Pyruvate Dehydrogenase Flux Research Articles

Ninerafaxstat in the Treatment of Diabetic Cardiomyopathy and Nonobstructive Hypertrophic Cardiomyopathy.

Ninerafaxstat is a novel mitotrope under investigation in 2 large clinical trials: IMPROVE-DiCE (a phase IIa trial investigating ninerafaxstat) and IMPROVE-hypertrophic cardiomyopathy (HCM). IMPROVE-DiCE is a single-center, open-label, phase 2a trial investigating the effectiveness of ninerafaxstat in diabetic cardiomyopathy. Ninerafaxstat significantly improved phosphocreatine/adenosine triphosphate median by 32% (P < 0.01) and reduced myocardial triglyceride content by 34% (P = 0.026). Magnetic resonance imaging (MRI) analysis showed improved left ventricular peak circumferential diastolic strain rate by 15% (P < 0.047) and peak left ventricular filling rate by 11% (P < 0.05). Pyruvate dehydrogenase flux was increased in 7 of 9 patients (P = 0.08), consistent with improved glucose utilization. IMPROVE-HCM (ninerafaxstat safe, effective for nonobstructive hypertrophic cardiomyopathy patients) is a phase 2, multicenter, randomized controlled and double-blinded study. From baseline to 12 weeks, ninerafaxstat was associated with a significantly improved ventilatory efficiency slope compared with placebo (P = 0.006). In a post hoc analysis with 35 patients with baseline Kansas City Cardiomyopathy Questionnaire score ≤80, changes in ventilatory efficiency slope favored ninerafaxstat versus placebo (P = 0.02). Left atrial size, a surrogate marker of diastolic dysfunction, was significantly decreased in patients on ninerafaxstat versus placebo (P = 0.01). These findings support a larger phase 3 study in symptomatic nonobstructive HCM patients to further investigate ninerafaxstat. Several drugs that also improve glucose utilization including fatty acid oxidation inhibitors, carnitine palmitoyltransferase I inhibitors, and glucagon-like peptide-1 receptor agonists are presently under investigation in clinical trials.

Hyperpolarized 13C and 31P MRS detects differences in cardiac energetics, metabolism, and function in obesity, and responses following treatment.

Obesity is associated with important changes in cardiac energetics and function, and an increased risk of adverse cardiovascular outcomes. Multi-nuclear MRS and MRI techniques have the potential to provide a comprehensive non-invasive assessment of cardiac metabolic perturbation in obesity. A rat model of obesity was created by high-fat diet feeding. This model was characterized using in vivo hyperpolarized [1-13C]pyruvate and [2-13C]pyruvate MRS, echocardiography and perfused heart 31P MRS. Two groups of obese rats were subsequently treated with either caloric restriction or the glucagon-like peptide-1 analogue/agonist liraglutide, prior to reassessment. The model recapitulated cardiovascular consequences of human obesity, including mild left ventricular hypertrophy, and diastolic, but not systolic, dysfunction. Hyperpolarized 13C and 31P MRS demonstrated that obesity was associated with reduced myocardial pyruvate dehydrogenase flux, altered cardiac tricarboxylic acid (TCA) cycle metabolism, and impaired myocardial energetic status (lower phosphocreatine to adenosine triphosphate ratio and impaired cardiac ΔG~ATP). Both caloric restriction and liraglutide treatment were associated with normalization of metabolic changes, alongside improvement in cardiac diastolic function. In this model of obesity, hyperpolarized 13C and 31P MRS demonstrated abnormalities in cardiac metabolism at multiple levels, including myocardial substrate selection, TCA cycle, and high-energy phosphorus metabolism. Metabolic changes were linked with impairment of diastolic function and were reversed in concert following either caloric restriction or liraglutide treatment. With hyperpolarized 13C and 31P techniques now available for human use, the findings support a role for multi-nuclear MRS in the development of new therapies for obesity.

Functional activation of pyruvate dehydrogenase in human brain using hyperpolarized [1-13 C]pyruvate.

Pyruvate, produced from either glucose, glycogen, or lactate, is the dominant precursor of cerebral oxidative metabolism. Pyruvate dehydrogenase (PDH) flux is a direct measure of cerebral mitochondrial function and metabolism. Detection of [13 C]bicarbonate in the brain from hyperpolarized [1-13 C]pyruvate using carbon-13 (13 C) MRI provides a unique opportunity for assessing PDH flux in vivo. This study is to assess changes in cerebral PDH flux in response to visual stimuli using in vivo 13 C MRS with hyperpolarized [1-13 C]pyruvate. From seven sedentary adults in good general health, time-resolved [13 C]bicarbonate production was measured in the brain using 90° flip angles with minimal perturbation of its precursors, [1-13 C]pyruvate and [1-13 C]lactate, to test the hypothesis that the appearance of [13 C]bicarbonate signals in the brain reflects the metabolic changes associated with neuronal activation. With a separate group of healthy participants (n = 3), the likelihood of the bolus-injected [1-13 C]pyruvate being converted to [1-13 C]lactate prior to decarboxylation was investigated by measuring [13 C]bicarbonate production with and without [1-13 C]lactate saturation. In the course of visual stimulation, the measured [13 C]bicarbonate signal normalized to the total 13 C signal in the visual cortex increased by 17.1% ± 15.9% (p = 0.017), whereas no significant change was detected in [1-13 C]lactate. Proton BOLD fMRI confirmed the regional activation in the visual cortex with the stimuli. Lactate saturation decreased bicarbonate-to-pyruvate ratio by 44.4% ± 9.3% (p < 0.01). We demonstrated the utility of 13 C MRS with hyperpolarized [1-13 C]pyruvate for assessing the activation of cerebral PDH flux via the detection of [13 C]bicarbonate production.

1592-P: Evaluating Metabolic Effects of Nicotinamide Riboside on Hepatic Substrate Metabolism in CDAA-Induced NAFLD Mice

Nonalcoholic fatty liver disease (NAFLD) is associated with insulin resistance and altered hepatic energy metabolism, with imbalanced glucose and lipid metabolism. Nicotinamide riboside (NR) has recently emerged as a potential therapeutic for a broad range of metabolic abnormalities. Here, we evaluate the effect of NR treatment on hepatic substrate metabolism in mice with NAFLD. NAFLD was induced by feeding the animals with a choline-deficient, amino acid-define (CDAA) diet for 4 weeks. Mice (C57BL/6) in the NR treatment group (CDAA+NR) were then put on a NR-containing CDAA diet for 10 weeks while the non-treated animals remained on CDAA. Both groups of animals had similar food consumption and weight gain rates, with comparable liver-to-body weight ratios. At the conclusion of the treatment, isolated livers were perfused via the portal vein with 13C metabolic tracers, i.e. 5.5 mM [1,6-13C]glucose + 0.4 mM [U-13C]long chain fatty acids (LCFA) suspended in bovine serum albumin. Fractional substrate oxidations and flux analyses were done using 13C nuclear magnetic resonance (NMR) spectroscopy. An increasing trend of oxygen consumption rates was observed as a result of NR treatment (1.50±0.43 µmol/min/liver) compared to the CDAA-treated group (0.68±0.15 µmol/min/liver). However, the increase is not statistically different. 13C NMR isotopomer flux analyses revealed that pyruvate dehydrogenase flux relative to citrate synthase flux are comparable across the 2 groups (CDAA: 30±4%; CDAA+NR: 33±10%). No changes in fractional oxidation of 13C-long-chain fatty acids and 13C-glucose were observed as a result of nicotinamide riboside treatment, with 44±2% and 48±3% of Ac-CoA derived from 13C-LCFA in CDAA and CDAA+NR livers, respectively. 13C-glucose contributed as a minor source of Ac-CoA (~4-5%) in both groups. These findings suggest that nicotinamide riboside treatment did not affect substrate, fats vs glucose, utilization in CDAA-induced NAFLD mouse livers. Disclosure Q. Shen: None. J.K. Pugmire: None. U. Suwannasual: None. N.R. Maptue: None. C. Khemtong: None. Funding University of Florida

Hyperpolarized Metabolic and Parametric CMR Imaging of Longitudinal Metabolic-Structural Changes in Experimental Chronic Infarction

BackgroundProlonged ischemia and myocardial infarction are followed by a series of dynamic processes that determine the fate of the affected myocardium toward recovery or necrosis. Metabolic adaptions are considered to play a vital role in the recovery of salvageable myocardium in the context of stunned and hibernating myocardium. ObjectivesThe potential of hyperpolarized pyruvate cardiac magnetic resonance (CMR) alongside functional and parametric CMR as a tool to study the complex metabolic-structural interplay in a longitudinal study of chronic myocardial infarction in an experimental pig model is investigated. MethodsMetabolic imaging using hyperpolarized [1-13C] pyruvate and proton-based CMR including cine, T1/T2 relaxometry, dynamic contrast-enhanced, and late gadolinium enhanced imaging were performed on clinical 3.0-T and 1.5-T MR systems before infarction and at 6 days and 5 and 9 weeks postinfarction in a longitudinal study design. Chronic myocardial infarction in pigs was induced using catheter-based occlusion and compared with healthy controls. ResultsMetabolic image data revealed temporarily elevated lactate-to-bicarbonate ratios at day 6 in the infarcted relative to remote myocardium. The temporal changes of lactate-to-bicarbonate ratios were found to correlate with changes in T2 and impaired local contractility. Assessment of pyruvate dehydrogenase flux via the hyperpolarized [13C] bicarbonate signal revealed recovery of aerobic cellular respiration in the hibernating myocardium, which correlated with recovery of local radial strain. ConclusionsThis study demonstrates the potential of hyperpolarized CMR to longitudinally detect metabolic changes after cardiac infarction over days to weeks. Viable myocardium in the area at risk was identified based on restored pyruvate dehydrogenase flux.

A phase 2a trial investigating ninerafaxstat – a novel cardiac mitotrope for the treatment of diabetic cardiomyopathy (IMPROVE-DiCE)

Abstract Background Type 2 diabetes (T2D) is a significant, independent contributor to the development of heart failure (HF), driven by energetic, metabolic, structural and functional myocardial changes. The T2D heart is characterised by over-reliance on fatty acid utilisation, shows reduced glucose oxidation and inhibition of pyruvate dehydrogenase (PDH). This results in a diminished myocardial energy reserve and blunted adenosine triphosphate (ATP) generation as well as cardiac steatosis, contributing to lipotoxicity, and diastolic dysfunction. Purpose We assessed the effects of ninerafaxstat – a novel cardiac mitotrope designed to shift myocardial substrate utilisation in favour of glucose and thus, restore myocardial energy homeostasis – on cardiac metabolism &amp; diastolic function in patients with T2D and obesity. Methods In this open-label, mechanistic phase 2a trial, we enrolled 21 patients with T2D &amp; obesity (HbA1c median 7.0% (IQR 6.6, 7.8), weight 97kg (90, 102)) and subsequently treated them with 200mg ninerafaxstat twice daily for 4 or 8 weeks; (Fig. 1). Cardiac metabolism and function were assessed pre- &amp; post-treatment using magnetic resonance imaging (MRI), 31P-, 1H- and, in a subset of n=9, hyperpolarized [1-13C]pyruvate MR spectroscopy. Results T2D patients at baseline presented with impaired myocardial energetics with a markedly reduced PCr/ATP (1.6 [1.4, 2.1]), myocardial steatosis (myocardial triglycerides 2.2% [1.5, 3.2]) left ventricular (LV) hypertrophy (LV mass 130g [98, 152]), and diastolic dysfunction (peak diastolic strain rate 0.86 1/s [0.82, 1.06]). Ninerafaxstat significantly improved myocardial energetics (PCr/ATP median by 32%, p&amp;lt;0.01), reduced myocardial triglyceride content (by 34%, p=0.03) and improved LV diastolic function (peak circumferential diastolic strain rate by 10%, peak LV filling rate by 11%, both p&amp;lt;0.05) (Fig. 2). PDH flux was increased in 7/9 subjects (mean 45%, p=0.08), consistent with improved glucose utilisation. Left ventricular volumes and mass, heart rate and blood pressure remained unchanged. Conclusions Treatment with ninerafaxstat significantly improves myocardial energetics, reduces myocardial steatosis and improves diastolic function in patients with T2D and obesity. Funding Acknowledgement Type of funding sources: Private company. Main funding source(s): Imbria Pharmaaceuticals

Detection of increased pyruvate dehydrogenase flux in the human heart during adenosine stress test using hyperpolarized [1-13C]pyruvate cardiovascular magnetic resonance imaging

BackgroundHyperpolarized (HP) [1-13C]pyruvate cardiovascular magnetic resonance (CMR) imaging can visualize the uptake and intracellular conversion of [1-13C]pyruvate to either [1-13C]lactate or 13C-bicarbonate depending on the prevailing metabolic state. The aim of the present study was to combine an adenosine stress test with HP [1-13C]pyruvate CMR to detect cardiac metabolism in the healthy human heart at rest and during moderate stress.MethodsA prospective descriptive study was performed between October 2019 and August 2020. Healthy human subjects underwent cine CMR and HP [1-13C]pyruvate CMR at rest and during adenosine stress. HP [1-13C]pyruvate CMR images were acquired at the mid-left-ventricle (LV) level. Semi-quantitative assessment of first-pass myocardial [1-13C]pyruvate perfusion and metabolism were assessed. Paired t-tests were used to compare mean values at rest and during stress.ResultsSix healthy subjects (two female), age 29 ± 7 years were studied and no adverse reactions occurred. Myocardial [1-13C]pyruvate perfusion was significantly increased during stress with a reduction in time-to-peak from 6.2 ± 2.8 to 2.7 ± 1.3 s, p = 0.02. This higher perfusion was accompanied by an overall increased myocardial uptake and metabolism. The conversion rate constant (kPL) for lactate increased from 11 ± 9 *10–3 to 20 ± 10 * 10–3 s−1, p = 0.04. The pyruvate oxidation rate (kPB) increased from 4 ± 4 *10–3 to 12 ± 7 *10–3 s−1, p = 0.008. This increase in carbohydrate metabolism was positively correlated with heart rate (R2 = 0.44, p = 0.02).ConclusionsAdenosine stress testing combined with HP [1-13C]pyruvate CMR is feasible and well-tolerated in healthy subjects. We observed an increased pyruvate oxidation during cardiac stress. The present study is an important step in the translation of HP [1-13C]pyruvate CMR into clinical cardiac imaging.Trial registration EUDRACT, 2018-003533-15. Registered 4th of December 2018, https://www.clinicaltrialsregister.eu/ctr-search/search?query=2018-003533-15

Assessment of Aspartate and Bicarbonate Produced From Hyperpolarized [1-13C]Pyruvate as Markers of Renal Gluconeogenesis.

As both a consumer and producer of glucose, the kidney plays a significant role in glucose homeostasis. Measuring renal gluconeogenesis requires invasive techniques, and less invasive methods would allow renal gluconeogenesis to be measured more routinely. Magnetic resonance spectroscopy and imaging of infused substrates bearing hyperpolarized carbon-13 spin labels allows metabolism to be detected within the body with excellent sensitivity. Conversion of hyperpolarized 1-13C pyruvate in the fasted rat liver is associated with gluconeogenic flux through phosphoenolpyruvate carboxykinase (PEPCK) rather than pyruvate dehydrogenase (PDH), and this study tested whether this was also the case in the kidney. The left kidney was scanned in fed and overnight-fasted rats either with or without prior treatment by the PEPCK inhibitor 3-mercaptopicolinic acid (3-MPA) following infusion of hyperpolarized 1-13C pyruvate. The 13C-bicarbonate signal normalized to the total metabolite signal was 3.2-fold lower in fasted rats (p = 0.00073) and was not significantly affected by 3-MPA treatment in either nutritional state. By contrast, the normalized [1-13C]aspartate signal was on average 2.2-fold higher in the fasted state (p = 0.038), and following 3-MPA treatment it was 2.8-fold lower in fed rats and 15-fold lower in fasted rats (p = 0.001). These results confirm that, unlike in the liver, most of the pyruvate-to-bicarbonate conversion in the fasted kidney results from PDH flux. The higher conversion to aspartate in fasted kidney and the marked drop following PEPCK inhibition demonstrate the potential of this metabolite as a marker of renal gluconeogenesis.

Hyperpolarized NMR study of the impact of pyruvate dehydrogenase kinase inhibition on the pyruvate dehydrogenase and TCA flux in type 2 diabetic rat muscle.

The role of pyruvate dehydrogenase in mediating lipid-induced insulin resistance stands as a central question in the pathogenesis of type 2 diabetes mellitus. Many researchers have invoked the Randle hypothesis to explain the reduced glucose disposal in skeletal muscle by envisioning an elevated acetyl CoA pool arising from increased oxidation of fatty acids. Over the years, in vivo NMR studies have challenged that monolithic view. The advent of the dissolution dynamic nuclear polarization NMR technique and a unique type 2 diabetic rat model provides an opportunity to clarify. Dynamic nuclear polarization enhances dramatically the NMR signal sensitivity and allows the measurement of metabolic kinetics in vivo. Diabetic muscle has much lower pyruvate dehydrogenase activity than control muscle, as evidenced in the conversion of [1-13C]lactate and [2-13C]pyruvate to HCO3- and acetyl carnitine. The pyruvate dehydrogenase kinase inhibitor, dichloroacetate, restores rapidly the diabetic pyruvate dehydrogenase activity to control level. However, diabetic muscle has a much larger dynamic change in pyruvate dehydrogenase flux than control. The dichloroacetate-induced surge in pyruvate dehydrogenase activity produces a differential amount of acetyl carnitine but does not affect the tricarboxylic acid flux. Further studies can now proceed with the dynamic nuclear polarization approach and a unique rat model to interrogate closely the biochemical mechanism interfacing oxidative metabolism with insulin resistance and metabolic inflexibility.

Hyperpolarized 13C MR Spectroscopy Depicts in Vivo Effect of Exercise on Pyruvate Metabolism in Human Skeletal Muscle.

Background Pyruvate dehydrogenase (PDH) and lactate dehydrogenase are essential for adenosine triphosphate production in skeletal muscle. At the onset of exercise, oxidation of glucose and glycogen is quickly enabled by dephosphorylation of PDH. However, direct measurement of PDH flux in exercising human muscle is daunting, and the net effect of covalent modification and other control mechanisms on PDH flux has not been assessed. Purpose To demonstrate the feasibility of assessing PDH activation and changes in pyruvate metabolism in human skeletal muscle after the onset of exercise using carbon 13 (13C) MRI with hyperpolarized (HP) [1-13C]-pyruvate. Materials and Methods For this prospective study, sedentary adults in good general health (mean age, 42 years ± 18 [standard deviation]; six men) were recruited from August 2019 to September 2020. Subgroups of the participants were injected with HP [1-13C]-pyruvate at resting, during plantar flexion exercise, or 5 minutes after exercise during recovery. In parallel, hydrogen 1 arterial spin labeling MRI was performed to estimate muscle tissue perfusion. An unpaired t test was used for comparing 13C data among the states. Results At rest, HP [1-13C]-lactate and [1-13C]-alanine were detected in calf muscle, but [13C]-bicarbonate was negligible. During moderate flexion-extension exercise, total HP 13C signals (tC) increased 2.8-fold because of increased muscle perfusion (P = .005), and HP [1-13C]-lactate-to-tC ratio increased 1.7-fold (P = .04). HP [13C]-bicarbonate-to-tC ratio increased 8.4-fold (P = .002) and returned to the resting level 5 minutes after exercise, whereas the lactate-to-tC ratio continued to increase to 2.3-fold as compared with resting (P = .008). Conclusion Lactate and bicarbonate production from hyperpolarized (HP) [1-carbon 13 {13C}]-pyruvate in skeletal muscle rapidly reflected the onset and the termination of exercise. These results demonstrate the feasibility of imaging skeletal muscle metabolism using HP [1-13C]-pyruvate MRI and the sensitivity of in vivo pyruvate metabolism to exercise states. © RSNA, 2021 Online supplemental material is available for this article.

335-OR: Lipid-Induced Insulin Resistance in the Renal Cortex Is Associated with Plasma Membrane Sn-1,2-diacylglycerol Accumulation and PKCe Translocation

While there is substantial evidence that the kidney is insulin responsive, it is unclear whether the organ becomes insulin resistant. Here, we investigate whether the kidney is susceptible to lipid-induced insulin resistance and, if so, whether insulin resistance may be mediated by increases in plasma membrane sn-1,2-diacylglycerol (DAG) content and activation of Protein Kinase Cε (PKCε). We studied 14 week old C57BL/6J mice fed either regular chow (RC) or a high fat diet (HFD) for three weeks. Infusion of [13C6]glucose under hyperinsulinemic-euglycemic clamp (HEC) conditions revealed HFD fed mice to have an ~40% reduction (P&amp;lt;0.01) in renal cortical mitochondrial pyruvate dehydrogenase flux relative to citrate synthase flux (VPDH/VCS) compared to RC mice. Next, we infused RC mice with a lipid emulsion (Intralipid® 20%) (RC+IL) during HEC. Despite matched plasma NEFAs at the clamp conclusion, renal cortical VPDH/VCS was significantly higher in RC+IL mice than in HFD mice (P&amp;lt;0.02). These findings suggest that reductions in insulin-stimulated mitochondrial VPDH/VCS observed in HFD mice are intrinsic to the kidney and cannot be attributed to alterations in fatty acid flux to the kidney. Consistent with our mitochondrial flux data, we found that feeding a HFD induced a defect in insulin signaling at the level of the insulin receptor (INSR) as evidenced by ~50% reductions in both insulin-stimulated INSRY1162 (P&amp;lt;0.05) and AKTS473 phosphorylation (P&amp;lt;0.001). HFD-induced reductions in insulin signaling were associated with an ~2-fold increase (P&amp;lt;0.01) in plasma membrane sn-1,2-DAG content, which was associated with an ~4-fold increase (P&amp;lt;0.05) in PKCε translocation. Conclusion: HFD feeding induces insulin resistance in the renal cortex at the level of INSRY1162 phosphorylation. This defect in INSR signaling is associated with reduced insulin-stimulated pyruvate oxidation and may be mediated by a sn-1,2-DAG-PKCε-INSR mechanism. Disclosure B. T. Hubbard: None. R. C. Gaspar: None. D. Zhang: None. M. Kahn: None. A. Nasiri: Employee; Spouse/Partner; Medtronic. X. Zhang: None. G. Cline: None. G. I. Shulman: Consultant; Self; 89bio, Inc., BridgeBio, Ionis Pharmaceuticals, Maze Therapeutics, Novo Nordisk, Other Relationship; Self; AstraZeneca, Esperion Therapeutics, Inc, Generian Pharmaceuticals, Inc., Gilead Sciences, Inc., iMetabolic Biopharma Corporation, Janssen Research &amp; Development, LLC, Merck &amp; Co., Inc., The Liver Company.

L-Carnitine Stimulates In Vivo Carbohydrate Metabolism in the Type 1 Diabetic Heart as Demonstrated by Hyperpolarized MRI.

The diabetic heart is energetically and metabolically abnormal, with increased fatty acid oxidation and decreased glucose oxidation. One factor contributing to the metabolic dysfunction in diabetes may be abnormal handling of acetyl and acyl groups by the mitochondria. L-carnitine is responsible for their transfer across the mitochondrial membrane, therefore, supplementation with L-carnitine may provide a route to improve the metabolic state of the diabetic heart. The primary aim of this study was to use hyperpolarized magnetic resonance imaging (MRI) to investigate the effects of L-carnitine supplementation on the in vivo metabolism of [1-13C]pyruvate in diabetes. Male Wistar rats were injected with either vehicle or streptozotocin (55 mg/kg) to induce type-1 diabetes. Three weeks of daily i.p. treatment with either saline or L-carnitine (3 g/kg/day) was subsequently undertaken. In vivo cardiac function and metabolism were assessed with CINE and hyperpolarized MRI, respectively. L-carnitine supplementation prevented the progression of hyperglycemia, which was observed in untreated streptozotocin injected animals and led to reductions in plasma triglyceride and ß-hydroxybutyrate concentrations. Hyperpolarized MRI revealed that L-carnitine treatment elevated pyruvate dehydrogenase flux by 3-fold in the diabetic animals, potentially through increased buffering of excess acetyl-CoA units in the mitochondria. Improved functional recovery following ischemia was also observed in the L-carnitine treated diabetic animals.

Hyperpolarized magnetic resonance shows that the anti-ischemic drug meldonium leads to increased flux through pyruvate dehydrogenase in vivo resulting in improved post-ischemic function in the diabetic heart.

The diabetic heart has a decreased ability to metabolize glucose. The anti‐ischemic drug meldonium may provide a route to counteract this by reducing l‐carnitine levels, resulting in improved cardiac glucose utilization. Therefore, the aim of this study was to use the novel technique of hyperpolarized magnetic resonance to investigate the in vivo effects of treatment with meldonium on cardiac metabolism and function in control and diabetic rats. Thirty‐six male Wistar rats were injected either with vehicle, or with streptozotocin (55 mg/kg) to induce a model of type 1 diabetes. Daily treatment with either saline or meldonium (100 mg/kg/day) was undertaken for three weeks. in vivo cardiac function and metabolism were assessed with CINE MRI and hyperpolarized magnetic resonance respectively. Isolated perfused hearts were challenged with low‐flow ischemia/reperfusion to assess the impact of meldonium on post‐ischemic recovery. Meldonium had no significant effect on blood glucose concentrations or on baseline cardiac function. However, hyperpolarized magnetic resonance revealed that meldonium treatment elevated pyruvate dehydrogenase flux by 3.1‐fold and 1.2‐fold in diabetic and control animals, respectively, suggesting an increase in cardiac glucose oxidation. Hyperpolarized magnetic resonance further demonstrated that meldonium reduced the normalized acetylcarnitine signal by 2.1‐fold in both diabetic and control animals. The increase in pyruvate dehydrogenase flux in vivo was accompanied by an improvement in post‐ischemic function ex vivo, as meldonium elevated the rate pressure product by 1.3‐fold and 1.5‐fold in the control and diabetic animals, respectively. In conclusion, meldonium improves in vivo pyruvate dehydrogenase flux in the diabetic heart, contributing to improved cardiac recovery after ischemia.

NLRX1 Deletion Increases Ischemia-Reperfusion Damage and Activates Glucose Metabolism in Mouse Heart.

BackgroundNOD-like receptors (NLR) are intracellular sensors of the innate immune system, with the NLRP3 being a pro-inflammatory member that modulates cardiac ischemia-reperfusion injury (IRI) and metabolism. No information is available on a possible role of anti-inflammatory NLRs on IRI and metabolism in the intact heart. Here we hypothesize that the constitutively expressed, anti-inflammatory mitochondrial NLRX1, affects IRI and metabolism of the isolated mouse heart.MethodsIsolated C57Bl/6J and NLRX1 knock-out (KO) mouse hearts were perfused with a physiological mixture of the essential substrates (lactate, glucose, pyruvate, fatty acid, glutamine) and insulin. For the IRI studies, hearts were subjected to either mild (20 min) or severe (35 min) ischemia and IRI was determined at 60 min reperfusion. Inflammatory mediators (IL-6, TNFα) and survival pathways (mito-HKII, p-Akt, p-AMPK, p-STAT3) were analyzed at 5 min of reperfusion. For the metabolism studies, hearts were perfused for 35 min with either 5.5 mM 13C-glucose or 0.4 mM 13C-palmitate under normoxic conditions, followed by LC-MS analysis and integrated, stepwise, mass-isotopomeric flux analysis (MIMOSA).ResultsNLRX1 KO significantly increased IRI (infarct size from 63% to 73%, end-diastolic pressure from 59 mmHg to 75 mmHg, and rate-pressure-product recovery from 15% to 6%), following severe, but not mild, ischemia. The increased IRI in NLRX1 KO hearts was associated with depressed Akt signaling at early reperfusion; other survival pathways or inflammatory parameters were not affected. Metabolically, NLRX1 KO hearts displayed increased lactate production and glucose oxidation relative to fatty acid oxidation, associated with increased pyruvate dehydrogenase flux and 10% higher cardiac oxygen consumption.ConclusionDeletion of the mitochondrially-located NOD-like sensor NLRX1 exacerbates severe cardiac IR injury, possibly through impaired Akt signaling, and increases cardiac glucose metabolism.

Imaging Acute Metabolic Changes in Patients with Mild Traumatic Brain Injury Using Hyperpolarized [1-13C]Pyruvate.

SummaryTraumatic brain injury (TBI) involves complex secondary injury processes following the primary injury. The secondary injury is often associated with rapid metabolic shifts and impaired brain function immediately after the initial tissue damage. Magnetic resonance spectroscopic imaging (MRSI) coupled with hyperpolarization of 13C-labeled substrates provides a unique opportunity to map the metabolic changes in the brain after traumatic injury in real-time without invasive procedures. In this report, we investigated two patients with acute mild TBI (Glasgow coma scale 15) but no anatomical brain injury or hemorrhage. Patients were imaged with hyperpolarized [1-13C]pyruvate MRSI 1 or 6 days after head trauma. Both patients showed significantly reduced bicarbonate (HCO3–) production, and one showed hyperintense lactate production at the injured sites. This study reports the feasibility of imaging altered metabolism using hyperpolarized pyruvate in patients with TBI, demonstrating the translatability and sensitivity of the technology to cerebral metabolic changes after mild TBI.

Nicotinic acid receptor agonists impair myocardial contractility by energy starvation.

Nicotinic acid receptor agonists have previously been shown to cause acute reductions in cardiac contractility. We sought to uncover the changes in cardiac metabolism underlying these alterations in function. In nine humans, we recorded cardiac energetics and function before and after a single oral dose of nicotinic acid using cardiac MRI to demonstrate contractile function and Phosphorus-31 (31 P) magnetic resonance spectroscopy to demonstrate myocardial energetics. Nicotinic Acid 400mg lowered ejection fraction by 4% (64±8% to 60±7%, P=.03), and was accompanied by a fall in phosphocreatine/ATP ratio by 0.4 (2.2±0.4 to 1.8±0.1, P=.04). In four groups of eight Wistar rats, we used pyruvate dehydrogenase (PDH) flux studies to demonstrate changes in carbohydrate metabolism induced by the nicotinic acid receptor agonist, Acipimox, using hyperpolarized Carbon-13 (13 C) magnetic resonance spectroscopy. In rats which had been starved overnight, Acipimox caused a fall in ejection fraction by 7.8% (67.5±8.9 to 60±3.1, P=.03) and a nearly threefold rise in flux through PDH (from 0.182±0.114 to 0.486±0.139, P=.002), though this rise did not match pyruvate dehydrogenase flux observed in rats fed carbohydrate rich chow (0.726±0.201). In fed rats, Acipimox decreased pyruvate dehydrogenase flux (to 0.512±0.13, P=.04). Concentration of plasma insulin fell by two-thirds in fed rats administered Acipimox (from 1695±891ng/L to 550±222ng/L, P=.005) in spite of glucose concentrations remaining the same. In conclusion, we demonstrate that nicotinic acid receptor agonists impair cardiac contractility associated with a decline in cardiac energetics and show that the mechanism is likely a combination of reduced fatty acid availability and a failure to upregulate carbohydrate metabolism, essentially starving the heart of fuel.

Assessment of hepatic pyruvate carboxylase activity using hyperpolarized [1-13 C]-l-lactate.

To evaluate the utility of hyperpolarized [1-13 C]-l-lactate to detect hepatic pyruvate carboxylase activity in vivo under fed and fasted conditions. [1-13 C]-labeled sodium L-lactate was polarized using a dynamic nuclear polarizer. Polarization level and the T1 were measured in vitro in a 3 Telsa MR scanner. Two groups of healthy rats (fasted vs. fed) were prepared for in vivo studies. Each rat was anesthetized and intravenously injected with 60-mM hyperpolarized [1-13 C]-l-lactate, immediately followed by dynamic acquisition of 13 C (carbon-13) MR spectra from the liver at 3 Tesla. The dosage-dependence of the 13 C-products was also investigated by performing another injection of an equal volume of 30-mM hyperpolarized [1-13 C]-l-lactate. T1 and liquid polarization level of [1-13 C]-l-lactate were estimated as 67.8 s and 40.0%, respectively. [1-13 C]pyruvate and [1-13 C]alanine, [13 C]bicarbonate ( ) and [1-13 C]aspartate were produced from hyperpolarized [1-13 C]-l-lactate in rat liver. Smaller and larger aspartate were measured in the fed group compared to the fasted group. Pyruvate and alanine production were increased in proportion to the lactate concentration, whereas the amount of and aspartate production was consistent between 30-mM and 60-mM lactate injections. This study demonstrates that a unique biomarker of pyruvate carboxylase flux, the appearance of [1-13 C]aspartate from [1-13 C]-l-lactate, is sensitive to nutritional state and may be monitored in vivo at 3 Tesla. Because [13 C] is largely produced by pyruvate dehydrogenase flux, these results suggest that the ratio of [1-13 C]aspartate and [13 C] (aspartate/ ) reflects the saturable pyruvate carboxylase/pyruvate dehydrogenase enzyme activities.

The Importance of the Fatty Acid Transporter L-Carnitine in Non-Alcoholic Fatty Liver Disease (NAFLD).

L-carnitine transports fatty acids into the mitochondria for oxidation and also buffers excess acetyl-CoA away from the mitochondria. Thus, L-carnitine may play a key role in maintaining liver function, by its effect on lipid metabolism. The importance of L-carnitine in liver health is supported by the observation that patients with primary carnitine deficiency (PCD) can present with fatty liver disease, which could be due to low levels of intrahepatic and serum levels of L-carnitine. Furthermore, studies suggest that supplementation with L-carnitine may reduce liver fat and the liver enzymes alanine aminotransferase (ALT) and aspartate transaminase (AST) in patients with Non-Alcoholic Fatty Liver Disease (NAFLD). L-carnitine has also been shown to improve insulin sensitivity and elevate pyruvate dehydrogenase (PDH) flux. Studies that show reduced intrahepatic fat and reduced liver enzymes after L-carnitine supplementation suggest that L-carnitine might be a promising supplement to improve or delay the progression of NAFLD.

Increasing carbohydrate oxidation improves contractile reserves and prevents hypertrophy in porcine right heart failure

In heart failure, myocardial overload causes vast metabolic changes that impair cardiac energy production and contribute to deterioration of contractile function. However, metabolic therapy is not used in heart failure care. We aimed to investigate the interplay between cardiac function and myocardial carbohydrate metabolism in a large animal heart failure model. Using magnetic resonance spectroscopy with hyperpolarized pyruvate and magnetic resonance imaging at rest and during pharmacological stress, we investigated the in-vivo cardiac pyruvate metabolism and contractility in a porcine model of chronic pulmonary insufficiency causing right ventricular volume overload. To assess if increasing the carbohydrate metabolic reserve improves the contractile reserve, a group of animals were fed dichloroacetate, an activator of pyruvate oxidation. Volume overload caused heart failure with decreased pyruvate dehydrogenase flux and poor ejection fraction reserve. The animals treated with dichloroacetate had a larger contractile response to dobutamine stress than non-treated animals. Further, dichloroacetate prevented myocardial hypertrophy. The in-vivo metabolic data were validated by mitochondrial respirometry, enzyme activity assays and gene expression analyses. Our results show that pyruvate dehydrogenase kinase inhibition improves the contractile reserve and decreases hypertrophy by augmenting carbohydrate metabolism in porcine heart failure. The approach is promising for metabolic heart failure therapy.

Abstract 797: Obesity-associated, but not obesity-independent, tumors respond to insulin by increasing mitochondrial glucose oxidation

Abstract Obesity is associated with increased incidence and worse prognosis of more than one dozen tumor types; however, the molecular mechanisms for this association are unknown. We hypothesized that insulin, which is elevated in obesity-driven insulin resistance, would increase tumor glucose oxidation in obesity-associated tumors. To test this hypothesis, we developed a stable isotope method in which tumor cells are incubated in physiological concentrations (5 mM) of [13C6] glucose and the ratio of pyruvate dehydrogenase flux to citrate synthase flux (VPDH/VCS, i.e. the percent of total mitochondrial oxidation fueled by glucose oxidation) is given as [4,5-13C2]glutamate/[13C3]alanine, measured by gas chromatography/mass spectrometry and liquid chromatography-tandem mass spectrometry respectively. Using this method, we found that both breast cancer (4T1) and colon cancer (MC38) cells, tumors that are associated with obesity, exhibit greater relative rates of glucose oxidation to total mitochondrial oxidation (VPDH/VCS 61±3% and 60±2%, respectively) than melanoma (YUMM) and Renca (renal cell carcinoma) cells (41±4% and 37±2%, respectively; P&amp;lt;0.05 for all comparisons between obesity-associated and non-obesity-associated tumors). In addition, both 4T1 and MC38 cells increased VPDH/VCS upon incubation in 100 nM insulin (75±3% and 81±2%, respectively, P&amp;lt;0.01 vs. the same tumor type without insulin), whereas there was no change in glucose oxidation with insulin in YUMM or Renca cells (35±3% and 36±2%, respectively). These data reveal that a shift in substrate preference may comprise a metabolic signature of obesity-associated tumors that differs from that of those not associated with obesity. In addition, contrary to the conventionally held view that glucose oxidation in tumor cells is constitutively high, these data reveal that obesity-driven tumors respond acutely to insulin by increasing mitochondrial glucose oxidation, whereas obesity-independent tumor cells do not. CONCLUSION: These data demonstrate that obesity associated tumors (breast and colon), in contrast to tumors that are not associated with obesity (melanoma, renal cell), are insulin-responsive and have higher rates of basal and insulin-stimulated mitochondrial glucose oxidation. Therefore assessment of in vitro tumor mitochondrial substrate preference may predict whether interventions to reverse hyperinsulinemia or reduce insulin-responsive glucose oxidation in tumors may be attractive therapeutic modalities in cancer types associated with obesity. Citation Format: Aviva Rabin-Court, Gerald I. Shulman, Rachel J. Perry. Obesity-associated, but not obesity-independent, tumors respond to insulin by increasing mitochondrial glucose oxidation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 797.