Racial Differences in Vaginal Fluid Metabolites and Association with Systemic Inflammation Markers among Ovarian Cancer Patients: A Pilot Study.

The vaginal microbiome differs by race and contributes to inflammation by directly producing or consuming metabolites or by indirectly inducing host immune response, but its potential contributions to ovarian cancer (OC) disparities remain unclear. In this exploratory cross-sectional study, we examine whether vaginal fluid metabolites differ by race among patients with OC, if they are associated with systemic inflammation, and if such associations differ by race. Study participants were recruited from the Ovarian Cancer Epidemiology, Healthcare Access, and Disparities Study between March 2021 and September 2022. Our study included 36 study participants with ovarian cancer who provided biospecimens; 20 randomly selected White patients and all 16 eligible Black patients, aged 50-70 years. Acylcarnitines (n = 45 species), sphingomyelins (n = 34), and ceramides (n = 21) were assayed on cervicovaginal fluid, while four cytokines (IL-1ß, IL-10, TNF-α, and IL-6) were assayed on saliva. Seven metabolites showed >2-fold differences, two showed significant differences using the Wilcoxon rank-sum test (p < 0.05; False Discovery Rate > 0.05), and 30 metabolites had coefficients > ±0.1 in a Penalized Discriminant Analysis that achieved two distinct clusters by race. Arachidonoylcarnitine, the carnitine adduct of arachidonic acid, appeared to be consistently different by race. Thirty-eight vaginal fluid metabolites were significantly correlated with systemic inflammation biomarkers, irrespective of race. These findings suggest that vaginal fluid metabolites may differ by race, are linked with systemic inflammation, and hint at a potential role for mitochondrial dysfunction and sphingolipid metabolism in OC disparities. Larger studies are needed to verify these findings and further establish specific biological mechanisms that may link the vaginal microbiome with OC racial disparities.

Targeted Metabolomic Profiling of Dapagliflozin in Heart Failure With Preserved Ejection Fraction: The PRESERVED-HF Trial.

BACKGROUND: Although sodium glucose co-transporter 2 inhibitors (SGLT2is) improve heart failure (HF)-related symptoms and outcomes in HF with preserved ejection fraction (HFpEF), underlying mechanisms remain unclear. In HF with reduced EF, dapagliflozin altered ketone and fatty acid metabolites vs placebo; however, metabolite signatures of SGLT2is have not been well elucidated in HFpEF. OBJECTIVES: The goal of this study was to assess whether SGLT2i treatment altered systemic metabolic pathways and their relationship to outcomes in HFpEF. METHODS: Targeted profiling of 64 metabolites was performed from 293 participants in PRESERVED-HF (Dapagliflozin in PRESERVED Ejection Fraction Heart Failure), a 12-week, placebo-controlled trial of dapagliflozin. Linear regression assessed changes in metabolite factors defined by principal components analysis (PCA) with dapagliflozin vs placebo. The relationship between changes in metabolite factors with changes in study endpoints was also assessed. RESULTS: The mean age was 70 ± 11 years, 58% were female, and 29% were Black. There were no significant differences in 12 PCA-derived metabolite factors between treatment arms, including metabolites reflecting ketone, fatty acid, or branched-chain amino acid (BCAA) pathways. Combining treatment arms, changes in BCAAs and branched-chain ketoacids were negatively associated with changes in N-terminal pro-B-type natriuretic peptide; changes in medium-/long-chain acylcarnitines were positively associated with changes in N-terminal pro-B-type natriuretic peptide and negatively associated with changes in 6-minute walk test distance; and changes in ketones were negatively associated with changes in weight, without treatment interaction. CONCLUSIONS: Leveraging targeted metabolomics in a placebo-controlled SGLT2i trial of HFpEF, dapagliflozin did not alter systemic metabolic as reflected by circulating metabolites, in contrast with reported effects in HF with reduced ejection fraction. Metabolite biomarkers reflecting BCAA, ketone, and fatty acid metabolism were associated with markers of disease severity, suggesting a role for potential novel treatment targets. (Dapagliflozin in PRESERVED Ejection Fraction Heart Failure [PRESERVED-HF]; NCT03030235).

Optical imaging reveals chemotherapy-induced metabolic reprogramming of residual disease and recurrence.

Fewer than 20% of triple-negative breast cancer patients experience long-term responses to mainstay chemotherapy. Resistant tumor subpopulations use alternative metabolic pathways to escape therapy, survive, and eventually recur. Here, we show in vivo, longitudinal metabolic reprogramming in residual disease and recurrence of triple-negative breast cancer xenografts with varying sensitivities to the chemotherapeutic drug paclitaxel. Optical imaging coupled with metabolomics reported an increase in non-glucose-driven mitochondrial metabolism and an increase in intratumoral metabolic heterogeneity during regression and residual disease in resistant MDA-MB-231 tumors. Conversely, sensitive HCC-1806 tumors were primarily reliant on glucose uptake and minimal changes in metabolism or heterogeneity were observed over the tumors' therapeutic life cycles. Further, day-matched resistant HCC-1806 tumors revealed a higher reliance on mitochondrial metabolism and elevated metabolic heterogeneity compared to sensitive HCC-1806 tumors. Together, metabolic flexibility, increased reliance on mitochondrial metabolism, and increased metabolic heterogeneity are defining characteristics of persistent residual disease, features that will inform the appropriate type and timing of therapies.

Prevotella copri and microbiota members mediate the beneficial effects of a therapeutic food for malnutrition.

Microbiota-directed complementary food (MDCF) formulations have been designed to repair the gut communities of malnourished children. A randomized controlled trial demonstrated that one formulation, MDCF-2, improved weight gain in malnourished Bangladeshi children compared to a more calorically dense standard nutritional intervention. Metagenome-assembled genomes from study participants revealed a correlation between ponderal growth and expression of MDCF-2 glycan utilization pathways by Prevotella copri strains. To test this correlation, here we use gnotobiotic mice colonized with defined consortia of age- and ponderal growth-associated gut bacterial strains, with or without P. copri isolates closely matching the metagenome-assembled genomes. Combining gut metagenomics and metatranscriptomics with host single-nucleus RNA sequencing and gut metabolomic analyses, we identify a key role of P. copri in metabolizing MDCF-2 glycans and uncover its interactions with other microbes including Bifidobacterium infantis. P. copri-containing consortia mediated weight gain and modulated energy metabolism within intestinal epithelial cells. Our results reveal structure-function relationships between MDCF-2 and members of the gut microbiota of malnourished children with potential implications for future therapies.

Chronic exercise improves hepatic acylcarnitine handling.

Exercise mediates tissue metabolic function through direct and indirect adaptations to acylcarnitine (AC) metabolism, but the exact mechanisms are unclear. We found that circulating medium-chain acylcarnitines (AC) (C12-C16) are lower in active/endurance trained human subjects compared to sedentary controls, and this is correlated with elevated cardiorespiratory fitness and reduced adiposity. In mice, exercise reduced serum AC and increased liver AC, and this was accompanied by a marked increase in expression of genes involved in hepatic AC metabolism and mitochondrial ß-oxidation. Primary hepatocytes from high-fat fed, exercise trained mice had increased basal respiration compared to hepatocytes from high-fat fed sedentary mice, which may be attributed to increased Ca2+ cycling and lipid uptake into mitochondria. The addition of specific medium- and long-chain AC to sedentary hepatocytes increased mitochondrial respiration, mirroring the exercise phenotype. These data indicate that AC redistribution is an exercise-induced mechanism to improve hepatic function and metabolism.

Metabolomic and genetic architecture of gestational diabetes subtypes.

AIMS/HYPOTHESIS: Physiological gestational diabetes mellitus (GDM) subtypes that may confer different risks for adverse pregnancy outcomes have been defined. The aim of this study was to characterise the metabolome and genetic architecture of GDM subtypes to address the hypothesis that they differ between GDM subtypes. METHODS: This was a cross-sectional study of participants in the Hyperglycemia and Adverse Pregnancy Outcome (HAPO) study who underwent an OGTT at approximately 28 weeks' gestation. GDM was defined retrospectively using International Association of Diabetes and Pregnancy Study Groups/WHO criteria, and classified as insulin-deficient GDM (insulin secretion <25th percentile with preserved insulin sensitivity) or insulin-resistant GDM (insulin sensitivity <25th percentile with preserved insulin secretion). Metabolomic analyses were performed on fasting and 1 h serum samples in 3463 individuals (576 with GDM). Genome-wide genotype data were obtained for 8067 individuals (1323 with GDM). RESULTS: Regression analyses demonstrated striking differences between the metabolomes for insulin-deficient or insulin-resistant GDM compared to those with normal glucose tolerance. After adjustment for covariates, 33 fasting metabolites, including 22 medium- and long-chain acylcarnitines, were uniquely associated with insulin-deficient GDM; 23 metabolites, including the branched-chain amino acids and their metabolites, were uniquely associated with insulin-resistant GDM; two metabolites (glycerol and 2-hydroxybutyrate) were associated with the same direction of association with both subtypes. Subtype differences were also observed 1 h after a glucose load. In genome-wide association studies, variants within MTNR1B (rs10830963, p=3.43×10-18, OR 1.55) and GCKR (rs1260326, p=5.17×10-13, OR 1.43) were associated with GDM. Variants in GCKR (rs1260326, p=1.36×10-13, OR 1.60) and MTNR1B (rs10830963, p=1.22×10-9, OR 1.49) demonstrated genome-wide significant association with insulin-resistant GDM; there were no significant associations with insulin-deficient GDM. The lead SNP in GCKR, rs1260326, was associated with the levels of eight of the 25 fasting metabolites that were associated with insulin-resistant GDM and ten of 41 1 h metabolites that were associated with insulin-resistant GDM. CONCLUSIONS/INTERPRETATION: This study demonstrates that physiological GDM subtypes differ in their metabolome and genetic architecture. These findings require replication in additional cohorts, but suggest that these differences may contribute to subtype-related adverse pregnancy outcomes.

APOL1-mediated monovalent cation transport contributes to APOL1-mediated podocytopathy in kidney disease.

Two coding variants of apolipoprotein L1 (APOL1), called G1 and G2, explain much of the excess risk of kidney disease in African Americans. While various cytotoxic phenotypes have been reported in experimental models, the proximal mechanism by which G1 and G2 cause kidney disease is poorly understood. Here, we leveraged 3 experimental models and a recently reported small molecule blocker of APOL1 protein, VX-147, to identify the upstream mechanism of G1-induced cytotoxicity. In HEK293 cells, we demonstrated that G1-mediated Na+ import/K+ efflux triggered activation of GPCR/IP3-mediated calcium release from the ER, impaired mitochondrial ATP production, and impaired translation, which were all reversed by VX-147. In human urine-derived podocyte-like epithelial cells (HUPECs), we demonstrated that G1 caused cytotoxicity that was again reversible by VX-147. Finally, in podocytes isolated from APOL1 G1 transgenic mice, we showed that IFN-γ-mediated induction of G1 caused K+ efflux, activation of GPCR/IP3 signaling, and inhibition of translation, podocyte injury, and proteinuria, all reversed by VX-147. Together, these results establish APOL1-mediated Na+/K+ transport as the proximal driver of APOL1-mediated kidney disease.

Circulating metabolic profile in idiopathic pulmonary fibrosis: data from the IPF-PRO Registry.

BACKGROUND: The circulating metabolome, reflecting underlying cellular processes and disease biology, has not been fully characterized in patients with idiopathic pulmonary fibrosis (IPF). We evaluated whether circulating levels of metabolites correlate with the presence of IPF, with the severity of IPF, or with the risk of clinically relevant outcomes among patients with IPF. METHODS: We analyzed enrollment plasma samples from 300 patients with IPF in the IPF-PRO Registry and 100 individuals without known lung disease using a set of targeted metabolomics and clinical analyte modules. Linear regression was used to compare metabolite and clinical analyte levels between patients with IPF and controls and to determine associations between metabolite levels and measures of disease severity in patients with IPF. Unadjusted and adjusted univariable Cox regression models were used to evaluate associations between circulating metabolites and the risk of mortality or disease progression among patients with IPF. RESULTS: Levels of 64 metabolites and 5 clinical analytes were significantly different between patients with IPF and controls. Among analytes with greatest differences were non-esterified fatty acids, multiple long-chain acylcarnitines, and select ceramides, levels of which were higher among patients with IPF versus controls. Levels of the branched-chain amino acids valine and leucine/isoleucine were inversely correlated with measures of disease severity. After adjusting for clinical factors known to influence outcomes, higher levels of the acylcarnitine C:16-OH/C:14-DC were associated with all-cause mortality, lower levels of the acylcarnitine C16:1-OH/C14:1DC were associated with all-cause mortality, respiratory death, and respiratory death or lung transplant, and higher levels of the sphingomyelin d43:2 were associated with the risk of respiratory death or lung transplantation. CONCLUSIONS: IPF has a distinct circulating metabolic profile characterized by increased levels of non-esterified fatty acids, long-chain acylcarnitines, and ceramides, which may suggest a more catabolic environment that enhances lipid mobilization and metabolism. We identified select metabolites that were highly correlated with measures of disease severity or the risk of disease progression and that may be developed further as biomarkers. TRIAL REGISTRATION: ClinicalTrials.gov; No: NCT01915511; URL: www. CLINICALTRIALS: gov .

Sex Differences in Branched-chain Amino Acid and Tryptophan Metabolism and Pathogenesis of Youth-onset Type 2 Diabetes.

OBJECTIVES: Insulin resistance is associated with elevations in plasma branched-chain amino acids (BCAAs). BCAAs compete with aromatic amino acids including tryptophan for uptake into ß cells. To explore relationships between BCAAs and tryptophan metabolism, adiposity, and glucose tolerance, we compared urine metabolites in overweight/obese youth with type 2 diabetes (T2D) with those in nondiabetic overweight/obese and lean youth. METHODS: Metabolites were measured in 24-hour and first-morning urine samples of 56 nondiabetic adolescents with overweight/obesity, 42 adolescents with T2D, and 43 lean controls, aged 12 to 21 years. Group differences were assessed by Kruskal Wallis or ANOVA. RESULTS: Groups were comparable for age, pubertal status, and ethnicity. Youth with T2D were predominantly female and had highest percent body fat. BCAAs, branched-chain ketoacids (BCKAs), tryptophan, and kynurenine were higher in urine of subjects with T2D. There were no differences between lean controls and nondiabetic youth with overweight/obesity. T2D was associated with diversion of tryptophan from the serotonin to the kynurenine pathway, with higher urinary kynurenine/serotonin ratio and lower serotonin/tryptophan and 5-HIAA/kynurenine ratios. Urinary BCAAs, BCKAs, tryptophan, and ratios reflecting diversion to the kynurenine pathway correlated positively with metrics of body fat and hemoglobin A1c. Increases in these metabolites in the obese T2D group were more pronounced and statistically significant only in adolescent girls. CONCLUSION: Increases in urinary BCAAs and BCKAs in adolescent females with T2D are accompanied by diversion of tryptophan metabolism from the serotonin to the kynurenine pathway. These adaptations associate with higher risks of T2D in obese adolescent females than adolescent males.

Metabolite signature of diabetes remission in individuals with obesity undergoing weight loss interventions.

OBJECTIVE: This observational study investigated metabolomic changes in individuals with type 2 diabetes (T2D) after weight loss. We hypothesized that metabolite changes associated with T2D-relevant phenotypes are signatures of improved health. METHODS: Fasting plasma samples from individuals undergoing bariatric surgery (n = 71 Roux-en-Y gastric bypass [RYGB], n = 22 gastric banding), lifestyle intervention (n = 66), or usual care (n = 14) were profiled for 139 metabolites before and 2 years after weight loss. Principal component analysis grouped correlated metabolites into factors. Association of preintervention metabolites was tested with preintervention clinical features and changes in T2D markers. Association between change in metabolites/metabolite factors and change in T2D remission markers, homeostasis model assessment of ß-cell function, homeostasis model assessment of insulin resistance, and glycated hemoglobin (HbA1c) was assessed. RESULTS: Branched-chain amino acids (BCAAs) were associated with preintervention adiposity. Changes in BCAAs (valine, leucine/isoleucine) and branched-chain ketoacids were positively associated with change in HbA1c (false discovery rate q value ≤ 0.001) that persisted after adjustment for percentage weight change and RYGB (p ≤ 0.02). In analyses stratified by RYGB or other weight loss method, some metabolites showed association with non-RYGB weight loss. CONCLUSIONS: This study confirmed known metabolite associations with obesity/T2D and showed an association of BCAAs with HbA1c change after weight loss, independent of the method or magnitude of weight loss.

Metabolomic profiling during ex situ normothermic perfusion before heart transplantation defines patterns of substrate utilization and correlates with markers of allograft injury.

BACKGROUND: Cardiac metabolism is altered in heart failure and ischemia-reperfusion injury states. We hypothesized that metabolomic profiling during ex situ normothermic perfusion before heart transplantation (HT) would lend insight into myocardial substrate utilization and report on subclinical and clinical allograft dysfunction risk. METHODS: Metabolomic profiling was performed on serial samples of ex situ normothermic perfusate assaying biomarkers of myocardial injury in lactate and cardiac troponin I (TnI) as well as metabolites (66 acylcarnitines, 15 amino acids, nonesterified fatty acids [NEFA], ketones, and 3-hydroxybutyrate). We tested for change over time in injury biomarkers and metabolites, along with differential changes by recovery strategy (donation after circulatory death [DCD] vs donation after brain death [DBD]). We examined associations between metabolites, injury biomarkers, and primary graft dysfunction (PGD). Analyses were performed using linear mixed models adjusted for recovery strategy, assay batch, donor-predicted heart mass, and time. RESULTS: A total of 176 samples from 92 ex situ perfusion runs were taken from donors with a mean age of 35 (standard deviation 11.3) years and a median total ex situ perfusion time of 234 (interquartile range 84) minutes. Lactate trends over time differed significantly by recovery strategy, while TnI increased during ex situ perfusion regardless of DCD vs DBD status. We found fuel substrates were rapidly depleted during ex situ perfusion, most notably the branched-chain amino acids leucine/isoleucine, as well as ketones, 3-hydroxybutyrate, and NEFA (least squares [LS] mean difference from the first to last time point -1.7 to -4.5, false discovery rate q < 0.001). Several long-chain acylcarnitines (LCAC), including C16, C18, C18:1, C18:2, C18:3, C20:3, and C20:4, increased during the perfusion run (LS mean difference 0.42-0.67, q < 0.001). Many LCACs were strongly associated with lactate and TnI. The change over time of many LCACs was significantly different for DCD vs DBD, suggesting differential trends in fuel substrate utilization by ischemic injury pattern. Changes in leucine/isoleucine, arginine, C12:1-OH/C10:1-DC, and C16-OH/C14-DC were associated with increased odds of moderate-severe PGD. Neither end-of-run nor change in lactate or TnI was associated with PGD. CONCLUSIONS: Metabolomic profiling of ex situ normothermic perfusion solution reveals a pattern of fuel substrate utilization that correlates with subclinical and clinical allograft dysfunction. This study highlights a potential role for interventions focused on fuel substrate modification in allograft conditioning during ex situ perfusion to improve allograft outcomes.

Catabolism of fats and branched-chain amino acids in children with Type 1 diabetes: Association with glycaemic control and total daily insulin dose.

OBJECTIVE: Hyperglycaemia in Type 1 diabetes (T1D) results from an absolute insulin deficiency. However, insulin resistance (IR) may exacerbate glycaemic instability in T1D and contribute to long-term cardiovascular complications. We previously showed that IR in teenagers with obesity is associated with sex-dependent derangements in the catabolism of branched-chain amino acids (BCAA) and fatty acids. Here we hypothesized that byproducts of BCAA and fatty acid metabolism may serve as biomarkers or determinants of glycaemic control and IR in prepubertal or early pubertal children with T1D. METHODS: Metabolites, hormones and cytokines from fasting blood samples were analysed in 28 children (15 females, 13 males; age 6-11 years) with T1D. Principal components analysis (PCA) and multiple linear regression models were used to correlate metabolites of interest with glycaemic control, total daily insulin dose (TDD, units/kg/d), adiponectin and the triglyceride (TG) to high-density lipoprotein (HDL) ratio. RESULTS: Males and females were comparable in age, BMI-z, insulin sensitivity, glycaemic control, inflammatory markers, BCAAs and C2/C3/C5-acylcarnitines. The majority of components retained in PCA were related to fatty acid oxidation (FAO) and BCAA catabolism. HbA1c correlated positively with Factor 2 (acylcarnitines, incomplete FAO) and Factor 9 (fasting glucose). TDD correlated negatively with C3 and C5 and Factor 10 (BCAA catabolism) and positively with the ratio of C2 to C3 + C5 and Factor 9 (fasting glucose). CONCLUSIONS: These findings suggest that glucose intolerance in prepubertal or early pubertal children with T1D is accompanied by incomplete FAO while TDD is associated with preferential catabolism of fats relative to amino acids.

Mitochondrial metabolites predict adverse cardiovascular events in individuals with diabetes.

Metabolic mechanisms underlying the heterogeneity of major adverse cardiovascular (CV) event (MACE) risk in individuals with type 2 diabetes mellitus (T2D) remain unclear. We hypothesized that circulating metabolites reflecting mitochondrial dysfunction predict incident MACE in T2D. Targeted mass-spectrometry profiling of 60 metabolites was performed on baseline plasma samples from the Trial Evaluating Cardiovascular Outcomes with Sitagliptin (TECOS; discovery cohort) and Exenatide Study of Cardiovascular Event Lowering (EXSCEL; validation cohort) biomarker substudy cohorts. A principal components analysis metabolite factor comprising medium-chain acylcarnitines (MCACs) was associated with MACE in TECOS and validated in EXSCEL, with higher levels associated with higher MACE risk. Meta-analysis showed that long-chain acylcarnitines (LCACs) and dicarboxylacylcarnitines were also associated with MACE. Metabolites remained associated with MACE in multivariate models and favorably changed with exenatide therapy. A third cohort (Cardiac Catheterization Genetics [CATHGEN]) with T2D was assessed to determine whether these metabolites improved discriminative capability of multivariate models for MACE. Nine metabolites (MCACs and LCACs and 1 dicarboxylacylcarnitine) were associated with time to MACE in the CATHGEN cohort. Addition of these metabolites to clinical models minimally improved the discriminative capability for MACE but did significantly down reclassify risk. Thus, metabolites reporting on dysregulated mitochondrial fatty acid oxidation are present in higher levels in individuals with T2D who experience subsequent MACE. These biomarkers may improve CV risk prediction models, be therapy responsive, and highlight emerging risk mechanisms.

Prevotella copri-related effects of a therapeutic food for malnutrition.

Preclinical and clinical studies are providing evidence that the healthy growth of infants and children reflects, in part, healthy development of their gut microbiomes1-5. This process of microbial community assembly and functional maturation is perturbed in children with acute malnutrition. Gnotobiotic animals, colonized with microbial communities from children with severe and moderate acute malnutrition, have been used to develop microbiome-directed complementary food (MDCF) formulations for repairing the microbiomes of these children during the weaning period5. Bangladeshi children with moderate acute malnutrition (MAM) participating in a previously reported 3-month-long randomized controlled clinical study of one such formulation, MDCF-2, exhibited significantly improved weight gain compared to a commonly used nutritional intervention despite the lower caloric density of the MDCF6. Characterizing the 'metagenome assembled genomes' (MAGs) of bacterial strains present in the microbiomes of study participants revealed a significant correlation between accelerated ponderal growth and the expression by two Prevotella copri MAGs of metabolic pathways involved in processing of MDCF-2 glycans1. To provide a direct test of these relationships, we have now performed 'reverse translation' experiments using a gnotobiotic mouse model of mother-to-offspring microbiome transmission. Mice were colonized with defined consortia of age- and ponderal growth-associated gut bacterial strains cultured from Bangladeshi infants/children in the study population, with or without P. copri isolates resembling the MAGs. By combining analyses of microbial community assembly, gene expression and processing of glycan constituents of MDCF-2 with single nucleus RNA-Seq and mass spectrometric analyses of the intestine, we establish a principal role for P. copri in mediating metabolism of MDCF-2 glycans, characterize its interactions with other consortium members including Bifidobacterium longum subsp. infantis, and demonstrate the effects of P. copri-containing consortia in mediating weight gain and modulating the activities of metabolic pathways involved in lipid, amino acid, carbohydrate plus other facets of energy metabolism within epithelial cells positioned at different locations in intestinal crypts and villi. Together, the results provide insights into structure/function relationships between MDCF-2 and members of the gut communities of malnourished children; they also have implications for developing future prebiotic, probiotic and/or synbiotic therapeutics for microbiome restoration in children with already manifest malnutrition, or who are at risk for this pervasive health challenge.

Maternal and Cord Blood Serum Metabolite Associations with Childhood Adiposity and Body Composition Outcomes.

Maternal metabolites influence the size of newborns independently of maternal body mass index (BMI) and glycemia, highlighting the importance of maternal metabolism on offspring outcomes. This study examined associations of maternal metabolites during pregnancy with childhood adiposity, and cord blood metabolites with childhood adiposity using phenotype and metabolomic data from the Hyperglycemia and Adverse Pregnancy Outcome (HAPO) Study and the HAPO Follow-Up Study. The maternal metabolites analyses included 2324 mother-offspring pairs, while the cord blood metabolites analyses included 937 offspring. Multiple logistic and linear regression were used to examine associations between primary predictors, maternal or cord blood metabolites, and childhood adiposity outcomes. Multiple maternal fasting and 1 hr metabolites were significantly associated with childhood adiposity outcomes in Model 1 but were no longer significant after adjusting for maternal BMI and/or maternal glycemia. In the fully adjusted model, fasting lactose levels were negatively associated with child BMI z-scores and waist circumference, while fasting urea levels were positively associated with waist circumference. One-hour methionine was positively associated with fat-free mass. There were no significant associations between cord blood metabolites and childhood adiposity outcomes. Few metabolites were associated with childhood adiposity outcomes after adjusting for maternal BMI and glucose, suggesting that maternal BMI accounts for the association between maternal metabolites and childhood adiposity.

Acetyl-CoA carboxylase 1 is a suppressor of the adipocyte thermogenic program.

Disruption of adipocyte de novo lipogenesis (DNL) by deletion of fatty acid synthase (FASN) in mice induces browning in inguinal white adipose tissue (iWAT). However, adipocyte FASN knockout (KO) increases acetyl-coenzyme A (CoA) and malonyl-CoA in addition to depletion of palmitate. We explore which of these metabolite changes triggers adipose browning by generating eight adipose-selective KO mouse models with loss of ATP-citrate lyase (ACLY), acetyl-CoA carboxylase 1 (ACC1), ACC2, malonyl-CoA decarboxylase (MCD) or FASN, or dual KOs ACLY/FASN, ACC1/FASN, and ACC2/FASN. Preventing elevation of acetyl-CoA and malonyl-CoA by depletion of adipocyte ACLY or ACC1 in combination with FASN KO does not block the browning of iWAT. Conversely, elevating malonyl-CoA levels in MCD KO mice does not induce browning. Strikingly, adipose ACC1 KO induces a strong iWAT thermogenic response similar to FASN KO while also blocking malonyl-CoA and palmitate synthesis. Thus, ACC1 and FASN are strong suppressors of adipocyte thermogenesis through promoting lipid synthesis rather than modulating the DNL intermediates acetyl-CoA or malonyl-CoA.

Association of Maternal Metabolites and Metabolite Networks with Newborn Outcomes in a Multi-Ancestry Cohort.

The in utero environment is important for newborn size at birth, which is associated with childhood adiposity. We examined associations between maternal metabolite levels and newborn birthweight, sum of skinfolds (SSF), and cord C-peptide in a multinational and multi-ancestry cohort of 2337 mother-newborn dyads. Targeted and untargeted metabolomic assays were performed on fasting and 1 h maternal serum samples collected during an oral glucose tolerance test performed at 24-32 week gestation in women participating in the Hyperglycemia and Adverse Pregnancy Outcome (HAPO) Study. Anthropometric measurements were obtained on newborns at birth. Following adjustment for maternal BMI and glucose, per-metabolite analyses demonstrated significant associations between maternal metabolite levels and birthweight, SSF, and cord C-peptide. In the fasting state, triglycerides were positively associated and several long-chain acylcarnitines were inversely associated with birthweight and SSF. At 1 h, additional metabolites including branched-chain amino acids, proline, and alanine were positively associated with newborn outcomes. Network analyses demonstrated distinct clusters of inter-connected metabolites significantly associated with newborn phenotypes. In conclusion, numerous maternal metabolites during pregnancy are significantly associated with newborn birthweight, SSF, and cord C-peptide independent of maternal BMI and glucose, suggesting that metabolites in addition to glucose contribute to newborn size at birth and adiposity.

Metabolic factors associated with incident fracture among older adults with type 2 diabetes mellitus: a nested case-control study.

Older adults with type 2 diabetes mellitus have an increased risk of fracture despite a paradoxically higher average bone mineral density. This study identified additional markers of fracture risk in this at-risk population. Non-esterified fatty acids and the amino acids glutamine/glutamate and asparagine/aspartate were associated with incident fractures. PURPOSE: Type 2 diabetes mellitus (T2D) is associated with an increased risk of fracture despite a paradoxically higher bone mineral density. Additional markers of fracture risk are needed to identify at-risk individuals. METHOD: The MURDOCK study is an ongoing study, initiated in 2007, of residents in central North Carolina. At enrollment, participants completed health questionnaires and provided biospecimen samples. In this nested case-control analysis, incident fractures among adults with T2D, age ≥ 50 years, were identified by self-report and electronic medical record query. Fracture cases were matched 1:2 by age, gender, race/ethnicity, and BMI to those without incident fracture. Stored sera were analyzed for conventional metabolites and targeted metabolomics (amino acids and acylcarnitines). The association between incident fracture and metabolic profile was assessed using conditional logistic regression, controlled for multiple confounders including tobacco and alcohol use, medical comorbidities, and medications. RESULTS: 107 incident fractures were identified with 210 matched controls. Targeted metabolomics analysis included 2 amino acid factors, consisting of: 1) the branched chain amino acids, phenylalanine and tyrosine; and 2) glutamine/glutamate, asparagine/aspartate, arginine, and serine [E/QD/NRS]. After controlling for multiple risk factors, E/QD/NRS was significantly associated with incident fracture (OR 2.50, 95% CI: 1.36-4.63). Non-esterified fatty acids were associated with lower odds of fracture (OR 0.17, 95% CI: 0.03-0.87). There were no associations with fracture among other conventional metabolites, acylcarnitine factors, nor the other amino acid factors. CONCLUSION: Our results indicate novel biomarkers, and suggest potential mechanisms, of fracture risk among older adults with T2D.

De novo lipogenesis fuels adipocyte autophagosome and lysosome membrane dynamics.

Adipocytes robustly synthesize fatty acids (FA) from carbohydrate through the de novo lipogenesis (DNL) pathway, yet surprisingly DNL contributes little to their abundant triglyceride stored in lipid droplets. This conundrum raises the hypothesis that adipocyte DNL instead enables membrane expansions to occur in processes like autophagy, which requires an abundant supply of phospholipids. We report here that adipocyte Fasn deficiency in vitro and in vivo markedly impairs autophagy, evident by autophagosome accumulation and severely compromised degradation of the autophagic substrate p62. Our data indicate the impairment occurs at the level of autophagosome-lysosome fusion, and indeed, loss of Fasn decreases certain membrane phosphoinositides necessary for autophagosome and lysosome maturation and fusion. Autophagy dependence on FA produced by Fasn is not fully alleviated by exogenous FA in cultured adipocytes, and interestingly, imaging studies reveal that Fasn colocalizes with nascent autophagosomes. Together, our studies identify DNL as a critical source of FAs to fuel autophagosome and lysosome maturation and fusion in adipocytes.

Fitm2 is required for ER homeostasis and normal function of murine liver.

The endoplasmic reticulum (ER)-resident protein fat storage-inducing transmembrane protein 2 (FIT2) catalyzes acyl-CoA cleavage in vitro and is required for ER homeostasis and normal lipid storage in cells. The gene encoding FIT2 is essential for the viability of mice and worms. Whether FIT2 acts as an acyl-CoA diphosphatase in vivo and how this activity affects the liver, where the protein was discovered, are unknown. Here, we report that hepatocyte-specific Fitm2 knockout (FIT2-LKO) mice fed a chow diet exhibited elevated acyl-CoA levels, ER stress, and signs of liver injury. These mice also had more triglycerides in their livers than control littermates due, in part, to impaired secretion of triglyceride-rich lipoproteins and reduced capacity for fatty acid oxidation. We found that challenging FIT2-LKO mice with a high-fat diet worsened hepatic ER stress and liver injury but unexpectedly reversed the steatosis phenotype, similar to what is observed in FIT2-deficient cells loaded with fatty acids. Our findings support the model that FIT2 acts as an acyl-CoA diphosphatase in vivo and is crucial for normal hepatocyte function and ER homeostasis in the murine liver.