Changes In Pyruvate Metabolism Research Articles

Adaptative response to changes in pyruvate metabolism on the epigenetic landscapes and transcriptomics of bovine embryos

The epigenetic reprogramming that occurs during the earliest stages of embryonic development has been described as crucial for the initial events of cell specification and differentiation. Recently, the metabolic status of the embryo has gained attention as one of the main factors coordinating epigenetic events. In this work, we investigate the link between pyruvate metabolism and epigenetic regulation by culturing bovine embryos from day 5 in the presence of dichloroacetate (DCA), a pyruvate analog that increases the pyruvate to acetyl-CoA conversion, and iodoacetate (IA), which inhibits the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), leading to glycolysis inhibition. After 8 h of incubation, both DCA and IA-derived embryos presented higher mitochondrial membrane potential. Nevertheless, in both cases, lower levels of acetyl-CoA, ATP-citrate lyase and mitochondrial membrane potential were found in blastocysts, suggesting an adaptative metabolic response, especially in the DCA group. The metabolic alteration found in blastocysts led to changes in the global pattern of H3K9 and H3K27 acetylation and H3K27 trimethylation. Transcriptome analysis revealed that such alterations resulted in molecular differences mainly associated to metabolic processes, establishment of epigenetic marks, control of gene expression and cell cycle. The latter was further confirmed by the alteration of total cell number and cell differentiation in both groups when compared to the control. These results corroborate previous evidence of the relationship between the energy metabolism and the epigenetic reprogramming in preimplantation bovine embryos, reinforcing that the culture system is decisive for precise epigenetic reprogramming, with consequences for the molecular control and differentiation of cells.

Hyperpolarized 13C MRI Reveals Large Changes in Pyruvate Metabolism During Digestion in Snakes

PurposeHyperpolarized 13C MRI is a powerful technique to study dynamic metabolic processes in vivo; but it has predominantly been used in mammals, mostly humans, pigs, and rodents.MethodsIn the present study, we use this technique to characterize the metabolic fate of hyperpolarized [1‐13C]pyruvate in Burmese pythons (Python bivittatus), a large species of constricting snake that exhibits a four‐ to tenfold rise in metabolism and large growth of the visceral organs within 24–48 h of ingestion of their large meals.ResultsWe demonstrate a fivefold elevation of the whole‐body lactate‐to‐pyruvate ratio in digesting snakes, pointing to a large rise in lactate production from pyruvate. Consistent with the well‐known metabolic stimulation of digestion, measurements of mitochondrial respiration in hepatocytes in vitro indicate a marked postprandial upregulation of mitochondrial respiration. We observed that a low SNR of the hyperpolarized 13C produced metabolites in the python, and this lack of signal was possibly due to the low metabolism of reptiles compared with mammals, preventing quantification of alanine and bicarbonate production with the experimental setup used in this study. Spatial quantification of the [1‐13C]lactate was only possible in postprandial snakes (with high metabolism), where a statistically significant difference between the heart and liver was observed.ConclusionWe confirm the large postprandial rise in the wet mass of most visceral organs, except for the heart, and demonstrated that it is possible to image the [1‐13C]pyruvate uptake and intracellular conversion to [1‐13C]lactate in ectothermic animals.

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.

150 Changes in pyruvate metabolism alters the epigenetic and molecular maturation of bovine oocytes

During invitro maturation (IVM), bovine oocytes undergo important metabolic, epigenetic, and transcriptional changes for the acquisition of developmental competence. Particularly, metabolic changes that alter the availability of cytoplasmic acetyl-CoA, the main substrate for histones acetylation, may alter the epigenetic profile of the oocyte, with consequences for correct molecular maturation. To test this hypothesis, cumulus–oocyte complexes (COCs) were IVM in three experimental groups: Control [IVM medium (TCM-199-Bicarbonate, 10% fetal bovine serum, 1µg mL−1 oestradiol, 10µg mL−1 FSH, and 10µg mL−1 human chorionic gonadotrophin)], DCA (IVM medium supplemented with 1.5mM sodium dichloroacetate, a pyruvate to acetyl-CoA conversion stimulator) and IA (IVM medium supplemented with 5µM sodium iodoacetate, a glycolysis inhibitor). Cumulus cells (CC) and oocytes (Oo) were analysed separately at 24h (mitochondrial activity, MA; MitoTracker Red CMXRos, ThermoFisher Scientific] and at 0, 4, 8, 16, and 24h of IVM [lysine 9 histone 3 acetylation (H3K9ac immunofluorescence) and new transcript synthesis (only CC; Click-iT® RNA, ThermoFisher Scientific). The images were acquired using a fluorescence microscope and analysed by Image J software. The results from at least 3 replicates were compared by Student’s t-test (treatment vs. control) or by ANOVA followed by Tukey’s test (comparison within the same group in different time points) considering P<0.05. As expected, DCA treatment led to an increase in MA in CC and oocytes (CC control vs. DCA, P=0.003; Oo control vs. DCA, P=0.003). In CC, during the first 4h, H3K9ac increased significantly in the treated group and decreased in the control group. At 8, 16, and 24h, both groups presented similar tendencies, although H3K9ac levels remained higher in DCA compared with control at all time points (P<0.001). The synthesis of new transcripts in CC was stimulated by DCA compared with control at 8h (P=0.02) and particularly at 16h (P=0.002), when acetylation levels were at the lowest point. Interestingly, in oocytes, the initial trend was reversed. An increase was observed in the H3K9ac levels of the control group (P=0.014), whereas no difference was observed for DCA in the first 4h. Moreover, although acetylation levels followed a downward tendency with time in both groups, oocytes treated with DCA showed lower H3K9ac levels at 4 and 8h and a higher level at 24h (P=0.04) compared with control. Regarding IA, lower MA were verified in CC whereas oocytes had the opposite profile (CC control vs. IA: P=0.0035; Oc control vs. IA: P<0.001). In CC, this decrease in MA was not accompanied by a decrease in H3K9ac. In contrast, H3K9ac increased compared with the control group at 8 and 16h (control 8h vs. IA 8 h: P=0.019 and control 16h vs. IA 16 h: P=0.019). These changes were accompanied by an increase in the synthesis of new transcripts in the IA group over the time of IVM. Based on these data, we can conclude that changes in pyruvate metabolism caused by manipulation of the IVM system lead to epigenetic and molecular changes in both CC and oocytes.

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.

Sampling Hyperpolarized Molecules Utilizing a 1 Tesla Permanent Magnetic Field.

Hyperpolarized magnetic resonance spectroscopy (HP MRS) using dynamic nuclear polarization (DNP) is a technique that has greatly enhanced the sensitivity of detecting 13C nuclei. However, the HP MRS polarization decays in the liquid state according to the spin-lattice relaxation time (T1) of the nucleus. Sampling of the signal also destroys polarization, resulting in a limited temporal ability to observe biologically interesting reactions. In this study, we demonstrate that sampling hyperpolarized signals using a permanent magnet at 1 Tesla (1T) is a simple and cost-effective method to increase T1s without sacrificing signal-to-noise. Biologically-relevant information may be obtained with a permanent magnet using enzyme solutions and in whole cells. Of significance, our findings indicate that changes in pyruvate metabolism can also be quantified in a xenograft model at this field strength.

Simultaneous assessment of cardiac metabolism and perfusion using copolarized [1-13 C]pyruvate and 13 C-urea.

PurposeTo demonstrate the feasibility of imaging a bolus of co‐polarized [1‐13C]pyruvate and 13C‐urea to simultaneously assess both metabolism and perfusion in the rodent heart.MethodsCopolarized [1‐13C]pyruvate and 13C‐urea was imaged using a multi‐echo, flow‐sensitized spiral pulse sequence. Healthy rats were scanned in a two‐factor factorial design (n = 12 total; metabolism: overnight fasting versus fed with dichloroacetate injection; perfusion: rest versus adenosine stress‐induced hyperemia).ResultsAlterations in metabolism were detected by changes in pyruvate metabolism into 13C‐bicarbonate. Statistically independent alterations in perfusion were detected by changes in myocardial pyruvate and urea signals.ConclusionThe new pulse sequence was used to obtain maps of metabolism and perfusion in the rodent heart in a single acquisition. This hyperpolarized 13C imaging test is expected to enable new studies in which the cardiac metabolism/perfusion mismatch can be studied in the acute environment. Magn Reson Med 77:151–158, 2017. © 2016 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine

Hyperpolarized Metabolic MR Imaging of Acute Myocardial Changes and Recovery after Ischemia-Reperfusion in a Small-Animal Model.

To implement hyperpolarized magnetic resonance (MR) imaging in an animal model of ischemia-reperfusion and to assess in vivo the regional changes in pyruvate metabolism within the 1st hour and at 1 week after a brief episode of coronary occlusion and reperfusion. All animal experiments were performed with adherence to the Swiss Animal Protection law and were approved by the regional veterinary office. A closed-chest rat model was implemented by using an inflatable balloon secured around the left coronary artery. Animals were placed in an MR system 5-7 days after surgery. [1-(13)C]pyruvate was polarized by using a home-built multisample hyperpolarizer. Hyperpolarized pyruvate was injected at five stages: at baseline; at reperfusion after 15 minutes of coronary occlusion; and at 30 minutes, 60 minutes, and 1 week after ischemia reperfusion. The conversion of pyruvate into lactate and bicarbonate was imaged by using dedicated MR sequences alongside wall motion and delayed enhancement imaging. After imaging, the heart was removed and stained to delineate the area at risk (AAR). Differences between AAR and remote myocardium were assessed by using a repeated measures analysis of variance and a post hoc Bonferroni multiple comparison test. Data were collected in 12 animals. Occlusion led to hypokinesia of the anterior or anterolateral segments of the myocardium. At reperfusion, the average lactate-to-bicarbonate ratio increased in the AAR relative to that at baseline (from 1.93 ± 0.48 to 3.01 ± 0.74, P < .001) and was significantly higher when compared with that in the remote area (1.91 ± 0.38, P < .001). In the 60 minutes after occlusion, the lactate-to-bicarbonate ratio in the AAR recovered but was still elevated relative to that in the remote area. One week after ischemia-reperfusion, no difference between AAR and remote area could be detected. Hyperpolarized metabolic MR imaging can be used to successfully detect acute changes in [1-(13)C]pyruvate metabolism after ischemia-reperfusion, thereby enabling in vivo monitoring of the metabolic effects of reperfusion strategies.

P0406 : Deletion of Cdk1 in the liver induces changes in pyruvate metabolism

P0406 : Deletion of Cdk1 in the liver induces changes in pyruvate metabolism

Changes in pyruvate metabolism detected by magnetic resonance imaging are linked to DNA damage and serve as a sensor of temozolomide response in glioblastoma cells.

Recent findings show that exposure to temozolomide (TMZ), a DNA-damaging drug used to treat glioblastoma (GBM), can suppress the conversion of pyruvate to lactate. To understand the mechanistic basis for this effect and its potential utility as a TMZ response biomarker, we compared the response of isogenic GBM cell populations differing only in expression of the DNA repair protein methyltransferase (MGMT), a TMZ-sensitivity determinant, after exposure to TMZ in vitro and in vivo. Hyperpolarized [1-((13))C]-pyruvate-based MRI was used to monitor temporal effects on pyruvate metabolism in parallel with DNA-damage responses and tumor cell growth. TMZ exposure decreased conversion of pyruvate to lactate only in MGMT-deficient cells. This effect coincided temporally with TMZ-induced increases in levels of the DNA-damage response protein pChk1. Changes in pyruvate to lactate conversion triggered by TMZ preceded tumor growth suppression and were not associated with changes in levels of NADH or lactate dehydrogenase activity in tumors. Instead, they were associated with a TMZ-induced decrease in the expression and activity of pyruvate kinase PKM2, a glycolytic enzyme that indirectly controls pyruvate metabolism. PKM2 silencing decreased PK activity, intracellular lactate levels, and conversion of pyruvate to lactate in the same manner as TMZ, and Chk1 silencing blocked the TMZ-induced decrease in PKM2 expression. Overall, our findings showed how TMZ-induced DNA damage is linked through PKM2 to changes in pyruvate metabolism, and how these changes can be exploited by MRI methods as an early sensor of TMZ therapeutic response.

Iron-induced changes in pyruvate metabolism of Tritrichomonas foetus and involvement of iron in expression of hydrogenosomal proteins.

The main function of the hydrogenosome, a typical organelle of trichomonads, is to convert malate or pyruvate to H(2), CO(2) and acetate by a pathway associated with ATP synthesis. This pathway relies on activity of iron-sulfur proteins such as pyruvate:ferredoxin oxidoreductase (PFOR), hydrogenase and ferredoxin. To examine the effect of iron availability on proper hydrogenosomal function, the metabolic activity of the hydrogenosome and expression of hydrogenosomal enzymes were compared in Tritrichomonas foetus maintained under iron-rich (150 microM iron nitrilotriacetate) or iron-restricted (180 microM 2,2-dipyridyl) conditions in vitro. The activities of PFOR and hydrogenase, and also production of acetate and H(2), were markedly decreased or absent in iron-restricted trichomonads. Moreover, a decrease in activity of the hydrogenosomal malic enzyme, which is a non-Fe-S protein, was also observed. Impaired function of hydrogenosomes under iron-restricted conditions was compensated for by activation of the cytosolic pathway, mediating conversion of pyruvate to ethanol via acetaldehyde. This metabolic switch was fully reversible. Production of hydrogen by iron-restricted trichomonads was restored to the level of organisms grown under iron-rich conditions within 3 h after addition of 150 microM iron nitrilotriacetate. Protein analysis of purified hydrogenosomes from iron-restricted cells showed decreased levels of proteins corresponding to PFOR, malic enzyme and ferredoxin. Accordingly, these cells displayed decreased steady-state level and synthesis of mRNAs encoding PFOR and hydrogenosomal malic enzyme. These data demonstrate that iron is essential for function of the hydrogenosome, show its involvement in the expression of hydrogenosomal proteins and indicate the presence of iron-dependent control of gene transcription in Tt. foetus.

Branched chain amino acid oxidation in cultured rat skeletal muscle cells. Selective inhibition by clofibric acid.

Leucine metabolism in skeletal muscle is linked to protein turnover. Since clofibrate is known both to cause myopathy and to decrease muscle protein content, the present investigations were designed to examine the effects of acute clofibrate treatment on leucine oxidation. Rat skeletal muscle cells in tissue culture were used in these studies because cultivated skeletal muscle cells, like muscle in vivo, have been shown to actively utilize branched chain amino acids and to produce alanine. The conversion of [1-(14)C]leucine to (14)CO(2) or to the [1-(14)C]keto-acid of leucine (alpha-keto-isocaproate) was linear for at least 2 h of incubation; the production of (14)CO(2) from [1-(14)C]leucine was saturable with a K(m) = 6.3 mM and a maximum oxidation rate (V(max)) = 31 nmol/mg protein per 120 min. Clofibric acid selectively inhibited the oxidation of [1-(14)C]leucine (K(i) = 0.85 mM) and [U-(14)C]isoleucine, but had no effect on the oxidation of [U-(14)C]glutamate, -alanine, -lactate, or -palmitate. The inhibition of [1-(14)C]leucine oxidation by clofibrate was also observed in the rat quarter-diaphragm preparation. Clofibrate primarily inhibited the production of (14)CO(2) and had relatively little effect on the production of [1-(14)C]keto-acid of leucine. A physiological concentration-3.0 g/100 ml-of albumin, which actively binds clofibric acid, inhibited but did not abolish the effects of a 2-mM concentration of clofibric acid on leucine oxidation. Clofibrate treatment stimulated the net consumption of pyruvate, and inhibited the net production of alanine. The drug also increased the cytosolic NADH/NAD(+) ratio as reflected by an increase in the lactate/pyruvate ratio, in association with a decrease in cell aspartate levels. The changes in pyruvate metabolism and cell redox state induced by the drug were delayed compared with the nearly immediate inhibition of leucine oxidation. These studies suggest that clofibric acid, in concentrations that approximate high therapeutic levels of the drug, selectively inhibits branched chain amino acid oxidation, possibly at the level of the branched chain keto-acid dehydrogenase.