRT3 expression in β cells, resulting in widespread SIRT3-dependent changes in acetylation of key metabolic enzymes but no appreciable changes in glucose- or pyruvate-stimulated insulin secretion or metabolomic profile during glucose stimulation. Moreover, these broad changes in the SIRT3-targeted acetylproteome did not affect responses to nutritional or ER stress. We also studied mice with global SIRT3 knockout fed either standard chow (STD) or high-fat and high-sucrose (HFHS) diets. Only when chronically fed HFHS diet do SIRT3 KO animals exhibit a modest reduction in insulin secretion. We conclude that broad changes in mitochondrial protein acetylation in response to manipulation of SIRT3 are not sufficient to cause changes in islet function or metabolism., In Brief Peterson et al. report that ablation of SIRT3 in 832/13 β cells dramatically alters the mitochondrial acetylproteome but does not affect insulin secretion, metabolomic profile, or β cell survival. Moreover, SIRT3 knockout causes a modest reduction in insulin secretion in mice fed a high-fat and high-sucrose but not a standard chow diet., Graphical Abstract

OBJECTIVE We used targeted metabolomics in pregnant mothers to compare maternal metabolite associations with maternal BMI, glycemia, and insulin sensitivity. RESEARCH DESIGN AND METHODS Targeted metabolomic assays of clinical metabolites, amino acids, and acylcarnitines were performed on fasting and 1-h postglucose serum samples from European ancestry, Afro-Caribbean, Thai, and Mexican American mothers (400 from each ancestry group) who participated in the Hyperglycemia and Adverse Pregnancy Outcome (HAPO) Study and underwent an oral glucose tolerance test at ∼28 weeks gestation. RESULTS K-means clustering, which identified patterns of metabolite levels across ancestry groups, demonstrated that, at both fasting and 1-h, levels of the majority of metabolites were similar across ancestry groups. Meta-analyses demonstrated association of a broad array of fasting and 1-h metabolites, including lipids and amino acids and their metabolites, with maternal BMI, glucose levels, and insulin sensitivity before and after adjustment for the different phenotypes. At fasting and 1 h, a mix of metabolites was identified that were common across phenotypes or associated with only one or two phenotypes. Partial correlation estimates, which allowed comparison of the strength of association of different metabolites with maternal phenotypes, demonstrated that metabolites most strongly associated with different phenotypes included some that were common across as well as unique to each phenotype. CONCLUSIONS Maternal BMI and glycemia have metabolic signatures that are both shared and unique to each phenotype. These signatures largely remain consistent across different ancestry groups and may contribute to the common and independent effects of these two phenotypes on adverse pregnancy outcomes.

Aims/hypothesis Our study aimed to integrate maternal metabolic and genetic data related to insulin sensitivity during pregnancy to provide novel insights into mechanisms underlying pregnancy-induced insulin resistance. Methods Fasting and 1 h serum samples were collected from women in the Hyperglycemia and Adverse Pregnancy Outcome Study who underwent an OGTT at ~28 weeks’ gestation. We obtained targeted and non-targeted metabolomics and genome-wide association data from 1600 and 4528 mothers, respectively, in four ancestry groups (Northern European, Afro-Caribbean, Mexican American and Thai); 1412 of the women had both metabolomics and genome-wide association data. Insulin sensitivity was calculated using a modified insulin sensitivity index that included fasting and 1 h glucose and C-peptide levels after a 75 g glucose load. Results Per-metabolite and network analyses across the four ancestries identified numerous metabolites associated with maternal insulin sensitivity before and 1 h after a glucose load, ranging from amino acids and carbohydrates to fatty acids and lipids. Genome-wide association analyses identified 12 genetic variants in the glucokinase regulatory protein gene locus that were significantly associated with maternal insulin sensitivity, including a common functional missense mutation, rs1260326 (β=−0.2004, p=4.67×10−12 in a meta-analysis across the four ancestries). This SNP was also significantly associated with multiple fasting and 1 h metabolites during pregnancy, including fasting and 1 h triacylglycerols and 2-hydroxybutyrate and 1 h lactate, 2-ketoleucine/ketoisoleucine and palmitoleic acid. Mediation analysis suggested that 1 h palmitoleic acid contributes, in part, to the association of rs1260326 with maternal insulin sensitivity, explaining 13.7% (95% CI 4.0%, 23.3%) of the total effect. Conclusions/interpretation The present study demonstrates commonalities between metabolites and genetic variants associated with insulin sensitivity in the gravid and non-gravid states and provides insights into mechanisms underlying pregnancy-induced insulin resistance., Graphical Abstract

Newborn adiposity is associated with a higher risk of childhood obesity and earlier onset of co-morbid metabolic diseases such as type 2 diabetes mellitus. The cord blood metabolome is one early life marker that provides mechanistic insight into fetal fat deposition, insulin sensitivity, and future obesity and metabolic disease risk and integrates in-utero genetic, nutritional and metabolic cues that contribute to newborn phenotype. We hypothesize that a unique cord blood metabolomic signature associated with newborn adiposity or hyperinsulinemia might emerge as a predictive tool to help identify at-risk children early in life, before disease develops. To evaluate this hypothesis, we performed a cross-sectional, observational study of 1600 newborns who participated in the Hyperglycemia and Adverse Pregnancy Outcome Study. Targeted and nontargeted metabolomics assays were performed on cord blood of newborns across four ancestry groups (Afro-Caribbean, Northern European, Thai, and Mexican American). Newborn birth weight and sum of skinfolds were measured by trained personnel and cord blood was additionally assayed for C-peptide. Associations of cord blood metabolites with newborn phenotype were investigated using 1) per-metabolite analyses within and across ancestries and 2) network analyses to identify interconnected metabolites associated with phenotypes. Several metabolites, including branched-chain amino acids, medium- and long-chain acylcarnitines, nonesterified fatty acids, and triglycerides were significantly negatively associated with cord C-peptide but positively associated with birth weight and/or sum of skinfolds. 1,5-Anhydroglucitol was significantly associated with birth weight and sum of skinfolds in nontargeted metabolite analyses. Network analyses revealed groups of interrelated amino acid, acylcarnitine, and fatty acid metabolites associated with all three newborn outcomes. In conclusion, cord blood metabolites are associated with newborn size and cord C-peptide, even after adjustment for maternal BMI and glucose during pregnancy. The paradoxical inverse association of metabolites with cord blood C-peptide suggests that the metabolite-insulin relationship reverses at a critical point in childhood to the well-described positive association seen in adolescents and adults. The well-known associations of branched chain amino acids and medium- and long-chain acylcarnitines with obesity in adolescents and adults appears to begin at birth and might serve as an early-life marker of obesity risk.

Persons living with HIV (PLWH) are at higher risk for cardiovascular disease (CVD) events than uninfected persons. Current risk-stratification methods to define PLWH at highest risk for CVD events are lacking. Using tandem flow injection mass spectrometry, we quantified plasma levels of 60 metabolites in 24 matched pairs of PLWH [1:1 with and without known coronary artery disease (CAD)]. Metabolite levels were reduced to interpretable factors using principal components analysis. Factors derived from short-chain dicarboxylacylcarnitines (SCDA) (p = 0.08) and glutamine/valine (p = 0.003) were elevated in CAD cases compared to controls. SCDAs and glutamine/valine may be valuable markers of cardiovascular risk among persons living with HIV in the future, pending validation in larger cohorts.

We aimed to identify plasma and urine metabolites altered by the Dietary Approaches to Stop Hypertension (DASH) diet in a post-hoc analysis of a pilot feeding trial. Twenty adult participants with un-medicated hypertension consumed a Control diet for one week followed by 2 weeks of random assignment to either Control or DASH diet. Non-missing fasting plasma (n = 56) and 24-h urine (n = 40) were used to profile metabolites using untargeted gas chromatography/mass spectrometry. Linear models were used to compare metabolite levels between the groups. In urine, 19 identifiable untargeted metabolites differed between groups at p < 0.05. These included a variety of phenolic acids and their microbial metabolites that were higher during the DASH diet, with many at false discovery rate (FDR) adjusted p < 0.2. In plasma, eight identifiable untargeted metabolites were different at p < 0.05, but only gamma-tocopherol was significantly lower on DASH at FDR adjusted p < 0.2. The results provide insights into the mechanisms of benefit of the DASH diet.

The HIV-1 envelope protein (Env) has evolved to subvert the host immune system, hindering viral control by the host. The tryptophan metabolic enzyme kynureninase (KYNU) is mimicked by a portion of the HIV Env gp41 membrane proximal region (MPER) and is cross-reactive with the HIV broadly neutralizing Ab (bnAb) 2F5. Molecular mimicry of host proteins by pathogens can lead to autoimmune disease. In this article, we demonstrate that neither the 2F5 bnAb nor HIV MPER-KYNU cross-reactive Abs elicited by immunization with an MPER peptide-liposome vaccine in 2F5 bnAb VHDJH and VLJL knock-in mice and rhesus macaques modified KYNU activity or disrupted tissue tryptophan metabolism. Thus, molecular mimicry by HIV-1 Env that promotes the evasion of host anti–HIV-1 Ab responses can be directed toward nonfunctional host protein epitopes that do not impair host protein function. Therefore, the 2F5 HIV Env gp41 region is a key and safe target for HIV-1 vaccine development.

Impaired GLUT4-dependent glucose uptake is a contributing factor in the development of whole-body insulin resistance in obese patients and obese animal models. Previously, we demonstrated that transgenic mice engineered to express the human GLUT4 gene under the control of the human GLUT4 promoter (i.e., transgenic [TG] mice) are resistant to obesity-induced insulin resistance. A likely mechanism underlying increased insulin sensitivity is increased glucose uptake in skeletal muscle. The purpose of this study was to investigate the broader metabolic consequences of enhanced glucose uptake into muscle. We observed that the expression of several nuclear and mitochondrially encoded mitochondrial enzymes was decreased in TG mice but that mitochondrial number, size, and fatty acid respiration rates were unchanged. Interestingly, both pyruvate and glutamate respiration rates were decreased in TG mice. Metabolomics analyses of skeletal muscle samples revealed that increased GLUT4 transgene expression was associated with decreased levels of some tricarboxylic acid intermediates and amino acids, whereas the levels of several glucogenic amino acids were elevated. Furthermore, fasting acyl carnitines in obese TG mice were decreased, indicating that increased GLUT4-dependent glucose flux decreases nutrient stress by altering lipid and amino acid metabolism in skeletal muscle.

Objective A branched-chain amino acid (BCAA)-related metabolic signature is strongly associated with insulin resistance and predictive of incident diabetes and intervention outcomes. To better understand the role that this metabolite cluster plays in obesity-related metabolic dysfunction, we studied the impact of BCAA restriction in a rodent model of obesity in which BCAA metabolism is perturbed in ways that mirror the human condition. Methods Zucker-lean rats (ZLR) and Zucker-fatty rats (ZFR) were fed either a custom control, low fat (LF) diet, or an isonitrogenous, isocaloric LF diet in which all three BCAA (Leu, Ile, Val) were reduced by 45% (LF-RES). We performed comprehensive metabolic and physiologic profiling to characterize the effects of BCAA restriction on energy balance, insulin sensitivity, and glucose, lipid and amino acid metabolism. Results LF-fed ZFR had higher levels of circulating BCAA and lower levels of glycine compared to LF-fed ZLR. Feeding ZFR with the LF-RES diet lowered circulating BCAA to levels found in LF-fed ZLR. Activity of the rate limiting enzyme in the BCAA catabolic pathway, branched chain keto acid dehydrogenase (BCKDH), was lower in liver but higher in skeletal muscle of ZFR compared to ZLR and was not responsive to diet in either tissue. BCAA restriction had very little impact on metabolites studied in liver of ZFR where BCAA content was low, and BCKDH activity was suppressed. However, in skeletal muscle of LF-fed ZFR compared to LF-fed ZLR, where BCAA content and BCKDH activity were increased, accumulation of fatty acyl CoAs was completely normalized by dietary BCAA restriction. BCAA restriction also normalized skeletal muscle glycine content and increased urinary acetyl glycine excretion in ZFR. These effects were accompanied by lower RER and improved skeletal muscle insulin sensitivity in LF-RES fed ZFR as measured by hyperinsulinemic-isoglycemic clamp. Conclusions Our data are consistent with a model wherein elevated circulating BCAA contribute to development of obesity-related insulin resistance by interfering with lipid oxidation in skeletal muscle. BCAA-dependent lowering of the skeletal muscle glycine pool appears to contribute to this effect by slowing acyl-glycine export to the urine., Highlights • Feeding a BCAA restricted diet improves skeletal muscle insulin sensitivity in Zucker fatty rats. • BCKDH activity is decreased in liver and increased in skeletal muscle in Zucker fatty versus lean rats. • High BCAA levels drive the obesity-associated decline in circulating and muscle glycine levels. • BCAA-driven glycine depletion restricts formation of acyl-glycine adducts for excretion in urine. • High BCAA/low glycine reduces efficiency of fat oxidation in muscle leading to acyl CoA buildup.

SummaryDysregulated mitochondrial metabolism during hepatic insulin resistance may contribute to pathophysiologies ranging from elevated glucose production to hepatocellular oxidative stress and inflammation. Given that obesity impairs insulin action but paradoxically activates mTORC1, we tested whether insulin action and mammalian target of rapamycin complex 1 (mTORC1) contribute to altered in vivo hepatic mitochondrial metabolism. Loss of hepatic insulin action for 2 weeks caused increased gluconeogenesis, mitochondrial anaplerosis, tricarboxylic acid (TCA) cycle oxidation, and ketogenesis. However, activation of mTORC1, induced by the loss of hepatic Tsc1, suppressed these fluxes. Only glycogen synthesis was impaired by both loss of insulin receptor and mTORC1 activation. Mice with a double knockout of the insulin receptor and Tsc1 had larger livers, hyperglycemia, severely impaired glycogen storage, and suppressed ketogenesis, as compared to those with loss of the liver insulin receptor alone. Thus, activation of hepatic mTORC1 opposes the catabolic effects of impaired insulin action under some nutritional states.

Millions of people worldwide are chronically exposed to arsenic through contaminated drinking water. Despite decades of research studying the carcinogenic potential of arsenic, the mechanisms by which arsenic causes cancer and other diseases remain poorly understood. Mitochondria appear to be an important target of arsenic toxicity. The trivalent arsenical, arsenite, can induce mitochondrial reactive oxygen species production, inhibit enzymes involved in energy metabolism, and induce aerobic glycolysis in vitro, suggesting that metabolic dysfunction may be important in arsenic-induced disease. Here, using the model organism Caenorhabditis elegans and a novel metabolic inhibition assay, we report an in vivo induction of aerobic glycolysis following arsenite exposure. Furthermore, arsenite exposure induced severe mitochondrial dysfunction, including altered pyruvate metabolism; reduced steady-state ATP levels, ATP-linked respiration and spare respiratory capacity; and increased proton leak. We also found evidence that induction of autophagy is an important protective response to arsenite exposure. Because these results demonstrate that mitochondria are an important in vivo target of arsenite toxicity, we hypothesized that deficiencies in mitochondrial electron transport chain genes, which cause mitochondrial disease in humans, would sensitize nematodes to arsenite. In agreement with this, nematodes deficient in electron transport chain complexes I, II, and III, but not ATP synthase, were sensitive to arsenite exposure, thus identifying a novel class of gene-environment interactions that warrant further investigation in the human populace.

Maternal metabolites and metabolic networks underlying associations between maternal glucose during pregnancy and newborn birth weight and adiposity demand fuller characterization. We performed targeted and nontargeted gas chromatography/mass spectrometry metabolomics on maternal serum collected at fasting and 1 h following glucose beverage consumption during an oral glucose tolerance test (OGTT) for 400 northern European mothers at ∼28 weeks' gestation in the Hyperglycemia and Adverse Pregnancy Outcome Study. Amino acids, fatty acids, acylcarnitines, and products of lipid metabolism decreased and triglycerides increased during the OGTT. Analyses of individual metabolites indicated limited maternal glucose associations at fasting, but broader associations, including amino acids, fatty acids, carbohydrates, and lipids, were found at 1 h. Network analyses modeling metabolite correlations provided context for individual metabolite associations and elucidated collective associations of multiple classes of metabolic fuels with newborn size and adiposity, including acylcarnitines, fatty acids, carbohydrates, and organic acids. Random forest analyses indicated an improved ability to predict newborn size outcomes by using maternal metabolomics data beyond traditional risk factors, including maternal glucose. Broad-scale association of fuel metabolites with maternal glucose is evident during pregnancy, with unique maternal metabolites potentially contributing specifically to newborn birth weight and adiposity.

Continual efferocytic clearance of apoptotic cells (ACs) by macrophages prevents necrosis and promotes injury resolution. How continual efferocytosis is promoted is not clear. Here, we show that the process is optimized by linking the metabolism of engulfed cargo from initial efferocytic events to subsequent rounds. We found that continual efferocytosis is enhanced by the metabolism of AC-derived arginine and ornithine to putrescine by macrophage arginase 1 (Arg1) and ornithine decarboxylase (ODC). Putrescine augments HuR-mediated stabilization of the mRNA encoding the GTP-exchange factor Dbl, which activates actin-regulating Rac1 to facilitate subsequent rounds of AC internalization. Inhibition of any step along this pathway after first-AC uptake suppresses second-AC internalization, whereas putrescine addition rescues this defect. Mice lacking myeloid Arg1 or ODC have defects in efferocytosis in vivo and in atherosclerosis regression, while treatment with putrescine promotes atherosclerosis resolution. Thus, macrophage metabolism of AC-derived metabolites allows for optimal continual efferocytosis and resolution of injury.

SUMMARY Acyl CoA metabolites derived from the catabolism of carbon fuels can react with lysine residues of mitochondrial proteins, giving rise to a large family of post-translational modifications (PTMs). Mass spectrometry-based detection of thousands of acyl-PTMs scattered throughout the proteome has established a strong link between mitochondrial hyperacylation and cardiometabolic diseases; however, the functional consequences of these modifications remain uncertain. Here, we use a comprehensive respiratory diagnostics platform to evaluate three disparate models of mitochondrial hyperacylation in the mouse heart caused by genetic deletion of malonyl CoA decarboxylase (MCD), SIRT5 demalonylase and desuccinylase, or SIRT3 deacetylase. In each case, elevated acylation is accompanied by marginal respiratory phenotypes. Of the >60 mitochondrial energy fluxes evaluated, the only outcome consistently observed across models is a ~15% decrease in ATP synthase activity. In sum, the findings suggest that the vast majority of mitochondrial acyl PTMs occur as stochastic events that minimally affect mitochondrial bioenergetics., Graphical Abstract, In Brief Fisher-Wellman et al. use a recently developed mitochondrial diagnostics platform for deep phenotyping of heart mitochondria derived from three disparate genetic models of protein hyperacylation. Their findings oppose the notion that hyperacylation of the mitochondrial proteome leads to broad-ranging vulnerabilities in respiratory function and bioenergetics.

We previously reported how the loss of CHIP expression (Carboxyl terminus of Hsc70-Interacting Protein) during pressure overload resulted in robust cardiac dysfunction, which was accompanied by a failure to maintain ATP levels in the face of increased energy demand. In this study, we analyzed the cardiac metabolome after seven days of pressure overload and found an increase in long-chain and medium-chain fatty acid metabolites in wild-type hearts. This response was attenuated in mice that lack expression of CHIP (CHIP&minus, /&minus, ). These findings suggest that CHIP may play an essential role in regulating oxidative metabolism pathways that are regulated, in part, by the nuclear receptor PPAR&alpha, (Peroxisome Proliferator-Activated Receptor alpha). Next, we challenged CHIP&minus, mice with the PPAR&alpha, agonist called fenofibrate. We found that treating CHIP&minus, mice with fenofibrate for five weeks under non-pressure overload conditions resulted in decreased skeletal muscle mass, compared to wild-type mice, and a marked increase in cardiac fibrosis accompanied by a decrease in cardiac function. Fenofibrate resulted in decreased mitochondrial cristae density in CHIP&minus, hearts as well as decreased expression of genes involved in the initiation of autophagy and mitophagy, which suggests that a metabolic challenge, in the absence of CHIP expression, impacts pathways that contribute to mitochondrial quality control. In conclusion, in the absence of functional CHIP expression, fenofibrate results in unexpected skeletal muscle and cardiac pathologies. These findings are particularly relevant to patients harboring loss-of-function mutations in CHIP and are consistent with a prominent role for CHIP in regulating cardiac metabolism.

Strong associations exist between branched chain amino acids (BCAA) and dysregulated glucose and lipid metabolism, but underlying mechanisms are not well understood. One factor contributing to elevated BCAA in obesity is inhibitory phosphorylation of the branched chain keto-acid dehydrogenase complex (BCKDH) in liver. We studied the impact of modulating the activity of the BCKDH kinase (BDK) and phosphatase (PPM1K) on amino acid, glucose and lipid metabolism in Zucker fatty rats (ZFR). Daily administration of a selective inhibitor of BDK, BT2 (20mg.kg-1, IP), for one week or expression of a recombinant adenovirus overexpressing PPM1K significantly lowered BCAA and branched chain keto acid (BCKA) levels in ZFR. Remarkably, these effects were accompanied by strong lowering of liver triacylglycerides and improved glucose tolerance in the absence of weight loss. Proteomics analysis of liver samples from these studies revealed that both inhibition of BDK with BT2 and adenoviral mediated overexpression of PPM1K results in reduced phosphorylation of the lipogenic enzyme ATP-citrate lyase (ACL) on its regulatory serine 454. Phosphorylation of ACL on this site is activating. Sequence analysis revealed the presence of a canonical BDK phosphorylation motif surrounding ser454 in ACL and incubation of ACL with purified BDK resulted in ACL phosphorylation in vitro. Furthermore, adenovirus-mediated overexpression of BDK increased ACL phosphorylation and activated de novo lipogenesis in liver of lean Wistar rats. Together these studies reveal a novel regulatory function for BDK and PPM1K in integration of BCAA and lipid metabolism. Moreover, we show that modulation of this node provides broad protection against metabolic abnormalities associated with obesity. Disclosure P.J. White: None. R.W. McGarrah: None. P.A. Grimsrud: None. W. Yang: None. G. Zhang: None. J.M. Haldeman: None. M. Arlotto: None. O. Ilkayeva: None. R.M. Wynn: None. D.T. Chuang: None. C.B. Newgard: Advisory Panel; Self; Eli Lilly and Company. Research Support; Self; Eli Lilly and Company.

Epitranscriptomic modifications of mRNA are important regulators of gene expression. While internal 2′-O-methylation (Nm) has been discovered on mRNA, questions remain about its origin and function in cells and organisms. Here, we show that internal Nm modification can be guided by small nucleolar RNAs (snoRNAs), and that these Nm sites can regulate mRNA and protein expression. Specifically, two box C/D snoRNAs (SNORDs) and the 2′-O-methyltransferase fibrillarin lead to Nm modification in the protein-coding region of peroxidasin (Pxdn). The presence of Nm modification increases Pxdn mRNA expression but inhibits its translation, regulating PXDN protein expression and enzyme activity both in vitro and in vivo. Our findings support a model in which snoRNA-guided Nm modifications of mRNA can regulate physiologic gene expression by altering mRNA levels and tuning protein translation., The post-transcriptional modification of mRNAs provides an additional layer to gene expression regulation. Here the authors show that 2′-O-methylation mediated by box C/D snoRNAs and fibrillarin can inhibit the translation of target mRNAs.

Current obesity interventions suffer from lack of durable effects and undesirable complications. Fumagillin, an inhibitor of methionine aminopeptidase-2, causes weight loss by reducing food intake, but with effects on weight that are superior to pair-feeding. Here, we show that feeding of rats on a high-fat diet supplemented with fumagillin (HF/FG) suppresses the aggressive feeding observed in pair-fed controls (HF/PF) and alters expression of circadian genes relative to the HF/PF group. Multiple indices of reduced energy expenditure are observed in HF/FG but not HF/PF rats. HF/FG rats also exhibit changes in gut hormones linked to food intake, increased energy harvest by gut microbiota, and caloric spilling in the urine. Studies in gnotobiotic mice reveal that effects of fumagillin on energy expenditure but not feeding behavior may be mediated by the gut microbiota. In sum, fumagillin engages weight loss-inducing behavioral and physiologic circuits distinct from those activated by simple caloric restriction.

Cardiac metabolism is highly adaptive in response to changes in substrate availability, as occur during fasting. This metabolic flexibility is essential to the maintenance of contractile function and is under the control of a group of select transcriptional regulators, notably the nuclear receptor family of factors member PPARα. However, the diversity of physiologic and pathologic states through which the heart must sustain function suggests the possible existence of additional transcriptional regulators that play a role in matching cardiac metabolism to energetic demand. Here we show that cardiac KLF15 is required for the normal cardiac response to fasting. Specifically, we find that cardiac function is impaired upon fasting in systemic and cardiac specific Klf15-null mice. Further, cardiac specific Klf15-null mice display a fasting-dependent accumulation of long chain acylcarnitine species along with a decrease in expression of the carnitine translocase Slc25a20. Treatment with a diet high in short chain fatty acids relieves the KLF15-dependent long chain acylcarnitine accumulation and impaired cardiac function in response to fasting. Our observations establish KLF15 as a critical mediator of the cardiac adaptive response to fasting through its regulation of myocardial lipid utilization.

The role of mitochondrial energy metabolism in maintaining lung function is not understood. We previously observed reduced lung function in mice lacking the fatty acid oxidation enzyme long-chain acyl-CoA dehydrogenase (LCAD). Here, we demonstrate that long-chain acylcarnitines, a class of lipids secreted by mitochondria when metabolism is inhibited, accumulate at the air-fluid interface in LCAD(-/-) lungs. Acylcarnitine accumulation is exacerbated by stress such as influenza infection or by dietary supplementation with l-carnitine. Long-chain acylcarnitines co-localize with pulmonary surfactant, a unique film of phospholipids and proteins that reduces surface tension and prevents alveolar collapse during breathing. In vitro, the long-chain species palmitoylcarnitine directly inhibits the surface adsorption of pulmonary surfactant as well as its ability to reduce surface tension. Treatment of LCAD(-/-) mice with mildronate, a drug that inhibits carnitine synthesis, eliminates acylcarnitines and improves lung function. Finally, acylcarnitines are detectable in normal human lavage fluid. Thus, long-chain acylcarnitines may represent a risk factor for lung injury in humans with dysfunctional fatty acid oxidation.

Objective Investigate the effects of obesity and high-fat diet (HFD) exposure on fatty acid oxidation and TCA cycle intermediates and amino acids in skeletal muscle to better characterize energy metabolism. Methods Plasma and skeletal muscle metabolomic profiles were measured from lean and obese males before and after a 5-day HFD in the 4 h postprandial condition. Results At both time points, plasma short-chain acylcarnitine species (SCAC) were higher in the obese subjects, while the amino acids glycine, histidine, methionine, and citrulline were lower in skeletal muscle of obese subjects. Skeletal muscle medium-chain acylcarnitines (MCAC) C6, C8, C10:2, C10:1, C10, and C12:1 increased in obese subjects, but decreased in lean subjects, from pre- to post-HFD. Plasma content of C10:1 was also decreased in the lean but increased in the obese subjects from pre- to post-HFD. CD36 increased from pre- to post-HFD in obese but not lean subjects. Conclusions Lower skeletal muscle amino acid content and accumulation of plasma SCAC in obese subjects could reflect increased anaplerosis for TCA cycle intermediates, while accumulation of MCAC suggests limitations in β-oxidation. These measures may be important markers of or contributors to dysregulated metabolism observed in skeletal muscle of obese humans.

Among its many breeds, dogs exhibit exceptional variation in their rate of aging and average lifespan, which presents an opportunity to identify longevity-associated traits via comparative analysis. Using primary dermal fibroblasts, we investigated mitochondrial dynamics, bioenergetics, and biomechanical properties in short and long-lived dog breeds. We demonstrated that intrinsic cellular properties correlate with canine breed longevity and likely underlie their aging diversity. More specifically, cells from longer lived-breeds have higher mitochondrial uncoupling and more efficient respiration dynamics, which appears to mitigate reactive oxygen species and promote stress tolerance. We also showed that fibroblasts of long-lived dogs had a more flexible cytoskeleton than their short-lived counterparts, which may similarly help protect cellular function. The benefits of these cellular characteristics likely translate upwards to the whole organism, slowing the rate of aging in long-lived dogs. Overall, our data support the uncoupling to survive hypothesis, where higher uncoupling in smaller breeds promotes longevity. The closeness of dogs and humans genetically and environmentally make these results pertinent to human biology.