Publications by authors named "Craig R Malloy"

144 Publications

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

Radiology 2021 Jun 22:204500. Epub 2021 Jun 22.

From the Advanced Imaging Research Center (J.M.P., C.E.H., J.M., J.C., J.R., J.L., G.D.R., A.C., C.R.M.), Department of Radiology (J.M.P., A.C., C.R.M.), Department of Neurology and Neurotherapeutics (R.G.H.), and Department of Internal Medicine (C.R.M.), University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390-8568; Department of Electrical and Computer Engineering, University of Texas at Dallas, Dallas, Tex (J.M.P.); Department of Diagnostic Imaging and Radiology, Developing Brain Institute, Children's National Hospital, Washington, DC (Z.Z.); Department of Pediatrics and Radiology, George Washington University, Washington, DC (Z.Z.); GE Healthcare, Dallas, Tex (G.D.R.); Department of Biochemistry and Molecular Medicine, University of California, Davis, Davis, Calif (T.J.); and Veterans Affairs North Texas Healthcare System, Dallas, Tex (C.R.M.).

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 (C) MRI with hyperpolarized (HP) [1-C]-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-C]-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 test was used for comparing C data among the states. Results At rest, HP [1-C]-lactate and [1-C]-alanine were detected in calf muscle, but [C]-bicarbonate was negligible. During moderate flexion-extension exercise, total HP C signals (tC) increased 2.8-fold because of increased muscle perfusion ( = .005), and HP [1-C]-lactate-to-tC ratio increased 1.7-fold ( = .04). HP [C]-bicarbonate-to-tC ratio increased 8.4-fold ( = .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 ( = .008). Conclusion Lactate and bicarbonate production from hyperpolarized (HP) [1-carbon 13 {C}]-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-C]-pyruvate MRI and the sensitivity of in vivo pyruvate metabolism to exercise states. © RSNA, 2021
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http://dx.doi.org/10.1148/radiol.2021204500DOI Listing
June 2021

Analysis of steady-state carbon tracer experiments using akaike information criteria.

Metabolomics 2021 Jun 19;17(7):61. Epub 2021 Jun 19.

Advanced Imaging Research Center, University of Texas Southwestern Medical Center, Dallas, TX, USA.

Introduction: Carbon isotope tracers have been used to determine relative rates of tricarboxylic acid cycle (TCA) cycle pathways since the 1950s. Steady-state experimental data are typically fit to a single mathematical model of metabolism to determine metabolic fluxes. Whether the chosen model is appropriate for the biological system has generally not been evaluated systematically. An overly-simple model omits known pathways while an overly-complex model may produce incorrect results due to overfitting.

Objectives: The objectives were to develop and study a method that systematically evaluates multiple TCA cycle mathematical models as part of the fitting process.

Methods: The problem of choosing overly-simple or overly-complex models was approached by developing software that automatically explores all possible combinations of flux through pyruvate dehydrogenase, pyruvate kinase, pyruvate carboxylase and anaplerosis at propionyl-CoA carboxylase, and equivalent pathways, all relative to TCA cycle flux. Typical TCA cycle metabolic tracer experiments that use C nuclear magnetic resonance for detection and quantification of C-enriched glutamate products were simulated and analyzed. By evaluating the multiple model fits with both the conventional sum-of-squares residual error (SSRE) and the Akaike Information Criterion (AIC), the software helps the investigator understand the interaction between model complexity and goodness of fit.

Results: When fitting alternative models of the TCA cycle metabolism, the SSRE may identify more than one model that fits the data well. Among those models, the AIC provides guidance as to which is the simplest of the candidate models is sufficient to describe the observed data. However under some conditions, AIC used alone inappropriately discriminates against necessary metabolic complexity.

Conclusion: In combination, the SSRE and AIC help the investigator identify the model that best describes the metabolism of a biological system.
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http://dx.doi.org/10.1007/s11306-021-01807-1DOI Listing
June 2021

PKM1 Exerts Critical Roles in Cardiac Remodeling Under Pressure Overload in the Heart.

Circulation 2021 Jun 9. Epub 2021 Jun 9.

Division of Cardiology, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX.

Metabolic remodeling precedes most alterations during cardiac hypertrophic growth under hemodynamic stress. The elevation of glucose utilization has been recognized as a hallmark of metabolic remodeling. However, its role in cardiac hypertrophic growth and heart failure in response to pressure overload remains to be fully illustrated. Here, we aimed to dissect the role of cardiac PKM1 (pyruvate kinase muscle isozyme 1) in glucose metabolic regulation and cardiac response under pressure overload. Cardiac specific deletion of PKM1 was achieved by crossing the floxed PKM1 mouse model with the cardiomyocyte-specific Cre transgenic mouse. PKM1 transgenic mice were generated under the control of tetracycline response elements, and cardiac specific overexpression of PKM1 was induced by doxycycline administration in adult mice. Pressure overload was triggered by transverse aortic constriction (TAC). Primary neonatal rat ventricular myocytes were used to dissect molecular mechanisms. Moreover, metabolomics and NMR spectroscopy analyses were conducted to determine cardiac metabolic flux in response to pressure overload. We found that PKM1 expression is reduced in failing human and mouse hearts. Importantly, cardiomyocyte-specific deletion of PKM1 exacerbates cardiac dysfunction and fibrosis in response to pressure overload. Inducible overexpression of PKM1 in cardiomyocytes protects the heart against TAC-induced cardiomyopathy and heart failure. At the mechanistic level, PKM1 is required for the augmentation of glycolytic flux, mitochondrial respiration, and ATP production under pressure overload. Furthermore, deficiency of PKM1 causes a defect in cardiomyocyte growth and a decrease in pyruvate dehydrogenase complex activity at both and levels. These findings suggest that PKM1 plays an essential role in maintaining a homeostatic response in the heart under hemodynamic stress.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.121.054885DOI Listing
June 2021

Spectral fitting strategy to overcome the overlap between 2-hydroxyglutarate and lipid resonances at 2.25 ppm.

Magn Reson Med 2021 10 12;86(4):1818-1828. Epub 2021 May 12.

Advanced Imaging Research Center, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

Purpose: H MRS provides a noninvasive tool for identifying mutations in isocitrate dehydrogenase (IDH). Quantification of the prominent 2-hydroxyglutarate (2HG) resonance at 2.25 ppm is often confounded by the lipid resonance at the same frequency in tumors with elevated lipids. We propose a new spectral fitting approach to separate these overlapped signals, therefore, improving 2HG evaluation.

Methods: TE 97 ms PRESS was acquired at 3T from 42 glioma patients. New lipid basis sets were created, in which the small lipid 2.25-ppm signal strength was preset with reference to the lipid signal at 0.9 ppm, incorporating published fat relaxation data. LCModel fitting using the new lipid bases (Fitting method 2) was conducted along with fitting using the LCModel built-in lipid basis set (Fitting method 1), in which the lipid 2.25-ppm signal is assessed with reference to the lipid 1.3-ppm signal. In-house basis spectra of low-molecular-weight metabolites were used in both fitting methods.

Results: Fitting method 2 showed marked improvement in identifying IDH mutational status compared with Fitting method 1. 2HG estimates from Fitting method 2 were overall smaller than those from Fitting method 1, which was because of differential assignment of the signal at 2.25 ppm to lipids. In receiver operating characteristic analysis, Fitting method 2 provided a complete distinction between IDH mutation and wild-type whereas Fitting method 1 did not.

Conclusion: The data suggest that H MR spectral fitting using the new lipid basis set provides a robust fitting strategy that improves 2HG evaluation in brain tumors with elevated lipids.
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http://dx.doi.org/10.1002/mrm.28829DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8295210PMC
October 2021

C NMR of glutamate for monitoring the pentose phosphate pathway in myocardium.

NMR Biomed 2021 Jul 26;34(7):e4533. Epub 2021 Apr 26.

Advanced Imaging Research Center, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

After administration of C-labeled glucose, the activity of the pentose phosphate pathway (PPP) is often assessed by the distribution of C in lactate. However, in some tissues, such as the well-oxygenated heart, the concentration of lactate may be too low for convenient analysis by NMR. Here, we examined C-labeled glutamate as an alternative biomarker of the PPP in the heart. Isolated rat hearts were perfused with media containing [2,3- C ]glucose and the tissue extracts were analyzed. Metabolism of [2,3- C ]glucose yields [1,2- C ]pyruvate via glycolysis and [2,3- C ]pyruvate via the PPP. Pyruvate is in exchange with lactate or is further metabolized to glutamate through pyruvate dehydrogenase and the TCA cycle. A doublet from [4,5- C ]glutamate, indicating flux through the PPP, was readily detected in C NMR of heart extracts even when the corresponding doublet from [2,3- C ]lactate was minimal. Benfotiamine, known to induce the PPP, caused an increase in production of [4,5- C ]glutamate. In rats receiving [2,3- C ]glucose, brain extracts showed well-resolved signals from both [2,3- C ]lactate and [4,5- C ]glutamate in C NMR spectra. Assessment of the PPP in the brain based on glutamate had a strong linear correlation with lactate-based assessment. In summary, C NMR analysis of glutamate enabled detection of the low PPP activity in isolated hearts. This analyte is an alternative to lactate for monitoring the PPP with the use of [2,3- C ]glucose.
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http://dx.doi.org/10.1002/nbm.4533DOI Listing
July 2021

Cardiac measurement of hyperpolarized C metabolites using metabolite-selective multi-echo spiral imaging.

Magn Reson Med 2021 09 6;86(3):1494-1504. Epub 2021 Apr 6.

Advanced Imaging Research Center, The University of Texas Southwestern Medical Center, Dallas, Texas, USA.

Purpose: Noninvasive imaging with hyperpolarized (HP) pyruvate can capture in vivo cardiac metabolism. For proper quantification of the metabolites and optimization of imaging parameters, understanding MR characteristics such as s of the HP signals is critical. This study is to measure in vivo cardiac s of HP [1- C]pyruvate and the products in rodents and humans.

Methods: A dynamic C multi-echo spiral imaging sequence that acquires [ C]bicarbonate, [1- C]lactate, and [1- C]pyruvate images in an interleaved manner was implemented for a clinical 3 Tesla system. of each metabolite was calculated from the multi-echo images by fitting the signal decay of each region of interest mono-exponentially. The performance of measuring using the sequence was first validated using a C phantom and then with rodents following a bolus injection of HP [1- C]pyruvate. In humans, of each metabolite was calculated for left ventricle, right ventricle, and myocardium.

Results: Cardiac s of HP [1- C]pyruvate, [1- C]lactate, and [ C]bicarbonate in rodents were measured as 24.9 ± 5.0, 16.4 ± 4.7, and 16.9 ± 3.4 ms, respectively. In humans, of [1- C]pyruvate was 108.7 ± 22.6 ms in left ventricle and 129.4 ± 8.9 ms in right ventricle. of [1- C]lactate was 40.9 ± 8.3, 44.2 ± 5.5, and 43.7 ± 9.0 ms in left ventricle, right ventricle, and myocardium, respectively. of [ C]bicarbonate in myocardium was 64.4 ± 2.5 ms. The measurements were reproducible and consistent over time after the pyruvate injection.

Conclusion: The proposed metabolite-selective multi-echo spiral imaging sequence reliably measures in vivo cardiac s of HP [1- C]pyruvate and products.
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http://dx.doi.org/10.1002/mrm.28796DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8212421PMC
September 2021

P-MRS of the healthy human brain at 7 T detects multiple hexose derivatives of uridine diphosphate glucose.

NMR Biomed 2021 Jul 27;34(7):e4511. Epub 2021 Mar 27.

Advanced Imaging Research Center, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

Nucleotide sugars are required for the synthesis of glycoproteins and glycolipids, which play crucial roles in many cellular functions such as cell communication and immune responses. Uridine diphosphate-glucose (UDP-Glc) was previously believed to be the only nucleotide sugar detectable in brain by P-MRS. Using spectra of high SNR and high resolution acquired at 7 T, we showed that multiple nucleotide sugars are coexistent in brain and can be measured simultaneously. In addition to UDP-Glc, these also include UDP-galactose (UDP-Gal), -N-acetyl-glucosamine (UDP-GlcNAc) and -N-acetyl-galactosamine (UDP-GalNAc), collectively denoted as UDP(G). Coexistence of these UDP(G) species is evident from a quartet-like multiplet at -9.8 ppm (M ), which is a common feature seen across a wide age range (24-64 years). Lineshape fitting of M allows an evaluation of all four UDP(G) components, which further aids in analysis of a mixed signal at -8.2 ppm (M ) for deconvolution of NAD and NADH. For a group of seven young healthy volunteers, the concentrations of UDP(G) species were 0.04 ± 0.01 mM for UDP-Gal, 0.07 ± 0.03 mM for UDP-Glc, 0.06 ± 0.02 mM for UDP-GalNAc and 0.08 ± 0.03 mM for UDP-GlcNA, in reference to ATP (2.8 mM). The combined concentration of all UDP(G) species (average 0.26 ± 0.06 mM) was similar to the pooled concentration of NAD and NADH (average 0.27 ± 0.06 mM, with a NAD /NADH ratio of 6.7 ± 2.1), but slightly lower than previously found in an older cohort (0.31 mM). The in vivo NMR analysis of UDP-sugar composition is consistent with those from tissue extracts by other modalities in the literature. Given that glycosylation is dependent on the availability of nucleotide sugars, assaying multiple nucleotide sugars may provide valuable insights into potential aberrant glycosylation, which has been implicated in certain diseases such as cancer and Alzheimer's disease.
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http://dx.doi.org/10.1002/nbm.4511DOI Listing
July 2021

The presence of 3-hydroxypropionate and 1,3-propanediol suggests an alternative path for conversion of glycerol to Acetyl-CoA.

Metabol Open 2021 Mar 26;9:100086. Epub 2021 Feb 26.

Advanced Imaging Research Center, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA.

Background: In our recent study using [U-C]glycerol, a small subset of hamsters showed an unusual profile of glycerol metabolism: negligible gluconeogenesis from glycerol plus conversion of glycerol to 1,3-propanediol (1,3PDO) and 3-hydroxypropionate (3HP) which were detected in the liver and blood. The purpose of the current study is to evaluate the association of these unusual glycerol products with other biochemical processes in the liver.

Methods: Fasted hamsters received acetaminophen (400 mg/kg; n = 16) or saline (n = 10) intraperitoneally. After waiting 2 h, all the animals received [U-C]glycerol intraperitoneally. Liver and blood were harvested 1 h after the glycerol injection for NMR analysis and gene expression assays.

Results: 1,3PDO and 3HP derived from [U-C]glycerol were detected in the liver and plasma of eight hamsters (two controls and six hamsters with acetaminophen treatment). Glycerol metabolism in the liver of these animals differed substantially from conventional metabolic pathways. [U-C]glycerol was metabolized to acetyl-CoA as evidenced with downstream products detected in glutamate and β-hydroxybutyrate, yet C labeling in pyruvate and glucose was minimal (p < 0.001, C labeling difference in each metabolite). Expression of aldehyde dehydrogenases was enhanced in hamster livers with 1,3PDO and 3HP (p < 0.05).

Conclusion: Detection of 1,3PDO and 3HP in the hamster liver was associated with unorthodox metabolism of glycerol characterized by conversion of 3HP to acetyl-CoA followed by ketogenesis and oxidative metabolism through the TCA cycle. Additional mechanistic studies are needed to determine the causes of unusual glycerol metabolism in a subset of these hamsters.
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http://dx.doi.org/10.1016/j.metop.2021.100086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7940983PMC
March 2021

Characterization and compensation of inhomogeneity artifact in spiral hyperpolarized C imaging of the human heart.

Magn Reson Med 2021 07 5;86(1):157-166. Epub 2021 Feb 5.

Advanced Imaging Research Center, University of Texas Southwestern Medical Center, Dallas, TX, USA.

Purpose: This study aimed to investigate the role of regional inhomogeneity in spiral hyperpolarized C image quality and to develop measures to alleviate these effects.

Methods: Field map correction of hyperpolarized C cardiac imaging using spiral readouts was evaluated in healthy subjects. Spiral readouts with differing duration (26 and 45 ms) but similar resolution were compared with respect to off-resonance performance and image quality. An map-based image correction based on the multifrequency interpolation (MFI) method was implemented and compared to correction using a global frequency shift alone. Estimation of an unknown frequency shift was performed by maximizing a sharpness objective based on the Sobel variance. The apparent full width half at maximum (FWHM) of the myocardial wall on [ C]bicarbonate was used to estimate blur.

Results: Mean myocardial wall FWHM measurements were unchanged with the short readout pre-correction (14.1 ± 2.9 mm) and post-MFI correction (14.1 ± 3.4 mm), but significantly decreased in the long waveform (20.6 ± 6.6 mm uncorrected, 17.7 ± 7.0 corrected, P = .007). Bicarbonate signal-to-noise ratio (SNR) of the images acquired with the long waveform were increased by 1.4 ± 0.3 compared to those acquired with the short waveform (predicted 1.32). Improvement of image quality was observed for all metabolites with correction.

Conclusions: -map correction reduced blur and recovered signal from dropouts, particularly along the posterior myocardial wall. The low image SNR of [ C]bicarbonate can be compensated with longer duration readouts but at the expense of increased artifacts, which can be partially corrected for with the proposed methods.
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http://dx.doi.org/10.1002/mrm.28691DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8049085PMC
July 2021

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

iScience 2020 Dec 30;23(12):101885. Epub 2020 Nov 30.

Advanced Imaging Research Center, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

Traumatic 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 C-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-C]pyruvate MRSI 1 or 6 days after head trauma. Both patients showed significantly reduced bicarbonate (HCO ) 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.
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http://dx.doi.org/10.1016/j.isci.2020.101885DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7736977PMC
December 2020

N-carnitine, a novel endogenous hyperpolarized MRI probe with long signal lifetime.

Magn Reson Med 2021 04 12;85(4):1814-1820. Epub 2020 Nov 12.

Department of Biochemistry, University of Florida, Gainesville, Florida, USA.

Purpose: The purpose of this study was to investigate hyperpolarization and in vivo imaging of [ N]carnitine, a novel endogenous MRI probe with long signal lifetime.

Methods: L-[ N]carnitine-d was hyperpolarized by the method of dynamic nuclear polarization followed by rapid dissolution. The T signal lifetimes were estimated in aqueous solution and in vivo following intravenous injection in rats, using a custom-built dual-tuned N/ H RF coil at 4.7 T. N chemical shift imaging and N fast spin-echo images of rat abdomen were acquired 3 minutes after [ N]carnitine injection.

Results: Estimated T times of [ N]carnitine at 4.7 T were 210 seconds (in H O) and 160 seconds (in vivo), with an estimated polarization level of 10%. Remarkably, the [ N]carnitine coherence was detectable in rat abdomen for 5 minutes after injection for the nonlocalized acquisition. No downstream metabolites were detected on localized or nonlocalized N spectra. Diffuse liver enhancement was detected on N fast spin-echo imaging 3 minutes after injection, with mean hepatic SNR of 18 ± 5 at a spatial resolution of 4 × 4 mm.

Conclusion: This study showed the feasibility of hyperpolarizing and imaging the biodistribution of HP [ N]carnitine.
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http://dx.doi.org/10.1002/mrm.28578DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7856872PMC
April 2021

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

Magn Reson Med 2021 03 16;85(3):1175-1182. Epub 2020 Sep 16.

Advanced Imaging Research Center, The University of Texas Southwestern Medical Center, Dallas, Texas, USA.

Purpose: To evaluate the utility of hyperpolarized [1- C]-l-lactate to detect hepatic pyruvate carboxylase activity in vivo under fed and fasted conditions.

Methods: [1- C]-labeled sodium L-lactate was polarized using a dynamic nuclear polarizer. Polarization level and the T 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- C]-l-lactate, immediately followed by dynamic acquisition of C (carbon-13) MR spectra from the liver at 3 Tesla. The dosage-dependence of the C-products was also investigated by performing another injection of an equal volume of 30-mM hyperpolarized [1- C]-l-lactate.

Results: T and liquid polarization level of [1- C]-l-lactate were estimated as 67.8 s and 40.0%, respectively. [1- C]pyruvate and [1- C]alanine, [ C]bicarbonate ( ) and [1- C]aspartate were produced from hyperpolarized [1- 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.

Conclusion: This study demonstrates that a unique biomarker of pyruvate carboxylase flux, the appearance of [1- C]aspartate from [1- C]-l-lactate, is sensitive to nutritional state and may be monitored in vivo at 3 Tesla. Because [ C] is largely produced by pyruvate dehydrogenase flux, these results suggest that the ratio of [1- C]aspartate and [ C] (aspartate/ ) reflects the saturable pyruvate carboxylase/pyruvate dehydrogenase enzyme activities.
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http://dx.doi.org/10.1002/mrm.28489DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7718288PMC
March 2021

Lactate Dehydrogenase A Governs Cardiac Hypertrophic Growth in Response to Hemodynamic Stress.

Cell Rep 2020 09;32(9):108087

Division of Cardiology, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA. Electronic address:

The heart manifests hypertrophic growth in response to high blood pressure, which may decompensate and progress to heart failure under persistent stress. Metabolic remodeling is an early event in this process. However, its role remains to be fully characterized. Here, we show that lactate dehydrogenase A (LDHA), a critical glycolytic enzyme, is elevated in the heart in response to hemodynamic stress. Cardiomyocyte-restricted deletion of LDHA leads to defective cardiac hypertrophic growth and heart failure by pressure overload. Silencing of LDHA in cultured cardiomyocytes suppresses cell growth from pro-hypertrophic stimulation in vitro, while overexpression of LDHA is sufficient to drive cardiomyocyte growth. Furthermore, we find that lactate is capable of rescuing the growth defect from LDHA knockdown. Mechanistically, lactate stabilizes NDRG3 (N-myc downregulated gene family 3) and stimulates ERK (extracellular signal-regulated kinase). Our results together suggest that the LDHA/NDRG3 axis may play a critical role in adaptive cardiomyocyte growth in response to hemodynamic stress.
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http://dx.doi.org/10.1016/j.celrep.2020.108087DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7520916PMC
September 2020

Divergent effects of glutathione depletion on isocitrate dehydrogenase 1 and the pentose phosphate pathway in hamster liver.

Physiol Rep 2020 08;8(16):e14554

Advanced Imaging Research Center, University of Texas Southwestern Medical Center, Dallas, TX, USA.

The liver regenerates NADPH via multiple pathways to maintain redox balance and reductive biosynthesis. The pentose phosphate pathway (PPP) contributes to hepatic lipogenesis by supplying NADPH, and it is thought to play a major role in response to oxidative stress. This study determined the significance of the PPP and related NADPH-regenerating enzymes in the liver under oxidative stress. Fasted hamsters received acetaminophen (400 mg/kg) to deplete glutathione in the liver and [U- C ]glycerol to measure the PPP activity by analysis of C distribution in plasma glucose. Blood and liver were harvested to assess NADPH-producing enzymes, antioxidant defense, PPP, and other relevant biochemical processes. Acetaminophen caused glutathione depletion and decreased activities of glutathione peroxidase and catalase in the liver, but it did not change triglyceride synthesis. Although the PPP is potentially an abundant source of NADPH, its activity was decreased and the expression of glucose 6-phosphate dehydrogenase remained unchanged after acetaminophen treatment. The effects of acetaminophen on other NADPH-producing enzymes were complex. Isocitrate dehydrogenase 1 was overexpressed, both isocitrate dehydrogenase 2 and malic enzyme 1 were underexpressed, and methylenetetrahydrofolate dehydrogenase 1 remained unchanged. In summary, isocitrate dehydrogenase 1 was most sensitive to glutathione depletion caused by acetaminophen, but glucose 6-phosphate dehydrogenase, the regulatory enzyme of PPP, was not.
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http://dx.doi.org/10.14814/phy2.14554DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7435027PMC
August 2020

A Frequency Translation System for Multi-Channel, Multi-Nuclear MR Spectroscopy.

IEEE Trans Biomed Eng 2021 01 21;68(1):109-118. Epub 2020 Dec 21.

Objective: Most MRI scanners are equipped to receive signals from H array coils but few support multi-channel reception for other nuclei. Using receive arrays can provide significant SNR benefits, usually exploited to enable accelerated imaging, but the extension of these arrays to non-H nuclei has received less attention because of the relative lack of broadband array receivers. Non-H nuclei often have low sensitivity and stand to benefit greatly from the increase in SNR that arrays can provide. This paper presents a cost-effective approach for adapting standard H multi-channel array receivers for use with other nuclei - in this case, C.

Methods: A frequency translation system has been developed that uses active mixers residing at the magnet bore to convert the received signal from a non-H array to the H frequency for reception by the host system receiver.

Results: This system has been demonstrated at 4.7T and 7T while preserving SNR and isolation. H decoupling, particularly important for C detection, can be straightforwardly accommodated.

Conclusion: Frequency translation can convert H-only multi-channel receivers for use with other nuclei while maintaining SNR and channel isolation while still enabling H decoupling.

Significance: This work allows existing multi-channel MRI receivers to be adapted to receive signals from nuclei other than H, allowing for the use of receive arrays for in vivo multi-nuclear NMR.
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http://dx.doi.org/10.1109/TBME.2020.2997770DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7872304PMC
January 2021

Mitochondrial Substrate Utilization Regulates Cardiomyocyte Cell Cycle Progression.

Nat Metab 2020 02 20;2(2):167-178. Epub 2020 Feb 20.

Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

The neonatal mammalian heart is capable of regeneration for a brief window of time after birth. However, this regenerative capacity is lost within the first week of life, which coincides with a postnatal shift from anaerobic glycolysis to mitochondrial oxidative phosphorylation, particularly towards fatty-acid utilization. Despite the energy advantage of fatty-acid beta-oxidation, cardiac mitochondria produce elevated rates of reactive oxygen species when utilizing fatty acids, which is thought to play a role in cardiomyocyte cell-cycle arrest through induction of DNA damage and activation of DNA-damage response (DDR) pathway. Here we show that inhibiting fatty-acid utilization promotes cardiomyocyte proliferation in the postnatatal heart. First, neonatal mice fed fatty-acid deficient milk showed prolongation of the postnatal cardiomyocyte proliferative window, however cell cycle arrest eventually ensued. Next, we generated a tamoxifen-inducible cardiomyocyte-specific, pyruvate dehydrogenase kinase 4 (PDK4) knockout mouse model to selectively enhance oxidation of glycolytically derived pyruvate in cardiomyocytes. Conditional PDK4 deletion resulted in an increase in pyruvate dehydrogenase activity and consequently an increase in glucose relative to fatty-acid oxidation. Loss of PDK4 also resulted in decreased cardiomyocyte size, decreased DNA damage and expression of DDR markers and an increase in cardiomyocyte proliferation. Following myocardial infarction, inducible deletion of PDK4 improved left ventricular function and decreased remodelling. Collectively, inhibition of fatty-acid utilization in cardiomyocytes promotes proliferation, and may be a viable target for cardiac regenerative therapies.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7331943PMC
February 2020

Effects of Empagliflozin Treatment on Glycerol-Derived Hepatic Gluconeogenesis in Adults with Obesity: A Randomized Clinical Trial.

Obesity (Silver Spring) 2020 07;28(7):1254-1262

Department of Internal Medicine, University of Texas Medical Center, Dallas, Texas, USA.

Objective: The aim of this study was to determine the effects of empagliflozin on glycerol-derived hepatic gluconeogenesis in adults with obesity without type 2 diabetes mellitus (T2DM) using oral carbon 13 ( C)-labeled glycerol.

Methods: A randomized, double-blind, placebo-controlled trial was performed in participants with magnetic resonance imaging assessment of body fat and measurement of glycerol-derived C enrichment in plasma glucose by nuclear magnetic resonance spectroscopy following ingestion of [U- C ]glycerol. Participants were randomized to oral empagliflozin 10 mg once daily or placebo for 3 months. Glycerol-derived C enrichment studies were repeated, and treatment differences in the mean percentage of C glycerol enrichment in glucose were compared using mixed linear models.

Results: Thirty-five participants completed the study. Empagliflozin increased glycerol-derived C enrichment between baseline and follow-up by 6.5% (P = 0.005), consistent with less glycerol from visceral adipose tissue (VAT). No difference was found with placebo. Glycerol-derived C enrichment was lower in participants with high VAT compared with low VAT by 12.6% (P = 0.04), but there was no heterogeneity of the treatment effect by baseline VAT. Glycerol-derived C enrichment was inversely correlated with VAT but was not correlated with weight loss.

Conclusions: VAT is associated with endogenous glycerol-derived hepatic gluconeogenesis, and empagliflozin reduces endogenous glycerol gluconeogenesis in adults with obesity without T2DM. These findings suggest a mechanism by which sodium-glucose cotransporter 2 inhibitors may prevent T2DM in obesity.
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http://dx.doi.org/10.1002/oby.22854DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7316140PMC
July 2020

Quantitative measurement of redox state in human brain by P MRS at 7T with spectral simplification and inclusion of multiple nucleotide sugar components in data analysis.

Magn Reson Med 2020 11 9;84(5):2338-2351. Epub 2020 May 9.

Advanced Imaging Research Center, University of Texas Southwestern Medical Center, Dallas, TX, USA.

Purpose: To develop a simplified method for quantitative measurement of NAD /NADH (nicotinamide adenine dinucleotides) levels in human brain by P MRS without interference from the α-ATP signal and with inclusion of multiple UDP-sugar components.

Methods: Simple pulse-acquire P MR spectra were collected at 7T with and without a frequency-selective inversion pulse to remove the dominant α-ATP signal from the underlying NAD(H) signal. Careful inspection of the P signal at -9.8 ppm previously assigned to UDP-glucose revealed multiple UDP-sugar components that must also be considered when deconvoluting the NAD(H) signal to quantify NAD and NADH. Finally, the overlapping NAD(H) and UDP(G) resonances were deconvoluted into individual components using Voigt lineshape analysis and UDP(G) modeling.

Results: The inversion-based spectral editing method enabled clean separation of the NAD(H) signal from the otherwise dominant α-ATP signal. In addition, the upfield signal near -9.8 ppm appears more "quartet-like" than a simple doublet consistent with contributions from other nucleotide sugars such as UDP-galactose, UDP-N-acetyl-galactosamine, and UDP-N-acetyl-glucosamine in addition to UDP-glucose. Deconvolution of the combined NAD(H) and UDP(G) signals showed that the measured NAD /NAD ratio was heavily influenced by UDP(G) modeling (7.5 ± 1.8 when the UDP(G) signal was fitted as multiple doublets versus 5.3 ± 0.6 when a simplified pseudo doublet model was used). In a test/re-test experiments separated by 2 weeks, consistent NAD /NADH ratios were measured in the brain of seven human subjects.

Conclusions: The NAD /NADH ratio in human brain can be measured using P MR spectra simplified by spectral editing and with inclusion of multiple UDP-sugar components in the data analysis.
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http://dx.doi.org/10.1002/mrm.28306DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7396304PMC
November 2020

Energetic Adaptations and Stress Reserve in the Obese Heart.

Circulation 2020 04 6;141(14):1164-1167. Epub 2020 Apr 6.

Department of Internal Medicine and Advanced Imaging Research Center, University of Texas Southwestern, Dallas, TX (C.R.M.).

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http://dx.doi.org/10.1161/CIRCULATIONAHA.120.045259DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7147721PMC
April 2020

Assessment of Rapid Hepatic Glycogen Synthesis in Humans Using Dynamic C Magnetic Resonance Spectroscopy.

Hepatol Commun 2020 Mar 4;4(3):425-433. Epub 2020 Jan 4.

Advanced Imaging Research Center University of Texas Southwestern Medical Center Dallas TX.

Carbon-13 magnetic resonance spectroscopy (MRS) following oral intake of C-labeled glucose is the gold standard for imaging glycogen metabolism in humans. However, the temporal resolution of previous studies has been >13 minutes. Here, we describe a high-sensitivity C MRS method for imaging hepatic glycogen synthesis with a temporal resolution of 1 minute or less. Nuclear magnetic resonance spectra were acquired from the liver of 3 healthy volunteers, using a C clamshell radiofrequency transmit and paddle-shaped array receive coils in a 3 Tesla magnetic resonance imaging system. Following a 15-minute baseline C MRS scan of the liver, [1-C]-glucose was ingested and C MRS data were acquired for an additional 1-3 hours. Dynamic change of the hepatic glycogen synthesis level was analyzed by reconstructing the acquired MRS data with temporal resolutions of 30 seconds to 15 minutes. Plasma levels of C-labeled glucose and lactate were measured using gas chromatography-mass spectrometry. While not detected at baseline C MRS, [1-C]-labeled α-glucose and β-glucose and glycogen peaks accumulated rapidly, beginning as early as ~2 minutes after oral administration of [1-C]-glucose. The [1-C]-glucose signals peaked at ~5 minutes, whereas [1-C]-glycogen peaked at ~25 minutes after [1-C]-glucose ingestion; both signals declined toward baseline levels over the next 1-3 hours. Plasma levels of C-glucose and C-lactate rose gradually, and approximately 20% of all plasma glucose and 5% of plasma lactate were C-labeled by 2 hours after ingestion. We observed rapid accumulation of hepatic [1-C]-glycogen following orally administered [1-C]-glucose, using a dynamic C MRS method with a temporal resolution of 1 minute or less. Commercially available technology allows high temporal resolution studies of glycogen metabolism in the human liver.
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http://dx.doi.org/10.1002/hep4.1458DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7049683PMC
March 2020

Glycine by MR spectroscopy is an imaging biomarker of glioma aggressiveness.

Neuro Oncol 2020 07;22(7):1018-1029

Advanced Imaging Research Center, University of Texas Southwestern Medical Center, Dallas, Texas.

Background: High-grade gliomas likely remodel the metabolic machinery to meet the increased demands for amino acids and nucleotides during rapid cell proliferation. Glycine, a non-essential amino acid and intermediate of nucleotide biosynthesis, may increase with proliferation. Non-invasive measurement of glycine by magnetic resonance spectroscopy (MRS) was evaluated as an imaging biomarker for assessment of tumor aggressiveness.

Methods: We measured glycine, 2-hydroxyglutarate (2HG), and other tumor-related metabolites in 35 glioma patients using an MRS sequence tailored for co-detection of glycine and 2HG in gadolinium-enhancing and non-enhancing tumor regions on 3T MRI. Glycine and 2HG concentrations as measured by MRS were correlated with tumor cell proliferation (MIB-1 labeling index), expression of mitochondrial serine hydroxymethyltransferase (SHMT2), and glycine decarboxylase (GLDC) enzymes, and patient overall survival.

Results: Elevated glycine was strongly associated with presence of gadolinium enhancement, indicating more rapidly proliferative disease. Glycine concentration was positively correlated with MIB-1, and levels higher than 2.5 mM showed significant association with shorter patient survival, irrespective of isocitrate dehydrogenase status. Concentration of 2HG did not correlate with MIB-1 index. A high glycine/2HG concentration ratio, >2.5, was strongly associated with shorter survival (P < 0.0001). GLDC and SHMT2 expression were detectable in all tumors with glycine concentration, demonstrating an inverse correlation with GLDC.

Conclusions: The data suggest that aggressive gliomas reprogram glycine-mediated one-carbon metabolism to meet the biosynthetic demands for rapid cell proliferation. MRS evaluation of glycine provides a non-invasive metabolic imaging biomarker that is predictive of tumor progression and clinical outcome.

Key Points: 1. Glycine and 2-hydroxyglutarate in glioma patients are precisely co-detected using MRS at 3T.2. Tumors with elevated glycine proliferate and progress rapidly.3. A high glycine/2HG ratio is predictive of shortened patient survival.
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http://dx.doi.org/10.1093/neuonc/noaa034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7339885PMC
July 2020

Does Tumor FDG-PET Avidity Represent Enhanced Glycolytic Metabolism in Non-Small Cell Lung Cancer?

Ann Thorac Surg 2020 04 14;109(4):1019-1025. Epub 2019 Dec 14.

Children's Medical Center Research Institute, University of Texas Southwestern, Dallas, Texas; Department of Pediatrics, University of Texas Southwestern, Dallas, Texas; Eugene McDermott Center for Human Growth and Development, University of Texas Southwestern, Dallas, Texas; Howard Hughes Medical Institute, Chevy Chase, Maryland.

Background: In non-small cell lung cancer (NSCLC), fluoro-2-deoxyglucose-positron emission tomography (FDG-PET) assists in diagnosis, staging, and evaluating treatment response. One variable of FDG-PET, the maximum standard uptake value (SUV), is considered an objective measure of glucose uptake. However, little is known about the fate of glucose in FDG-avid lung tumors in vivo. This study used stable glucose isotope tracing to determine whether the SUV predicts glycolytic metabolism or other glucose fates in tumors.

Methods: In this prospective Institutional Review Board-approved clinical trial, 52 untreated potentially resectable confirmed NSCLC patients underwent FDG-PET computed tomography. During the surgical procedure, the patients were infused with C-labeled glucose. Blood, tumor, and normal lung samples were analyzed by mass spectrometry to determine C enrichment in glycolytic intermediates. These values were compared with clinical variables, including SUV, maximum tumor diameter, stage, grade, and MIB-1/Ki67 proliferation index.

Results: For each patient, C enrichment in each metabolite was compared between tumor and adjacent lung. Although all tumors metabolized glucose, SUV did not correlate with glycolytic intermediate labeling. Rather, SUV correlated with markers indicating the use of other respiratory substrates, including lactate, and with the proliferation index.

Conclusions: SUV does not correlate with glycolytic metabolism in human NSCLC but does correlate with the proliferation index, suggesting that SUV predicts glucose use by pathways other than glycolysis. These pathways may offer alternative therapeutic targets, including biosynthetic pathways required for cell proliferation. The research techniques in this study offer the opportunity to understand the relationships between SUV, tumor metabolism, and therapeutic vulnerabilities in human NSCLCs.
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http://dx.doi.org/10.1016/j.athoracsur.2019.10.061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7370816PMC
April 2020

Real-time hyperpolarized C magnetic resonance detects increased pyruvate oxidation in pyruvate dehydrogenase kinase 2/4-double knockout mouse livers.

Sci Rep 2019 11 11;9(1):16480. Epub 2019 Nov 11.

Advanced Imaging Research Center, University of Texas Southwestern Medical Center, Dallas, TX, United States.

The pyruvate dehydrogenase complex (PDH) critically regulates carbohydrate metabolism. Phosphorylation of PDH by one of the pyruvate dehydrogenase kinases 1-4 (PDK1-4) decreases the flux of carbohydrates into the TCA cycle. Inhibition of PDKs increases oxidative metabolism of carbohydrates, so targeting PDKs has emerged as an important therapeutic approach to manage various metabolic diseases. Therefore, it is highly desirable to begin to establish imaging tools for noninvasive measurements of PDH flux in rodent models. In this study, we used hyperpolarized (HP) C-magnetic resonance spectroscopy to study the impact of a PDK2/PDK4 double knockout (DKO) on pyruvate metabolism in perfused livers from lean and diet-induced obese (DIO) mice and validated the HP observations with high-resolution C-nuclear magnetic resonance (NMR) spectroscopy of tissue extracts and steady-state isotopomer analyses. We observed that PDK-deficient livers produce more HP-bicarbonate from HP-[1-C]pyruvate than age-matched control livers. A steady-state C-NMR isotopomer analysis of tissue extracts confirmed that flux rates through PDH, as well as pyruvate carboxylase and pyruvate cycling activities, are significantly higher in PDK-deficient livers. Immunoblotting experiments confirmed that HP-bicarbonate production from HP-[1-C]pyruvate parallels decreased phosphorylation of the PDH E1α subunit (pE1α) in liver tissue. Our findings indicate that combining real-time hyperpolarized C NMR spectroscopy and C isotopomer analysis provides quantitative insights into intermediary metabolism in PDK-knockout mice. We propose that this method will be useful in assessing metabolic disease states and developing therapies to improve PDH flux.
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http://dx.doi.org/10.1038/s41598-019-52952-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6848094PMC
November 2019

Active pyruvate dehydrogenase and impaired gluconeogenesis in orthotopic hepatomas of rats.

Metabolism 2019 12 28;101:153993. Epub 2019 Oct 28.

Advanced Imaging Research Center, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Department of Internal Medicine, University of Texas Southwestern Medical Center, USA. Electronic address:

Background: Therapies targeting altered activity of pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC) have been proposed for hepatomas. However, the activities of these pathways in hepatomas in vivo have not been distinguished. Here we examined pyruvate entry into the tricarboxylic acid (TCA) cycle through PDH versus PC in vivo using hepatoma-bearing rats.

Methods: Hepatoma-bearing rats were generated by intrahepatic injection of H4IIE cells. Metabolism of C-labeled glycerol, a physiological substrate for both gluconeogenesis and energy production, was measured with C NMR analysis. The concentration of key metabolites and the expression of relevant enzymes were measured in hepatoma, surrounding liver, and normal liver.

Results: In orthotopic hepatomas, pyruvate entry into the TCA cycle occurred exclusively through PDH and the excess PDH activity compared to normal liver was attributed to downregulated pyruvate dehydrogenase kinase (PDK) 2/4. However, pyruvate carboxylation via PC and gluconeogenesis were minimal, which was linked to downregulated forkhead box O1 (FoxO1) by Akt activity. In contrast to many studies of cancer metabolism, lactate production in hepatomas was not increased which corresponded to reduced expression of lactate dehydrogenase. The production of serine and glycine in hepatomas was enhanced, but glycine decarboxylase was downregulated.

Conclusions: The combination of [U-C]glycerol and NMR analysis enabled investigation of multiple biochemical processes in hepatomas and surrounding liver. We demonstrated active PDH and other related metabolic alterations in orthotopic hepatomas that differed substantially not only from the host organ but also from many earlier studies with cancer cells.
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http://dx.doi.org/10.1016/j.metabol.2019.153993DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6892165PMC
December 2019

Remodeling of substrate consumption in the murine sTAC model of heart failure.

J Mol Cell Cardiol 2019 09 21;134:144-153. Epub 2019 Jul 21.

Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 32610-0245, United States of America. Electronic address:

Background: Energy metabolism and substrate selection are key aspects of correct myocardial mechanical function. Myocardial preference for oxidizable substrates changes in both hypertrophy and in overt failure. Previous work has shown that glucose oxidation is upregulated in overpressure hypertrophy, but its fate in overt failure is less clear. Anaplerotic flux of pyruvate into the tricarboxylic acid cycle (TCA) has been posited as a secondary fate of glycolysis, aside from pyruvate oxidation or lactate production.

Methods And Results: A model of heart failure that emulates both valvular and hypertensive heart disease, the severe transaortic constriction (sTAC) mouse, was assayed for changes in substrate preference using metabolomic and carbon-13 flux measurements. Quantitative measures of O consumption in the Langendorff perfused mouse heart were paired with C isotopomer analysis to assess TCA cycle turnover. Since the heart accommodates oxidation of all physiological energy sources, the utilization of carbohydrates, fatty acids, and ketones were measured simultaneously using a triple-tracer NMR method. The fractional contribution of glucose to acetyl-CoA production was upregulated in heart failure, while other sources were not significantly different. A model that includes both pyruvate carboxylation and anaplerosis through succinyl-CoA produced superior fits to the data compared to a model using only pyruvate carboxylation. In the sTAC heart, anaplerosis through succinyl-CoA is elevated, while pyruvate carboxylation was not. Metabolomic data showed depleted TCA cycle intermediate pool sizes versus the control, in agreement with previous results.

Conclusion: In the sTAC heart failure model, the glucose contribution to acetyl-CoA production was significantly higher, with compensatory changes in fatty acid and ketone oxidation not reaching a significant level. Anaplerosis through succinyl-CoA is also upregulated, and is likely used to preserve TCA cycle intermediate pool sizes. The triple tracer method used here is new, and can be used to assess sources of acetyl-CoA production in any oxidative tissue.
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http://dx.doi.org/10.1016/j.yjmcc.2019.07.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6704481PMC
September 2019

A simple method to monitor hepatic gluconeogenesis and triglyceride synthesis following oral sugar tolerance test in obese adolescents.

Am J Physiol Regul Integr Comp Physiol 2019 07 1;317(1):R134-R142. Epub 2019 May 1.

Department of Pediatrics, Division of Pediatric Endocrinology, University of Colorado Anschutz Medical Campus , Aurora, Colorado.

Hepatic energy metabolism is a key element in many metabolic diseases. Hepatic anaplerosis provides carbons for gluconeogenesis (GNG) and triglyceride (TG) synthesis. We aimed to optimize a protocol that measures hepatic anaplerotic contribution for GNG, TG synthesis, and hepatic pentose phosphate pathway (PPP) activity using a single dose of oral [UC]glycerol paired with an oral sugar tolerance test (OSTT) in a population with significant insulin resistance. The OSTT (75 g glucose + 25 g fructose) was administered to eight obese adolescents with polycystic ovarian syndrome (PCOS) followed by ingestion of [U-C]glycerol at = 180 or = 210 min. C-labeling patterns of serum glucose and TG-glycerol were determined by nuclear magnetic resonance. C enrichment in plasma TG-glycerol was detectable and stable from 240 to 390 min with the [U-C]glycerol drink at = 180 min(3.65 ± 2.3 to 4.47 ± 1.4%; > 0.4), but the enrichment was undetectable at 240 min with the glycerol drink at = 210 min. The relative contribution from anaplerosis was determined at the end of the OSTT [18.5 ±3.4% ( = 180 min) vs. 16.0 ± 3.5% ( = 210 min); = 0.27]. [U-C]glycerol was incorporated into GNG 390 min after the OSTT with an enrichment of 7.5-12.5%. Glucose derived from TCA cycle activity was 0.3-1%, and the PPP activity was 2.8-4.7%. In conclusion, it is possible to obtain relative measurements of hepatic anaplerotic contribution to both GNG and TG esterification following an OSTT in a highly insulin-resistant population using a minimally invasive technique. Tracer administration should be timed to allow enough de novo TG esterification and endogenous glucose release after the sugar drink.
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http://dx.doi.org/10.1152/ajpregu.00047.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6692761PMC
July 2019

Metabolism of hyperpolarized C-acetoacetate to β-hydroxybutyrate detects real-time mitochondrial redox state and dysfunction in heart tissue.

NMR Biomed 2019 06 10;32(6):e4091. Epub 2019 Apr 10.

Advanced Imaging Research Center, University of Texas Southwestern Medical Center, Dallas, Texas.

Mitochondrial dysfunction is considered to be an important component of many metabolic diseases yet there is no reliable imaging biomarker for monitoring mitochondrial damage in vivo. A large prior literature on inter-conversion of β-hydroxybutyrate and acetoacetate indicates that the process is mitochondrial and that the ratio reflects a specifically mitochondrial redox state. Therefore, the conversion of [1,3- C]acetoacetate to [1,3- C]β-hydroxybutyrate is expected to be sensitive to the abnormal redox state present in dysfunctional mitochondria. In this study, we examined the conversion of hyperpolarized (HP) C-acetoacetate (AcAc) to C-β-hydroxybutyrate (β-HB) as a potential imaging biomarker for mitochondrial redox and dysfunction in perfused rat hearts. Conversion of HP-AcAc to β-HB was investigated using C magnetic resonance spectroscopy in Langendorff-perfused rat hearts in four groups: control, global ischemic reperfusion, low-flow ischemic, and rotenone (mitochondrial complex-I inhibitor)-treated hearts. We observed that more β-HB was produced from AcAc in ischemic hearts and the hearts exposed to complex I inhibitor rotenone compared with controls, consistent with the accumulation of excess mitochondrial NADH. The increase in β-HB, as detected by C MRS, was validated by a direct measure of tissue β-HB by H nuclear magnetic resonance in tissue extracts. The redox ratio, NAD /NADH, measured by enzyme assays of homogenized tissue, also paralleled production of β-HB from AcAc. Transmission electron microscopy of tissues provided direct evidence for abnormal mitochondrial structure in each ischemic tissue model. The results suggest that conversion of HP-AcAc to HP-β-HB detected by C-MRS may serve as a useful diagnostic marker of mitochondrial redox and dysfunction in heart tissue in vivo.
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http://dx.doi.org/10.1002/nbm.4091DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6525062PMC
June 2019

Assessing the pentose phosphate pathway using [2, 3- C ]glucose.

NMR Biomed 2019 06 29;32(6):e4096. Epub 2019 Mar 29.

Advanced Imaging Research Center, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

The pentose phosphate pathway (PPP) is essential for reductive biosynthesis, antioxidant processes and nucleotide production. Common tracers such as [1,2- C ]glucose rely on detection of C in lactate and require assumptions to correct natural C abundance. Here, we introduce a novel and specific tracer of the PPP, [2,3- C ]glucose. C NMR analysis of the resulting isotopomers is informative because [1,2- C ]lactate arises from glycolysis and [2,3- C ]lactate arises exclusively through the PPP. A correction for natural abundance is unnecessary. In rats receiving [2,3- C ]glucose, the PPP was more active in the fed versus fasted state in the liver and the heart, consistent with increased expression of key enzymes in the PPP. Both the PPP and glycolysis were substantially increased in hepatoma compared with liver. In summary, [2,3- C ]glucose and C NMR simplify assessment of the PPP.
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http://dx.doi.org/10.1002/nbm.4096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6525052PMC
June 2019

Effects of deuteration on transamination and oxidation of hyperpolarized C-Pyruvate in the isolated heart.

J Magn Reson 2019 04 4;301:102-108. Epub 2019 Mar 4.

Advanced Imaging Research Center, University of Texas Southwestern Medical Center, Dallas, TX, United States; Department of Radiology, University of Texas Southwestern Medical Center, Dallas, TX, United States; Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, United States; Veterans Affairs North Texas Healthcare System, Dallas, TX, United States. Electronic address:

This study was designed to determine the effects of deuteration in pyruvate on exchange reactions in alanine aminotransferase (ALT), lactate dehydrogenase (LDH) and flux through pyruvate dehydrogenase (PDH). Although deuteration of a C enriched substrate is commonly used to increase the lifetime of a probe for hyperpolarization experiments, the potential impact of kinetic isotope effects on such substitutions has not been studied in detail. Metabolism of deuterated pyruvate was investigated in isolated rat hearts. Hearts were perfused with a 1:1 mixture of [U-C]pyruvate and [2-C]pyruvate or a 1:1 mixture of [U-C]pyruvate plus [2-C, U-H]pyruvate for 30 min before being freeze clamped. Another set of hearts received [2-C, U-H]pyruvate and was freeze-clamped at 3 min or 6 min. Tissue extracts were analyzed by H and C{H} NMR spectroscopy. The chemical shift isotope effect of H was monitored in the C NMR spectra of the C2 resonance of lactate and alanine plus the C5 of glutamate. There was little kinetic isotope effect of H in pyruvate on flux through PDH, LDH or ALT as detected by the distribution of C, but the distribution of H differed markedly between alanine and lactate. At steady-state, alanine was a mixture of deuterated species, while lactate was largely perdeuterated. Consistent with results at steady-state, hearts freeze-clamped at 3 min or 6 min showed rapid removal of deuterium in alanine but not in lactate. Metabolism of hyperpolarized [1-C]pyruvate was compared to [1-C,U-H]pyruvate in isolated hearts. Consistent with the results from tissue extracts, there was little effect of deuteration on the kinetics of appearance of lactate, alanine or bicarbonate, but there was a small, time-dependent upfield chemical shift in the HP[1-C]alanine signal reflecting exchange of methyl deuterons with water protons. Together, these results demonstrate that (1) the kinetics of pyruvate metabolism in hearts detected by C NMR are not affected by replacement of the pyruvate methyl protons with deuterons and (2) that the loss of deuterium from the methyl position occurs rapidly during the conversion of pyruvate to alanine. The majority of the deuterium atoms are lost on the time-scale of a hyperpolarization experiment.
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http://dx.doi.org/10.1016/j.jmr.2019.03.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6666394PMC
April 2019
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