Ketosis, the metabolic response to energy crisis, is a mechanism to sustain life by altering oxidative fuel selection. Often overlooked for its metabolic potential, ketosis is poorly understood outside of starvation or diabetic crisis. Thus, we studied the biochemical advantages of ketosis in humans using a ketone ester-based form of nutrition without the unwanted milieu of endogenous ketone body production by caloric or carbohydrate restriction. In five separate studies of 39 high-performance athletes, we show how this unique metabolic state improves physical endurance by altering fuel competition for oxidative respiration. Ketosis decreased muscle glycolysis and plasma lactate concentrations, while providing an alternative substrate for oxidative phosphorylation. Ketosis increased intramuscular triacylglycerol oxidation during exercise, even in the presence of normal muscle glycogen, co-ingested carbohydrate and elevated insulin. These findings may hold clues to greater human potential and a better understanding of fuel metabolism in health and disease.
Objective To estimate performance of a single-nucleotide-polymorphism–based noninvasive prenatal screen for fetal aneuploidy in high-risk and low-risk populations upon single venopuncture. Methods One thousand sixty-four maternal blood samples from 7 weeks of gestation and beyond were included; one thousand fifty-one were within specifications, 518 (49.3%) low-risk. Cell-free DNA was amplified, sequenced, and analyzed using the Next-generation Aneuploidy Test Using SNPs algorithm. Samples were called as trisomies 21, 18, 13, or monosomy X, or euploid, and male or female. Results Nine hundred sixty-six samples (91.9%) successfully generated a cell-free DNA result. Among these, sensitivity was 100% for trisomy 21 (58/58, CI: 93.8–100%), trisomy 13 (12/12, CI: 73.5–100%), and fetal sex (358/358 female, CI:99.0–100%; 418/418 male, CI: 99.1–100%), 96.0% for trisomy 18 (24/25, CI: 79.7–99.9%), and 90% for monosomy X (9/10, CI: 55.5–99.8%). Specificity for trisomies 21 and 13 was 100% (905/905 [CI: 99.6–100%] and 953/953 [CI: 99.6–100%], respectively) and for trisomy 18 and monosomy X was 99.9% (938/939 [CI: 99.4–100%] and 953/954 [CI: 99.4–100%], respectively). However, 16% (20/125) of aneuploid samples did not return a result; 50% (10/20) had a fetal fraction below the 1.5th percentile of euploid pregnancies. Aneuploidy rate was significantly higher in these samples (p<0.001, odds ratio: 9.2, CI: 4.4–19.0). Sensitivity and specificity did not differ in low-risk and high-risk populations. Conclusions This noninvasive prenatal screen performed with high sensitivity and specificity in high-risk and low-risk cohorts. Aneuploid samples were significantly more likely to not return a result; the number of aneuploidy samples was especially increased among samples with low fetal fraction. This underscores the importance of redraws or, in rare cases, invasive procedures based on low fetal fraction.
Objective Develop a non-invasive prenatal test based on analysis of cell-free DNA in maternal blood to detect fetal aneuploidy at chromosomes 13, 18, 21, X, and Y. Methods 166 samples from pregnant women, including eleven trisomy 21, three trisomy 18, two trisomy 13, two 45,X, and two 47,XXY samples were analyzed using an informatics-based method. Cell-free DNA from maternal blood was isolated and amplified using a multiplex PCR assay targeting 11,000 SNPs on chromosomes 13, 18, 21, X, and Y in a single reaction, then sequenced. A Bayesian-based Maximum Likelihood statistical method was applied to determine the chromosomal count of the five chromosomes interrogated in each sample, along with a sample-specific calculated accuracy for each test result. Results The algorithm correctly reported the chromosome copy number at all five chromosomes in 145 samples that passed a DNA quality test, for a total of 725/725 correct calls. The average calculated accuracy for these samples was 99.92%. Twenty-one samples did not pass the DNA quality test. Conclusions This informatics-based method non-invasively detected fetuses with trisomy 13, 18, and 21, 45,X, and 47,XXY with high sample-specific calculated accuracies for each individual chromosome and across all five chromosomes.
SummaryThe citric acid cycle (CAC) metabolite fumarate has been proposed to be cardioprotective; however, its mechanisms of action remain to be determined. To augment cardiac fumarate levels and to assess fumarate's cardioprotective properties, we generated fumarate hydratase (Fh1) cardiac knockout (KO) mice. These fumarate-replete hearts were robustly protected from ischemia-reperfusion injury (I/R). To compensate for the loss of Fh1 activity, KO hearts maintain ATP levels in part by channeling amino acids into the CAC. In addition, by stabilizing the transcriptional regulator Nrf2, Fh1 KO hearts upregulate protective antioxidant response element genes. Supporting the importance of the latter mechanism, clinically relevant doses of dimethylfumarate upregulated Nrf2 and its target genes, hence protecting control hearts, but failed to similarly protect Nrf2-KO hearts in an in vivo model of myocardial infarction. We propose that clinically established fumarate derivatives activate the Nrf2 pathway and are readily testable cytoprotective agents.
Rationale: The recent development of hyperpolarized 13 C magnetic resonance spectroscopy has made it possible to measure cellular metabolism in vivo, in real time. Objective: By comparing participants with and without type 2 diabetes mellitus (T2DM), we report the first case-control study to use this technique to record changes in cardiac metabolism in the healthy and diseased human heart. Methods and Results: Thirteen people with T2DM (glycated hemoglobin, 6.9±1.0%) and 12 age-matched healthy controls underwent assessment of cardiac systolic and diastolic function, myocardial energetics ( 31 P-magnetic resonance spectroscopy), and lipid content ( 1 H-magnetic resonance spectroscopy) in the fasted state. In a subset (5 T2DM, 5 control), hyperpolarized [1- 13 C]pyruvate magnetic resonance spectra were also acquired and in 5 of these participants (3 T2DM, 2 controls), this was successfully repeated 45 minutes after a 75 g oral glucose challenge. Downstream metabolism of [1- 13 C]pyruvate via PDH (pyruvate dehydrogenase, [ 13 C]bicarbonate), lactate dehydrogenase ([1- 13 C]lactate), and alanine transaminase ([1- 13 C]alanine) was assessed. Metabolic flux through cardiac PDH was significantly reduced in the people with T2DM (Fasted: 0.0084±0.0067 [Control] versus 0.0016±0.0014 [T2DM], Fed: 0.0184±0.0109 versus 0.0053±0.0041; P =0.013). In addition, a significant increase in metabolic flux through PDH was observed after the oral glucose challenge ( P <0.001). As is characteristic of diabetes mellitus, impaired myocardial energetics, myocardial lipid content, and diastolic function were also demonstrated in the wider study cohort. Conclusions: This work represents the first demonstration of the ability of hyperpolarized 13 C magnetic resonance spectroscopy to noninvasively assess physiological and pathological changes in cardiac metabolism in the human heart. In doing so, we highlight the potential of the technique to detect and quantify metabolic alterations in the setting of cardiovascular disease.
Aim Many diseases of the heart are characterised by changes in substrate utilisation, which is in part regulated by the activity of the enzyme pyruvate dehydrogenase (PDH). Consequently, there is much interest in the in vivo evaluation of PDH activity in a range of physiological and pathological states to obtain information regarding the metabolic mechanisms of cardiac diseases. Hyperpolarized [1-13C]pyruvate, detected using MRS, is a novel technique for evaluating PDH flux non-invasively. PDH flux has been assumed to directly reflect in vivo PDH activity, although to date this assumption remains unproven. Methods Control animals and animals undergoing interventions known to modulate PDH activity, namely high fat feeding and dichloroacetate infusion, were used to investigate the relationship between in vivo hyperpolarized MRS measurements of PDH flux and ex vivo measurements of PDH enzyme activity (PDHa). Further, the plasma concentrations of pyruvate and other important metabolites were evaluated following pyruvate infusion to assess the metabolic consequences of the pyruvate infusion during hyperpolarized MRS experiments. Results Hyperpolarized MRS measurements of PDH flux significantly correlated with ex vivo measurements of PDHa, confirming that PDH activity directly influences the in vivo flux of hyperpolarized pyruvate through cardiac PDH. The maximum plasma concentration of pyruvate reached during hyperpolarized MRS experiments was ~250 μM, equivalent to physiological pyruvate concentrations reached during exercise or with dietary interventions. Concentrations of other metabolites, including lactate, glucose and β-hydroxybutyrate (BHB), did not vary during the 60 s following pyruvate infusion. Hence, during the 60 s data acquisition period, metabolism was minimally affected by pyruvate infusion.
Background Carnitine acetyltransferase (CAT) catalyses the reversible conversion of acetyl-CoA into acetylcarnitine. The aim of this study was to use the metabolic tracer hyperpolarised [2-13C]pyruvate with magnetic resonance spectroscopy (MRS) to determine if CAT facilitates carbohydrate oxidation in the heart. Methods and Results Ex vivo, following hyperpolarised [2-13C]pyruvate infusion, the [1-13C]acetylcarnitine MR resonance was saturated with a radiofrequency pulse and the effect of this saturation on [1-13C]citrate and [5-13C]glutamate was observed. In vivo, [2-13C]pyruvate was infused into three groups of fed male Wistar rats; (a) controls, (b) rats in which dichloroacetate enhanced PDH flux and (c) rats in which dobutamine elevated cardiac workload. In the perfused heart, [1-13C]acetylcarnitine saturation reduced the [1-13C]citrate and [5-13C]glutamate resonances by 63% and 51%, indicating rapid exchange between pyruvate-derived acetyl-CoA and the acetylcarnitine pool. In vivo, dichloroacetate increased the rate of [1-13C]acetylcarnitine production by 35% and increased the overall acetylcarnitine pool size by 33%. Dobutamine decreased the rate of [1-13C]acetylcarnitine production by 37% and decreased the acetylcarnitine pool size by 40%. Conclusions Hyperpolarised 13C MRS has revealed that acetylcarnitine provides a route of disposal for excess acetyl-CoA, and also a means to replenish acetyl-CoA when cardiac workload is increased. Cycling of acetyl-CoA through acetylcarnitine appears key to matching instantaneous acetyl-CoA supply with metabolic demand, thereby helping to balance myocardial substrate supply and contractile function.
Chronic hypoxia decreases cardiomyocyte respiration, yet the mitochondrial mechanisms remain largely unknown. We investigated the mitochondrial metabolic pathways and enzymes that were decreased following in vivo hypoxia, and questioned whether hypoxic adaptation was protective for the mitochondria. Wistar rats were housed in hypoxia (7 days acclimatisation and 14 days at 11% oxygen), while control rats were housed in normoxia. Chronic exposure to physiological hypoxia increased haematocrit and cardiac vascular endothelial growth factor, in the absence of weight loss and changes in cardiac mass. In both subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria isolated from hypoxic hearts, state 3 respiration rates with fatty acid were decreased by 17-18%, and with pyruvate were decreased by 29-15%, respectively. State 3 respiration rates with electron transport chain (ETC) substrates were decreased only in hypoxic SSM, not in hypoxic IFM. SSM from hypoxic hearts had decreased activities of ETC complexes I, II and IV, which were associated with decreased reactive oxygen species generation and protection against mitochondrial permeability transition pore (MPTP) opening. In contrast, IFM from hypoxic hearts had decreased activity of the Krebs cycle enzyme, aconitase, which did not modify ROS production or MPTP opening. In conclusion, cardiac mitochondrial respiration was decreased following chronic hypoxia, associated with downregulation of different pathways in the two mitochondrial populations, determined by their subcellular location. Hypoxic adaptation was not deleterious for the mitochondria, in fact, SSM acquired increased protection against oxidative damage under the oxygen-limited conditions.
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