Long-chain Acylcarnitines Research Articles

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

BackgroundAlthough 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. ObjectivesThe goal of this study was to assess whether SGLT2i treatment altered systemic metabolic pathways and their relationship to outcomes in HFpEF. MethodsTargeted 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. ResultsThe 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. ConclusionsLeveraging 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)

Association of Acylcarnitines With Maternal Cardiometabolic Risk Factors Is Defined by Chain Length: The S-PRESTO Study

Abstract Context Due to the essential role of carnitine as an intermediary in amino acid, carbohydrate, and lipid metabolism, a detailed characterization of circulating and urinary carnitine concentrations will aid in elucidating the molecular basis of impaired maternal metabolic flexibility and facilitating timely intervention for expectant mothers. Objective To investigate the association of maternal plasma and urinary free carnitine and acylcarnitines with cardiometabolic risk factors. Methods Liquid chromatography tandem mass spectrometry–based quantification of free carnitine and acylcarnitines (C2-C18) was performed on 765 plasma and 702 urine samples collected at preconception, 26 to 28 weeks’ pregnancy, and 3 months postpartum in the Singapore PREconception Study of long-Term maternal and child Outcomes (S-PRESTO) cohort study. Results Plasma concentrations of free carnitine and acylcarnitines decreased coupled with increased renal clearance in pregnancy compared with preconception and postpartum. Renal clearance of carnitine increased with an increase in prepregnancy body mass index (ppBMI) and gestational weight gain. Plasma short-chain acylcarnitines were positively associated with ppBMI, irrespective of the physiological state, while medium- and long-chain acylcarnitines were negatively associated with ppBMI at preconception and postpartum but showed a positive association in pregnancy. Similarly, plasma short-chain acylcarnitines were positively associated with Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) whereas medium- and long-chain acylcarnitines were negatively associated with HOMA-IR at preconception and in pregnancy. Mothers who developed gestational diabetes mellitus during pregnancy had ∼10% higher plasma propionylcarnitine concentration and ∼18% higher urine tiglylcarnitine concentration than mothers with normal glucose metabolism at preconception. Conclusion This study provides the metabolic and physiological basis of maternal carnitine homeostasis, which can be used in assessment of maternal cardiometabolic health at preconception to improve pregnancy outcomes.

Carnitine palmitoyltransferase II (CPT II) deficiency responsible for refractory cardiac arrhythmias, acute multiorgan failure and early fatal outcome

BackgroundCarnitine palmitoyltransferase II (CPT II) deficiency is a rare inborn error of mitochondrial fatty acid metabolism with autosomal recessive pattern of inheritance. Its phenotype is highly variable (neonatal, infantile, and adult onset) on the base of mutations of the CPT II gene. In affected subjects, long-chain acylcarnitines cannot be subdivided into carnitine and acyl-CoA, leading to their toxic accumulation in different organs. Neonatal form is the most severe, and all the reported patients died within a few days to 6 months after birth. Hereby, we report on a male late-preterm newborn who presented refractory cardiac arrhythmias and acute multiorgan (hepatic, renal, muscular) injury, leading to cerebral hemorrhage, hydrocephalus, cardiovascular failure and early (day 5 of life) to death. Subsequently, extended metabolic screening and target next generation sequencing (NGS) analysis allowed the CPT II deficiency diagnosis.Case presentationThe male proband was born at 36+ 4 weeks of gestation by spontaneous vaginal delivery. Parents were healthy and nonconsanguineous, although both coming from Nigeria. Family history was unremarkable. Apgar score was 9/9. At birth, anthropometric measures were as follows: weight 2850 g (47th centile, -0.07 standard deviations, SD), length 50 cm (81st centile, + 0.89 SD) and occipitofrontal circumference (OFC) 35 cm (87th centile, + 1.14 SD). On day 2 of life our newborn showed bradycardia (heart rate around 80 bpm) and hypotonia, and was then transferred to the Neonatal Intensive Care Unit (NICU). There, he subsequently manifested many episodes of ventricular tachycardia, which were treated with pharmacological (magnesium sulfate) and electrical cardioversion. Due to the critical conditions of the baby (hepatic, renal and cardiac dysfunctions) and to guarantee optimal management of the arrythmias, he was transferred to the Pediatric Cardiology Reference Center of our region (Sicily, Italy), where he died 2 days later. Thereafter, the carnitines profile evidenced by the extended metabolic screening resulted compatible with a fatty acid oxidation defect (increased levels of acylcarnitines C16 and C18, and low of C2); afterwards, the targeted next generation sequencing (NGS) analysis revealed the known c.680 C > T p. (Pro227Leu) homozygous missense mutation of the CPTII gene, for diagnosis of CPT II deficiency. Genetic investigations have been, then, extended to the baby’s parents, who were identified as heterozygous carriers of the same variant. When we meet again the parents for genetic counseling, the mother was within the first trimester of her second pregnancy. Therefore, we offered to the couple and performed the prenatal target NGS analysis on chorionic villi sample, which did not detect any alterations, excluding thus the CPT II deficiency in their second child.ConclusionsCPTII deficiency may be suspected in newborns showing cardiac arrhythmias, associated or not with hypertrophic cardiomyopathy, polycystic kidneys, brain malformations, hepatomegaly. Its diagnosis should be even more suspected and investigated in cases of increased plasmatic levels of creatine phosphokinase and acylcarnitines in addition to kidney, heart and liver dysfunctions, as occurred in the present patient. Accurate family history, extended metabolic screening, and multidisciplinary approach are necessary for diagnosis and adequate management of affected subjects. Next generation sequencing (NGS) techniques allow the identification of the CPTII gene mutation, essential to confirm the diagnosis before or after birth, as well as to calculate the recurrence risk for family members. Our report broads the knowledge of the genetic and molecular bases of such rare disease, improving its clinical characterization, and provides useful indications for the treatment of patients.

Allele-specific dysregulation of lipid and energy metabolism in early-stage hypertrophic cardiomyopathy

IntroductionHypertrophic cardiomyopathy (HCM) results from pathogenic variants in sarcomeric protein genes that increase myocyte energy demand and lead to cardiac hypertrophy. However, it is unknown whether a common metabolic trait underlies cardiac phenotype at the early disease stage. To address this question and define cardiac biochemical pathology in early-stage HCM, we studied two HCM mouse models that express pathogenic variants in cardiac troponin T (Tnt2) or myosin heavy chain (Myh6) genes, and have marked differences in cardiac imaging phenotype, mitochondrial function at early disease stage. MethodsWe used a combination of echocardiography, transcriptomics, mass spectrometry-based untargeted metabolomics (GC-TOF, HILIC, CSH-QTOF), and computational modeling (CardioNet) to examine cardiac structural and metabolic remodeling at early disease stage (5 weeks of age) in R92W-TnT+/− and R403Q-MyHC+/− mutant mice. Data from mutants was compared with respective littermate controls (WT). ResultsAllele-specific differences in cardiac phenotype, gene expression and metabolites were observed at early disease stage. LV diastolic dysfunction was prominent in TnT mutants. Differentially-expressed genes in TnT mutant hearts were predominantly enriched in the Krebs cycle, respiratory electron transport, and branched-chain amino acid metabolism, whereas MyHC mutants were enriched in mitochondrial biogenesis, calcium homeostasis, and liver-X-receptor signaling. Both mutant hearts demonstrated significant alterations in levels of purine nucleosides, trisaccharides, dicarboxylic acids, acylcarnitines, phosphatidylethanolamines, phosphatidylinositols, ceramides and triglycerides; 40.4 % of lipids and 24.7 % of metabolites were significantly different in TnT mutants, whereas 10.4 % of lipids and 5.8 % of metabolites were significantly different in MyHC mutants. Both mutant hearts had a lower abundance of unsaturated long-chain acyl-carnitines (18:1, 18:2, 20:1), but only TnT mutants showed enrichment of FA18:0 in ceramide and cardiolipin species. CardioNet predicted impaired energy substrate metabolism and greater phospholipid remodeling in TnT mutants than in MyHC mutants. ConclusionsOur systems biology approach revealed marked differences in metabolic remodeling in R92W-TnT and R403Q-MyHC mutant hearts, with TnT mutants showing greater derangements than MyHC mutants, at early disease stage. Changes in cardiolipin composition in TnT mutants could contribute to impairment of energy metabolism and diastolic dysfunction observed in this study, and predispose to energetic stress, ventricular arrhythmias under high workloads such as exercise.

Metabolic profiling of aortic stenosis and hypertrophic cardiomyopathy identifies mechanistic contrasts in substrate utilization.

Aortic stenosis (AS) and hypertrophic cardiomyopathy (HCM) are distinct disorders leading to left ventricular hypertrophy (LVH), but whether cardiac metabolism substantially differs between these in humans remains to be elucidated. We undertook an invasive (aortic root, coronary sinus) metabolic profiling in patients with severe AS and HCM in comparison with non-LVH controls to investigate cardiac fuel selection and metabolic remodeling. These patients were assessed under different physiological states (at rest, during stress induced by pacing). The identified changes in the metabolome were further validated by metabolomic and orthogonal transcriptomic analysis, in separately recruited patient cohorts. We identified a highly discriminant metabolomic signature in severe AS in all samples, regardless of sampling site, characterized by striking accumulation of long-chain acylcarnitines, intermediates of fatty acid transport across the inner mitochondrial membrane, and validated this in a separate cohort. Mechanistically, we identify a downregulation in the PPAR-α transcriptional network, including expression of genes regulating fatty acid oxidation (FAO). In silico modeling of β-oxidation demonstrated that flux could be inhibited by both the accumulation of fatty acids as a substrate for mitochondria and the accumulation of medium-chain carnitines which induce competitive inhibition of the acyl-CoA dehydrogenases. We present a comprehensive analysis of changes in the metabolic pathways (transcriptome to metabolome) in severe AS, and its comparison to HCM. Our results demonstrate a progressive impairment of β-oxidation from HCM to AS, particularly for FAO of long-chain fatty acids, and that the PPAR-α signaling network may be a specific metabolic therapeutic target in AS.

Abstract P259: Favorable Ventricular-Arterial Mechanics Among Women is Associated With Better Mitochondrial Oxidation in the Left Ventricle

Background: Traditional risk factors insufficiently characterize the biological phenomena underlying sex differences in ventriculoarterial coupling (VAC). We hypothesize that sex differences in VAC are due to differences in left ventricular global longitudinal strain (LVGLS), a fuel-dependent chamber closely linked to mitochondrial fuel oxidation pathways. Methods: Community older adults without cardiovascular disease were prospectively recruited for cardiac magnetic resonance imaging and concurrent serum metabolomics sampling. VAC was determined as aortic pulse wave velocity (PWV) divided by LVGLS. Multivariable models adjusted for clinical risk factors and metabolites. Results: Among 202 participants (46.0% female), fewer women were smokers compared to men (3.2% vs 25.7% p<0.001). Despite similarities in age, (women: 69.7 vs men: 70.7 p=0.432) women had better VAC (-0.39 vs -0.47 p = 0.003), LVGLS (-21.9 vs -20.1% p <0.001), and LV mass index (LVMI) (41.7 vs 49.5 g/m 2 p <0.001) compared to men. Both sexes had similar PWV (8.41 vs 9.13 m/s p = 0.087). LVGLS was inversely related to LVMI (β = -0.09 p <0.001). Among women, metabolomic determinants of better LVGLS, independent of LVMI, were lower levels of long chain acylcarnitines (LCACs) [C12:2-OH/C10:2-DC (OR 0.830 adj. p=0.020), C16:3-OH/C14:3-DC (OR 0.334 adj. p=0.048), C16-OH (OR 0.756 adj. p=0.029) and higher levels of C20:2 (OR 1.987 adj. p=0.033)] (Figure A). Among men, determinants of better LVGLS included lower levels of C12:2-OH/C10:2-DC (OR 0.864 adj. p=0.020), C12:1-OH (OR 0.969 adj. p=0.016), C16:1 (OR 0.923 adj. p=0.025), C16:3-OH/C14:3-DC (OR 0.566 adj. p=0.028)] and higher levels of C5:1 (OR 1.096 adj. p=0.026). Conclusion: Better LV longitudinal function contributed to better VAC among older women. Lower LCAC fatty acid metabolism intermediaries was linked to better LVGLS, implying improved mitochondrial β-oxidation. Circulating metabolites may represent novel precision medicine factors for sex-based stratification of vascular risks.

Description of the fasted serum metabolomic signature of lean and obese cats at maintenance and of obese cats under energy restriction.

This study aimed to investigate the serum metabolomic profile of obese and lean cats as well as obese cats before and after energy restriction for weight loss. Thirty cats, 16 obese (body condition score 8 to 9/9) and 14 lean (body condition score 4 to 5/9), were fed a veterinary weight loss food during a 4-week period of weight maintenance (L-MAINT and O-MAINT). The 16 obese cats were then energy restricted by a 60% energy intake reduction with the same food for a 10-week period (O-RESTRICT). Fasted serum metabolites were measured using nuclear magnetic resonance and direct infusion mass spectrometry after the maintenance period for L-MAINT and O-MAINT cats and after the energy restriction period for O-RESTRICT and compared between groups using a two-sided t-test. Obese cats lost 672 g ± 303 g over the 10-week restriction period, representing a weight loss rate of 0.94 ± 0.28% per week. Glycine, l-alanine, l-histidine, l-glutamine, 2-hydroxybutyrate, isobutryric acid, citric acid, creatine, and methanol were greater in O-RESTRICT compared to O-MAINT. There was a greater concentration of long-chain acylcarnitines in O-RESTRICT compared to both O-MAINT and L-MAINT, and greater total amino acids compared to O-MAINT. Glycerol and 3-hydroxybutyric acid were greater in O-MAINT compared to L-MAINT, as were several lysophosphatidylcholines. Thus, energy restriction resulted in increased dispensable amino acids in feline serum which could indicate alterations in amino acid partitioning. An increase in lipolysis was not evident, though greater circulating acylcarnitines were observed, suggesting that fatty acid oxidation rates may have been greater under calorie restriction. More research is needed to elucidate energy metabolism and substrate utilization, specifically fatty acid oxidation and methyl status, during energy restriction in strict carnivorous cats to optimize weight loss.

Abstract 1949 Stearoyl-CoA desaturase-1 deficiency increases hepatic and plasma levels of Saturated Long-Chain Acylcarnitines under a high carbohydrate feeding

Abstract 1949 Stearoyl-CoA desaturase-1 deficiency increases hepatic and plasma levels of Saturated Long-Chain Acylcarnitines under a high carbohydrate feeding

Characteristics of pulmonary artery strain assessed by cardiovascular magnetic resonance imaging and associations with metabolomic pathways in human ageing.

Pulmonary artery (PA) strain is associated with structural and functional alterations of the vessel and is an independent predictor of cardiovascular events. The relationship of PA strain to metabolomics in participants without cardiovascular disease is unknown. In the current study, community-based older adults, without known cardiovascular disease, underwent simultaneous cine cardiovascular magnetic resonance (CMR) imaging, clinical examination, and serum sampling. PA global longitudinal strain (GLS) analysis was performed by tracking the change in distance from the PA bifurcation to the pulmonary annular centroid, using standard cine CMR images. Circulating metabolites were measured by cross-sectional targeted metabolomics analysis. Among n = 170 adults (mean age 71 ± 6.3 years old; 79 women), mean values of PA GLS were 16.2 ± 4.4%. PA GLS was significantly associated with age (β = -0.13, P = 0.017), heart rate (β = -0.08, P = 0.001), dyslipidemia (β = -2.37, P = 0.005), and cardiovascular risk factors (β = -2.49, P = 0.001). Alanine (β = -0.007, P = 0.01) and proline (β = -0.0009, P = 0.042) were significantly associated with PA GLS after adjustment for clinical risk factors. Medium and long-chain acylcarnitines were significantly associated with PA GLS (C12, P = 0.027; C12-OH/C10-DC, P = 0.018; C14:2, P = 0.036; C14:1, P = 0.006; C14, P = 0.006; C14-OH/C12-DC, P = 0.027; C16:3, P = 0.019; C16:2, P = 0.006; C16:1, P = 0.001; C16:2-OH, P = 0.016; C16:1-OH/C14:1-DC, P = 0.028; C18:1-OH/C16:1-DC, P = 0.032). By conventional CMR, PA GLS was associated with aging and vascular risk factors among a contemporary cohort of older adults. Metabolic pathways involved in PA stiffness may include gluconeogenesis, collagen synthesis, and fatty acid oxidation.

Influence of microbiota-associated metabolic reprogramming on clinical outcome in patients with melanoma from the randomized adjuvant dendritic cell-based MIND-DC trial

Tumor immunosurveillance plays a major role in melanoma, prompting the development of immunotherapy strategies. The gut microbiota composition, influencing peripheral and tumoral immune tonus, earned its credentials among predictors of survival in melanoma. The MIND-DC phase III trial (NCT02993315) randomized (2:1 ratio) 148 patients with stage IIIB/C melanoma to adjuvant treatment with autologous natural dendritic cell (nDC) or placebo (PL). Overall, 144 patients collected serum and stool samples before and after 2 bimonthly injections to perform metabolomics (MB) and metagenomics (MG) as prespecified exploratory analysis. Clinical outcomes are reported separately. Here we show that different microbes were associated with prognosis, with the health-related Faecalibacterium prausnitzii standing out as the main beneficial taxon for no recurrence at 2 years (p = 0.008 at baseline, nDC arm). Therapy coincided with major MB perturbations (acylcarnitines, carboxylic and fatty acids). Despite randomization, nDC arm exhibited MG and MB bias at baseline: relative under-representation of F. prausnitzii, and perturbations of primary biliary acids (BA). F. prausnitzii anticorrelated with BA, medium- and long-chain acylcarnitines. Combined, these MG and MB biomarkers markedly determined prognosis. Altogether, the host-microbial interaction may play a role in localized melanoma. We value systematic MG and MB profiling in randomized trials to avoid baseline differences attributed to host-microbe interactions.

Metabolomic and genetic architecture of gestational diabetes subtypes.

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. 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 1h serum samples in 3463 individuals (576 with GDM). Genome-wide genotype data were obtained for 8067 individuals (1323 with GDM). 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 1h 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 1h metabolites that were associated with insulin-resistant GDM. 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.

A novel preliminary metabolomic panel for IHD diagnostics and pathogenesis

Cardiovascular disease (CVD) represents one of the main causes of mortality worldwide and nearly a half of it is related to ischemic heart disease (IHD). The article represents a comprehensive study on the diagnostics of IHD through the targeted metabolomic profiling and machine learning techniques. A total of 112 subjects were enrolled in the study, consisting of 76 IHD patients and 36 non-CVD subjects. Metabolomic profiling was conducted, involving the quantitative analysis of 87 endogenous metabolites in plasma. A novel regression method of age-adjustment correction of metabolomics data was developed. We identified 36 significantly changed metabolites which included increased cystathionine and dimethylglycine and the decreased ADMA and arginine. Tryptophan catabolism pathways showed significant alterations with increased levels of serotonin, intermediates of the kynurenine pathway and decreased intermediates of indole pathway. Amino acid profiles indicated elevated branched-chain amino acids and increased amino acid ratios. Short-chain acylcarnitines were reduced, while long-chain acylcarnitines were elevated. Based on these metabolites data, machine learning algorithms: logistic regression, support vector machine, decision trees, random forest, and gradient boosting, were used for IHD diagnostic models. Random forest demonstrated the highest accuracy with an AUC of 0.98. The metabolites Norepinephrine; Xanthurenic acid; Anthranilic acid; Serotonin; C6-DC; C14-OH; C16; C16-OH; GSG; Phenylalanine and Methionine were found to be significant and may serve as a novel preliminary panel for IHD diagnostics. Further studies are needed to confirm these findings.

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

BackgroundThe 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.MethodsWe 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.ResultsLevels 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.ConclusionsIPF 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 registrationClinicalTrials.gov; No: NCT01915511; URL: www.clinicaltrials.gov.

APOE genotype dictates lipidomic signatures in primary human hepatocytes

Apolipoprotein E (APOE) genetic variants are most notably known for their divergent impact on the risk of developing Alzheimer’s disease. While APOE genotype has been consistently shown to modulate lipid metabolism in a variety of cellular contexts, the effect of APOE alleles on the lipidome in hepatocytes is unknown. In this study, we investigated the contribution of APOE alleles to lipidomic profiles of donor-derived primary human hepatocytes from 77 subjects. Lipidomic data obtained by liquid chromatography-mass spectrometry were analyzed across ε2/ε3, ε3/ε3, and ε3/ε4 genotypes to reveal how APOE modulates lipid relative levels over age and between groups. Hepatic APOE concentration, measured by ELISA, was assessed for correlation with lipid abundance in subjects grouped as per APOE genotype and sex. APOE genotype-specific differential lipidomic signatures associated with age for multiple lipid classes but did not differ between sexes. Compared to ε2/ε3, ε3/ε4 hepatocytes had higher abundance of acylcarnitines (AC) and acylphosphatidylglycerol (AcylPG) as a class, as well as higher medium and long-chain ACs, AcylPG, phosphatidylglycerol (PG), bis(monoacylglycerol)phosphate (BMP), monoacylglycerol (MG) and diacylglycerol (DG) species. The ε3/ε4 hepatocytes also exhibited a higher abundance of medium and long-chain ACs compared to the ε3/ε3 hepatocytes. Only in the ε3/ε4 hepatocytes, APOE concentration was lower and showed a negative correlation with BMP levels, specifically in females. APOE genotype dictates a differential lipidome in primary human hepatocytes. The lipids involved suggest mitochondrial dysfunction with accompanying alterations in neutral lipid storage, reflective of a general disturbance of free fatty acid metabolism in human hepatocytes with the ε4 allele.

Response of Neuropeptides to Hunger Signals in Teleost

Introduction: The perception of hunger is a complex physiological process that requires precise coordination between the central and peripheral tissues. Methods: In this study, tilapia fasted for 24 h was chosen to establish a hunger model to study the mechanism of homeostasis recovery under the joint regulation of the central nervous system (CNS) and peripheral tissues. Results: The gastric and intestinal contents of tilapia were predominantly depleted after a fasting period of 9 h and 24 h, respectively. The serum glucose level significantly decreased at the 9-h and 24-h fasting, respectively, and the glucokinase-dependent glucosensing mechanism in the liver was identified as well as the significant activation of phospho-AMPK. However, fasting for 24 h did not activate glucosensing mechanisms and AMPK signaling pathways in the hypothalamus. On the other hand, significant reductions were observed in the mRNA levels of the lipid synthesis-related genes fas and accα, and the serum triglyceride levels as well. The mRNA levels of npy, agrp, pomc, and cart in the hypothalamus fluctuated during the fasting period without significant differences. With in situ hybridization npy signals upregulated in the ventral zone of posterior periventricular nucleus after 24-h fasting, pomc signals enhanced in the lateral tuberal nucleus. Based on the serum metabolomic analysis, the levels of branched-chain amino acids, butyrate, and short-chain acylcarnitine decreased, while those of medium- and long-chain acylcarnitine increased. Conclusion: Fasting for 24 h resulted in changes in npy and pomc signals within the hypothalamus and triggered the glucosensing mechanism in the liver of tilapia. This study is beneficial for elucidating the response of neuropeptides in the CNS to the changes of nutritional factors when hungry.

Integrated plasma metabolomics and lipidomics profiling highlights distinctive signature of hepatocellular carcinoma in HCV patients

BackgroundEarly diagnosis of hepatocellular carcinoma (HCC) is essential towards the improvement of prognosis and patient survival. Circulating markers such as α-fetoprotein (AFP) and micro-RNAs represent useful tools but still have limitations. Identifying new markers can be fundamental to improve both diagnosis and prognosis. In this approach, we harness the potential of metabolomics and lipidomics to uncover potential signatures of HCC.MethodsA combined untargeted metabolomics and lipidomics plasma profiling of 102 HCV-positive patients was performed by HILIC and RP-UHPLC coupled to Mass Spectrometry. Biochemical parameters of liver function (AST, ALT, GGT) and liver cancer biomarkers (AFP, CA19.9 e CEA) were evaluated by standard assays.ResultsHCC was characterized by an elevation of short and long-chain acylcarnitines, asymmetric dimethylarginine, methylguanine, isoleucylproline and a global reduction of lysophosphatidylcholines. A supervised PLS-DA model showed that the predictive accuracy for HCC class of metabolomics and lipidomics was superior to AFP for the test set (100.00% and 94.40% vs 55.00%). Additionally, the model was applied to HCC patients with AFP values < 20 ng/mL, and, by using only the top 20 variables selected by VIP scores achieved an Area Under Curve (AUC) performance of 0.94.ConclusionThese exploratory findings highlight how metabo-lipidomics enables the distinction of HCC from chronic HCV conditions. The identified biomarkers have high diagnostic potential and could represent a viable tool to support and assist in HCC diagnosis, including AFP-negative patients.Graphical abstract

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

PURPOSECardiac 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. METHODSMetabolomic 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, non-esterified 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) v. 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. RESULTSA total of 176 samples from 92 ex-situ perfusion runs were taken from donors with a mean age of 35 years (SD 11.3) years and a median total ex-situ perfusion time of 234 (IQR 84) minutes. Lactate trends over time differed significantly by recovery strategy, while TnI increased during ex-situ perfusion regardless of DCD v. 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 timepoint -1.7 - -4.5, FDR 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. CONCLUSIONMetabolomic 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.

Involvement of the Mitochondrial Ca2+-Independent Phospholipase iPLA2 in the Induction of Mitochondrial Permeability Transition Pore by Long-Chain Acylcarnitines

Involvement of the Mitochondrial Ca2+-Independent Phospholipase iPLA2 in the Induction of Mitochondrial Permeability Transition Pore by Long-Chain Acylcarnitines

Exploiting the Metabolic Vulnerability of Leukemic Stem Cells (LSCs) in Inv(16) Acute Myeloid Leukemia (AML) with the Combination of Miristen, a Mir-126 Inhibitor, and Venetoclax

AML with inv(16)(p13q22) expresses the leukemogenic fusion gene CBFB-MYH11 and upregulates microRNA (miR)-126 that supports homeostasis, quiescence, and activity of LSCs. Genetic or pharmacologic depletion of miR-126 via miR-126 knock out (KO) or miRisten (a CpG-anti-miR-126 inhibitor) reduces LSCs in a Cbfb-MYH11 ( CM) knock-in (KI) AML model and inv(16) AML patient-derived xenografts (PDXs), respectively [PMID: 34686664]. However, the antileukemic underpinnings of miR-126 inhibition in inv(16) AML remain to be elucidated. LSCs reportedly rely on fatty acid metabolism/oxidative phosphorylation (OXPHOS) for their bioenergetic metabolic needs. To assess the role of miR-126 in metabolism, we performed metabolomic profiling for Lin-/c-Kit+ cells isolated from CM KI AML (miR-126 high) mice and CM with miR-126 KO mice ( CM/miR-126 Δ /Δ). Our data revealed that CM/miR-126 Δ /Δ had a significant decrease of short, medium as well as long-chain acyl carnitines and fatty acid (2 to 11 fold, p&amp;lt;0.05) compared to CM/miR-126 WT/ WT mice, suggesting that miR-126 deficit decreased fatty acid oxidation (FAO). Moreover, electron microscope imaging demonstrated mitochondrial fusion (i.e., larger and networked mitochondria) in CM Lin-/c-Kit+Sca1+ (LSK) cells vs syngeneic controls. In contrast, mitochondria fragmentation (fission) and decreased FAO/OXPHOS were observed in murine CM with miR-126 KO LSK. Accordingly, FAO and Seahorse assays demonstrated miRisten treatment led to mitochondria fragmentation and reduced FAO/OXPHOS and in human CD34 + CD38 ‒ inv(16) blasts (enriched for LSCs) vs normal CD34 + CD38 ‒ cells. Sprouty Related EVH1 Domain Containing 1 (SPRED1), a downstream target of miR-126, inhibits ERK/CREB activation and in turn increases antiapoptotic protein BCL-2 via phosphorylation and stabilization. In inv(16) AML cells, upregulated miR-126 downregulated SPRED1 and in turn increased ERK/CREB signaling and BCL-2 levels. This increase augments the ubiquitination of DRP1, a protein otherwise involved in mitochondrial fission, via NFE2-related factor 2 (NRF2), and increases Carnitine Palmitoyltransferase 1B (CPT1B), a rate-controlling protein for FAO and OXPHOS. In contrast, miR-126 KO or miR-126 inhibition by miRisten led to SPRED1 upregulation and decreased ERK/CREB signaling and BCL-2 levels, thereby resulting in increased Dynamin-related protein 1 (DRP1) and mitochondrial fission and a decrease in CPT1B, FAO, and OXPHOS. Thus, given the interplay of miR-126 with BCL-2 and its implication in mitochondrial fusion and increase of OXPHOS in inv(16) blasts and LSCs, we hypothesized a synergism between miRisten and the BCL-2 inhibitor venetoclax (VEN). In fact, combination of miRisten/VEN synergistically decreased FAO/OXPHOS, enhanced mitochondria fission and increased apoptosis of murine and human inv(16) blasts and LSCs [combination indices (CIs)&amp;lt;1, (ED50-ED95)]. Thus, we treated CM mice with scrambled oligonucleotides (sODN) (20 mg/kg i.v., daily for 3 weeks), miRisten (20 mg/kg i.v., daily for 3 weeks), VEN (100 mg/kg oral gavage, daily for 2 weeks), or miRisten/VEN (same dosages as single agents). Compared with sODN or single agents, miRisten/VEN significantly reduced bone marrow (BM) leukemic blast infiltration [miRisten/VEN 33.6±3.4% vs miRisten 49.8±2.6% (p=0.001), VEN 56.2±4.7% (p=0.001), or sODN 60.0±2.6% (p&amp;lt;0.0001)] and spleen weights [miRisten/VEN 0.228±0.038g vs miRisten 0.495±0.058g (p=0.001), VEN 0.479±0.06g (p=0.002), or sODN 0.853±0.039g (p&amp;lt;0.0001)] and prolonged survival of CM mice [median survival miRisten/VEN 125d vs miRisten 108d (p=0.005), VEN 106.5d (p=0.039), or sODN 91d (p&amp;lt;0.0001), respectively; secondary transplant median survival: miRisten/VEN 92d vs miRisten 70.5d (p=0.048), VEN 54d (p=0.006), or sODN 45d (p=0.0002)]. Similar results were also observed in inv(16) PDXs with significant reduction of BM huCD45+ leukemic blasts by miRisten/VEN compared to single agents or sODN [miRisten/VEN 13.7±1.9% vs miRisten 27.9±3.4% (p=0.003), VEN 25.3±4.6%(p=0.032), or sODN 68.6±5.3% (p&amp;lt;0.0001)]; survival analyses for treated PDX mice and secondary transplant recipients are in progress. In summary, our study provides the mechanism for combining miRisten with VEN in the treatment of inv(16) AML. While VEN is FDA approved for AML, miRisten has just undergone a successful toxicology evaluation and is underway to the clinic.

Biliverdin Reductase B Protects Against Primaquine Induced Hemolysis - Identification of a Novel Anti-Oxidant Pathway in Murine Erythrocytes

Background: Biliverdin Reductase B (BlvrB) is the fourth most abundant enzyme in red blood cells (RBCs) and is widely expressed in other tissues. In normal heme catabolism, porphyrin is converted to biliverdin-alpha (by hemoxygenase (HO)) and then reduced to bilirubin-alpha (by biliverdin reductase A (BlvrA)). Bilirubin-alpha is then glucuronidated and excreted. The large body of literature investigating heme metabolism, bilirubin-based pathologies, and the oxidative biology of bilirubin/biliverdin in adults focuses almost exclusively on BlvrA and the alpha isomers of biliverdin and bilirubin. However, porphyrin is a tetrapyrrole with 4 distinct meso-carbon bridges, each of which can be broken to generate a distinct biliverdin isomer (alpha, beta, gamma, or delta). Although HO is exquisitely specific for the alpha-bond (generating only biliverdin-alpha), chemical coupled oxidation of heme yields all four biliverdin isomers. BlvrB (and not BlvrA) can reduce each of non-alpha biliverdin isomers into their corresponding bilirubin isomers (bilirubin-beta is prevalent in neonatal jaundice and kernicterus). BlvrB also reduces flavins and additional quinone metabolites. Despite BlvrB's abundance and evolutionary conservation, its only known function is a subtle effect on hematopoietic lineage fate (megakaryocyte vs. erythroid). BlvrB has no known function in mature RBCs. We hypothesized that BlvrB plays an important antioxidant role that would only become obvious when other redundant anti-oxidant pathways were limited. Methods: A novel BlvrB knockout mouse was generated on a C57BL/6 background. A model of primaquine (PQ) induced oxidative hemolytic anemia in G6PD deficient mice was used as a clinically relevant scenario in which compromised antioxidant biology leads to treatment limiting pharmacotoxicity in RBCs. BlvrB-KO mice were crossed with two strains of mice that had murine G6PD replaced with either the A- deficient variant of human G6PD (hG6PD(A-)) or the non-deficient form of human G6PD (hG6PD(ND)). Four mouse strains (i.e., hG6PD(ND) and hG6PD(A-) mice with or without BlvrB deletion) received either a 4-day course of PQ or control PBS injections. Anemia was monitored by hematocrit (HCT). Reticulocyte count (RET) was monitored by flow cytometry as CD71+, thiazole orange+ erythrocytes. Peripheral blood was subjected to high resolution metabolomics to elucidate biochemical changes. Results: All mouse strains were healthy at baseline, with normal measured hematological parameters (i.e., Hct 40, RET 1.5%). No change in HCT or RET was observed in hG6PD(ND) or BlvrB-KOxhG6PD(ND) mice receiving PQ or in any strain receiving PBS. PQ induced hemolytic anemia in hG6PD(A-) mice, with an average HCT of 32.7 (18% drop) with RET of 3.1%. A significant exacerbation of PQ induced hemolysis was observed in BlvrB-KOxhG6PD(A-) mice that had an average HCT of 22.5 (44% drop) with RET of 5.3%. (n= 11 mice per group, p &amp;lt;0.001 between groups). Blood from PQ-treated hG6PD(A-)xBlvrBKO mice showed defective glycolysis at the level of glyceraldehyde 3-phosphate dehydrogenase, increased levels of multiple long-chain acylcarnitines, altered redox metabolism, and specific increases of hypoxanthine and sphingosine-1-phosphate (S1P). Three separate experiments were performed with similar results. Discussion: These data demonstrate that BlvrB deletion exacerbates oxidative injury and suggest that BlvrB provides a novel and hitherto undescribed antioxidant pathway in RBCs. Because approximately half a billion humans exhibit some form of G6PD deficiency, these data are also directly relevant to a large population of humans, which were modeled using hG6PD(A-) mice. These studies also define the metabolic lesion in peripheral blood. For example, GAPDH is well known to be highly sensitive to inactivation by oxidative damage, resulting in impaired glycolysis. Changes in hypoxanthine and S1P are known to be associated with decreased RBC function and survival. Finally, altered carnitine metabolism is associated with repair of lipid peroxidation in RBCs via the Lands Cycle. Although the mechanism by which BlvrB facilitates antioxidation is not yet clear, we hypothesize it involves redox cycling of non-alpha isomers of biliverdin and bilirubin. However, the mechanisms may be through reduction of flavins and/or other endogenous substrates. Ongoing studies are testing these hypotheses.