Defects In Glycolysis Research Articles

Pathogenesis and Management of Citrin Deficiency.

Citrin deficiency (CD) is a hereditary disorder caused by SLC25A13 mutations that manifests as neonatal intrahepatic cholestasis caused by CD (NICCD), failure to thrive and dyslipidemia caused by CD (FTTDCD), and adult-onset type 2 citrullinemia (CTLN2). Citrin, an aspartate-glutamate carrier primarily expressed in the liver, is a component of the malate-aspartate shuttle, which is essential for glycolysis. Citrin-deficient hepatocytes have primary defects in glycolysis and de novo lipogenesis and exhibit secondarily downregulated PPARα, leading to impaired β-oxidation. They are unable to utilize glucose and free fatty acids as energy sources, resulting in energy deficiencies. Medium-chain triglyceride (MCT) supplements are effective for treating CD by providing energy to hepatocytes, increasing lipogenesis, and activating the malate-citrate shuttle. However, patients with CD often exhibit growth impairment and irreversible brain and/or liver damage. To improve the quality of life and prevent irreversible damage, MCT supplementation with a diet containing minimal carbohydrates is recommended promptly after the diagnosis.

Sirtuin 2 up-regulation suppresses the anti-tumour activity of exhausted natural killer cells in mesenteric lymph nodes in murine colorectal carcinoma.

Natural killer (NK) cells inhibit colorectal carcinoma (CRC) initiation and progression through their tumoricidal activity. However, cumulative evidence suggests that NK cells become functionally exhausted in patients with CRC. To deepen the understanding of the mechanisms underlying CRC-associated NK cell exhaustion, we explored the expression and effect of Sirtuin 2 (Sirt2) in mesenteric lymph node (mLN) NK cells in a murine colitis-associated CRC model. Sirt2 was remarkably up-regulated in mLN NK cells after CRC induction. Particularly, Sirt2 was increased in mLN NK cells expressing high T cell immunoglobulin and mucin domain-3 (TIM3), high lymphocyte activation protein-3 (LAG3), high programmed death-1 (PD-1), high T cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT), high NK group 2 member A (NKG2A), but low tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), low interferon-gamma and low granzyme B. In addition, Sirt2 was also increased in NK cells after induction of exhaustion in vitro. Lentivirus-mediated Sirt2 silencing did not affect the acute activation and cytotoxicity of non-exhausted NK cells. However, Sirt2 silencing partially restored the expression of interferon-gamma, granzyme B and CD107a in exhausted NK cells. Meanwhile, Sirt2 silencing down-regulated TIM3, LAG3, TIGIT and NKG2A while up-regulated TRAIL on exhausted NK cells. Consequently, Sirt2 silencing restored the cytotoxicity of exhausted NK cells. Moreover, Sirt2 silencing partially ameliorates the defects in glycolysis and mitochondrial respiration of exhausted NK cells, as evidenced by increases in glycolytic capacity, glycolytic reserve, basal respiration, maximal respiration and spare respiration capacity. Accordingly, Sirt2 negatively regulates the tumoricidal activity of exhausted NK cells in CRC.

CDK7 controls E2F- and MYC-driven proliferative and metabolic vulnerabilities in multiple myeloma

AbstractTherapeutic targeting of CDK7 has proven beneficial in preclinical studies, yet the off-target effects of currently available CDK7 inhibitors make it difficult to pinpoint the exact mechanisms behind MM cell death mediated by CDK7 inhibition. Here, we show that CDK7 expression positively correlates with E2F and MYC transcriptional programs in cells from patients with multiple myeloma (MM); its selective targeting counteracts E2F activity via perturbation of the cyclin-dependent kinases/Rb axis and impairs MYC-regulated metabolic gene signatures translating into defects in glycolysis and reduced levels of lactate production in MM cells. CDK7 inhibition using the covalent small-molecule inhibitor YKL-5-124 elicits a strong therapeutic response with minimal effects on normal cells, and causes in vivo tumor regression, increasing survival in several mouse models of MM including a genetically engineered mouse model of MYC-dependent MM. Through its role as a critical cofactor and regulator of MYC and E2F activity, CDK7 is therefore a master regulator of oncogenic cellular programs supporting MM growth and survival, and a valuable therapeutic target providing rationale for development of YKL-5-124 for clinical use.

Transcriptional reprogramming from innate immune functions to a pro-thrombotic signature by monocytes in COVID-19

Although alterations in myeloid cells have been observed in COVID-19, the specific underlying mechanisms are not completely understood. Here, we examine the function of classical CD14+ monocytes in patients with mild and moderate COVID-19 during the acute phase of infection and in healthy individuals. Monocytes from COVID-19 patients display altered expression of cell surface receptors and a dysfunctional metabolic profile that distinguish them from healthy monocytes. Secondary pathogen sensing ex vivo leads to defects in pro-inflammatory cytokine and type-I IFN production in moderate COVID-19 cases, together with defects in glycolysis. COVID-19 monocytes switch their gene expression profile from canonical innate immune to pro-thrombotic signatures and are functionally pro-thrombotic, both at baseline and following ex vivo stimulation with SARS-CoV-2. Transcriptionally, COVID-19 monocytes are characterized by enrichment of pathways involved in hemostasis, immunothrombosis, platelet aggregation and other accessory pathways to platelet activation and clot formation. These results identify a potential mechanism by which monocyte dysfunction may contribute to COVID-19 pathology.

Mitochondrial and glucose metabolic dysfunctions in granulosa cells induce impaired oocytes of polycystic ovary syndrome through Sirtuin 3

Mitochondrial function and glucose metabolism play important roles in bidirectional signaling between granulosa cells (GCs) and oocytes. However, the factors associated with mitochondrial function and glucose metabolism of GCs in polycystic ovary syndrome (PCOS) are poorly understood, and their potential downstream effects on oocyte quality are still unknown. The aim of this study was to investigate whether there are alterations in mitochondrial-related functions and glucose metabolism in ovarian GCs of women with PCOS and the role of Sirtuin 3 (SIRT3) in this process. Here, we demonstrated that women with PCOS undergoing in vitro fertilization and embryo transfer had significantly lower rates of metaphase II oocytes, two-pronuclear fertilization, cleavage, and day 3 good-quality embryos. Germinal vesicle- and metaphase I-stage oocytes from women with PCOS exhibited increased mitochondrial reactive oxygen species (ROS), decreased mitochondrial membrane potential, and downregulation of glucose-6-phosphate dehydrogenase. GCs from women with PCOS presented significant alterations in mitochondrial morphology, amount, and localization, decreased membrane potential, reduced adenosine triphosphate (ATP) synthesis, increased mitochondrial ROS and oxidative stress, and insufficient oxidative phosphorylation (OXPHOS) together with decreased glycolysis. SIRT3 expression was significantly decreased in GCs of PCOS patients, and knockdown of SIRT3 in KGN cells could mimic the alterations in mitochondrial functions and glucose metabolism in PCOS GCs. SIRT3 knockdown changed the acetylation status of NDUFS1, which might induce altered mitochondrial OXPHOS, the generation of mitochondrial ROS, and eventually defects in the cellular insulin signaling pathway. These findings suggest that SIRT3 deficiency in GCs of PCOS patients may contribute to mitochondrial dysfunction, elevated oxidative stress, and defects in glucose metabolism, which potentially induce impaired oocytes in PCOS.

Glycolytic and Oxidative Phosphorylation Defects Precede the Development of Senescence in Primary Human Brain Microvascular Endothelial Cells.

Alterations of mitochondrial and glycolytic energy pathways related to aging could contribute to cerebrovascular dysfunction. We studied the impact of aging on energetics of primary human brain microvascular endothelial cells (HBMECs) by comparing the young (passages 7-9), pre-senescent (passages 13-15), and senescent (passages 20-21) cells. Pre-senescent HBMECs displayed decreased telomere length and undetectable telomerase activity although markers of senescence were unaffected. Bioenergetics in HBMECs were determined by measuring the oxygen consumption (OCR) and extracellular acidification (ECAR) rates. Cellular ATP production in young HBMECs was predominantly dependent on glycolysis with glutamine as the preferred fuel for mitochondrial oxidative phosphorylation (OXPHOS). In contrast, pre-senescent HBMECs displayed equal contribution to ATP production rate from glycolysis and OXPHOS with equal utilization of glutamine, glucose, and fatty acids as mitofuels. Compared to young, pre-senescent HBMECs showed a lower overall ATP production rate that was characterized by diminished contribution from glycolysis. Impairments of glycolysis displayed by pre-senescent cells included reduced basal glycolysis, compensatory glycolysis, and non-glycolytic acidification. Furthermore, impairments of mitochondrial respiration in pre-senescent cells involved the reduction of maximal respiration and spare respiratory capacity but intact basal and ATP production-related OCR. Proton leak and non-mitochondrial respiration, however, were unchanged in the pre-senescent HBMECs. HBMECS at passages 20-21 displayed expression of senescence markers and continued similar defects in glycolysis and worsened OXPHOS. Thus, for the first time, we characterized the bioenergetics of pre-senescent HBMECs comprehensively to identify the alterations of the energy pathways that could contribute to aging.

A Family Affected by a Life-Threatening Erythrocyte Defect Caused by Pyruvate Kinase Deficiency With Normal Iron Status: A Case Report.

BackgroundRed cell pyruvate kinase deficiency (PKD) is a defect of glycolysis causing congenital non-spherocytic hemolytic anemia. PKD is transmitted as an autosomal recessive trait. The clinical features of PKD are highly variable, from mild to life-threatening anemia which can lead to death in the neonatal period. Most patients with PKD must receive regular transfusions in early childhood and as a consequence suffer from iron overloading.PatientHere, we report a Polish family with life-threatening hemolytic anemia of unknown etiology. Whole exome sequencing identified two heterozygous mutations, c.1529 G > A (p.R510Q) and c.1495 T > C (p.S499P) in the PKLR gene. Molecular modeling showed that the both PKLR mutations are responsible for major disturbance of the protein structure and functioning. Despite frequent transfusions the patients do not show any signs of iron overload and hepcidin, a major regulator of iron uptake, is undetectable in their serum. The patients were homozygous for the rs855791 variant of the TMPRSS6 gene which has earlier been shown to down-regulate iron absorption and accumulation.ConclusionThe lack of iron overload despite a reduced level of hepcidin in two transfusion-dependent PKD patients suggests the existence of a hepcidin-independent mechanism of iron regulation preventing iron overloading.

Abstract PR14: Defining isoform-specific roles for AKTs in BRAFV600E-driven melanoma

Abstract The PI3K/AKT pathway is frequently dysregulated in cutaneous melanoma and impacts both tumor aggression and resistance to BRAFV600E/K inhibitors. While current clinical approaches to AKT inhibition are severely limited by the toxicity of pan-AKT inhibitors, selective inhibition of individual AKT isoforms (AKT1, AKT2, or AKT3) remains an attractive, if yet unattainable, approach. A critical gap in our understanding concerns how the three highly homologous yet functionally distinct AKT isoforms contribute to melanomagenesis and treatment response. To address this question, we are employing RNAi and gene editing approaches to interrogate the effect of selective suppression or CRISPR/Cas9-mediated deletion of each isoform in vitro, as well as the impact of AKT isoform loss on 1) the growth of melanoma xenografts and 2) the development of spontaneous tumors in a melanoma-prone mouse model. In addition, we are interrogating tumor-promoting functions of AKT isoform-selective substrates recently identified in a phospho-proteomic screen of mouse fibroblasts, a system that allows us to identify downstream actionable targets that may mediate the effects of AKT isoforms in tumorigenesis. Broadly, we are focusing on the importance of AKTs in tumor cell growth, metastasis, and response to inhibitors of BRAFV600E/K or CDK4/6. We find that loss of AKT1 impacts growth of BRAF mutant human melanoma cells both in vitro and in vivo. Additionally, AKT1 appears to play an isoform-specific role in response to pharmacologic CDK4/6 inhibition, impacting transcriptional and morphologic changes typically associated with permanent cell cycle arrest. In contrast, loss of AKT2 has a minimal impact on melanoma cell growth or response to CDK4/6 inhibition, but severely limits the development of metastatic disease, potentially through a combination of impaired seeding at the metastatic site and defects in glycolysis. While AKT3 loss does not appreciably impact the above-mentioned cellular phenotypes, the protumorigenic role of AKT3 may involve activation of broadly-acting neutral proteases previously implicated in cell cycle progression, cell migration, and CDK5 regulation. Taken together, we provide evidence for distinct roles for AKT isoforms in several aspects of the tumorigenic process as well as response to current therapies. Future studies will focus on identification and targeting of the relevant downstream mediators of these phenotypes. This abstract is also being presented as Poster A16. Citation Format: Jaymes Farrell, Jodie Pietruska, Siobhan McRee, Philip Tsichlis, Philip Hinds. Defining isoform-specific roles for AKTs in BRAFV600E-driven melanoma [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr PR14.

CD28 regulates T cell metabolic reprogramming through ARS2-dependent effects on alternative splicing

Abstract An adaptive immune response requires activation of naïve T cells via T cell receptor (TCR) ligation and engagement of costimulatory molecules such as CD28, which enables cell growth, rapid proliferation, and effector differentiation. To accomplish such bioenergetically demanding processes, T cells must dynamically regulate their metabolism upon activation. After initial TCR triggering of aerobic glycolysis, CD28 co-stimulation maintains uptake and catabolism of glucose required for CD8+ T cells to attain full effector status. Despite evidence that CD28 signaling through the PI3-kinase pathway induces glucose uptake via increased GLUT1 surface expression, knockin T cells expressing PI3-kinase signaling-deficient CD28 are functional in vivo and displayed only transient defects in glycolysis. In contrast, knockin T cells expressing Grb2/Vav signaling-deficient CD28 lacked in vivo function and displayed sustained defects in glycolysis. Here we identify a novel link between CD28-Grb2/Vav signaling and T cell glycolytic reprogramming through the RNA binding protein ARS2, a protein central to co-transcriptional processing of nascent RNA polymerase II transcripts. Data show that ARS2 is a critical downstream mediator of CD28-Grb2/Vav signaling that supports alternative splicing (AS) of the rate-limiting glycolytic enzyme pyruvate kinase M from the M1 to M2 isoform. Mechanistically, ARS2 binds known Pkm splicing factors and promotes their interaction with Pkm mRNA. Consistent with this, ARS2 knockout T cells displayed reduced Pkm2 AS, defective glycolytic reprogramming and lacked the ability to control in vivo tumor growth, suggesting a novel mechanism by which co-stimulation drives T cell metabolic reprogramming.

Metal-independent variants of phosphoglycerate mutase promote resistance to nutritional immunity and retention of glycolysis during infection.

The ability of Staphylococcus aureus and other pathogens to consume glucose is critical during infection. However, glucose consumption increases the cellular demand for manganese sensitizing S. aureus to host-imposed manganese starvation. The current investigations were undertaken to elucidate how S. aureus copes with the need to consume glucose when metal-limited by the host. A critical component of host defense is production of the manganese binding protein calprotectin. S. aureus has two variants of phosphoglycerate mutase, one of which is manganese-dependent, GpmI, and another that is manganese-independent, GpmA. Leveraging the ability to impose metal starvation in culture utilizing calprotectin revealed that the loss of GpmA, but not GpmI, sensitized S. aureus to manganese starvation. Metabolite feeding experiments revealed that the growth defect of GpmA when manganese-starved was due to a defect in glycolysis and not gluconeogenesis. Loss of GpmA reduces the ability of S. aureus to cause invasive disease in wild type mice. However, GpmA was dispensable in calprotectin-deficient mice, which have defects in manganese sequestration, indicating that this isozyme contributes to the ability of S. aureus to overcome manganese limitation during infection. Cumulatively, these observations suggest that expressing a metal-independent variant enables S. aureus to consume glucose while mitigating the negative impact that glycolysis has on the cellular demand for manganese. S. aureus is not the only bacterium that expresses manganese-dependent and -independent variants of phosphoglycerate mutase. Similar results were also observed in culture with Salmonella enterica serovar Typhimurium mutants lacking the metal-independent isozyme. These similar observations in both Gram-positive and Gram-negative pathogens suggest that expression of metal-independent glycolytic isozymes is a common strategy employed by bacteria to survive in metal-limited environments, such as the host.

Abstract LB-367: [18F]FAZA PET imaging reveals precise pharmacodynamics in vivo of the novel chemotherapeutic IACS-010759

Abstract Genetic deletions and mutations resulting in defects in glycolysis, force these tumors to depend on oxidative phosphorylation (OxPhos) for growth. IACS-010759 is a nanomolar inhibitor of Complex I, and in phase I clinical trials at MD Anderson for both AML and solid tumors. While ex vivo monitoring of inhibition of oxygen consumption in leukocytes was sufficient for AML, non-invasive methods of monitoring target engagement in solid tumors was desired. Tumors, particularly those that rely on oxidative phosphorylation, yield hypoxic and reducing environments. These conditions are ideal for trapping 2-nitroimidazole based imaging agents, such as F18-labeled fluoroazomycin arabinoside ([18F]FAZA). IACS-010759 inhibited oxygen consumption in vitro with IC50 values ranging from 1.3 nM to 6 nM across a variety of cell lines with a diversity of glycolysis defects. In H460 NSLC, SKMEL5 melanoma, A375R melanoma, and D423-Fluc orthotopic GBM in vivo, at the MTD 10 mg/kg, a robust, up to 6 fold, (2 way ANOVA, p<0.0001) decrease in FAZA T/B ratio was observed. A slight, 1.5-fold, but detectable increase in [18F]FDG was also observed at 10mg/kg in A375 and A375R cells (p=0.002), but smaller than the decrease [18F]FAZA retention. Thus neither cell death nor loss of perfusion could explain the reduction in [18F]FAZA retention. To further test the robustness of the mechanism of inhibition, the converse experiment was conducted. Both 2,4-dinitrophenol and pyruvate were utilized to stimulate oxygen consumption in vivo, and as predicted [18F]FAZA retention increases (p<0.05 in both cases). Finally, [18F]FAZA retention by PET yielded the same IC50 for IACS-010759 as by IHC in a dose-dependent manner, with an apparent IC50 of 1.4 mg/kg (95%CI 0.48 to 4.1 mg/kg, n=12 mice) for A375R melanoma tumors. The PET measurement was more precise than an independently scored IHC metric based upon staining for pimonidazole from the same mice. [18F]FAZA can be a powerful PD marker for the complex-I inhibitor IACS-010759 in preclinical models, and is translatable to upcoming clinical trials in patients. Citation Format: Seth T. Gammon, Federica Pisaneschi, Madhavi Bandi, Melinda Smith, Yi Rao, Vashisht G. Yennu Nanda, Yuting Sun, Michael Davies, Emilia Di Francesco, Joseph Marszalek, David Piwnica-Worms. [18F]FAZA PET imaging reveals precise pharmacodynamics in vivo of the novel chemotherapeutic IACS-010759 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-367.

The p53‐specific inhibitor pifithrin‐α prevents diabetic cardiomyopathy by attenuating the early‐stage apoptosis and improving late‐stage defects in glycolysis and angiogenesis

RationalElevated expression levels of the tumor suppressor p53 have been associated with various heart diseases, including diabetic cardiomyopathy (DCM). However, its underlying mechanisms are unclear.ObjectEarly cardiac cell apoptosis, the persistent reduction in glucose uptake and utilization, and an inadequate neovascularization may play important roles in the pathogenesis of DCM. The present study observed an upregulation of p53 in the heart of streptozotocin‐induced type 1 diabetes mice compared to nondiabetic mice. The aim of this study was to determine the protective mechanism and effect of pifithrin‐α in DCM.Methods and ResultsFVB mice were treated with five daily injections of 50 mg/kg streptozotocin to induce type 1 diabetes. Both hyperglycemic and age‐matched control mice were treated with or without pifithrin‐α five times a week for 2 months and then kept without treatment until 3 and 6 months. Treatment with pifithrin‐α significantly improved the progression of diabetes‐induced cardiac remodeling and dysfunction (i.e., DCM). Mechanistically, the inhibition of p53 prevented cardiac mitochondrial cell death during early‐stage diabetes (0.5 month) as well as prevented diabetes‐induced impairment of glucose metabolism and angiogenesis by increasing hypoxia‐induced factor (HIF)‐1α protein stability and upregulating the HIF‐1α transcription of specific protective genes at the later stages (3–6 months).ConclusionsThese results demonstrate for the first time that the inhibition of p53‐mediated early‐stage apoptosis and late‐stage defective glycolysis and angiogenesis effectively prevents the development of DCM, thereby suggesting that an intervention involving P53 may be a potential approach for the prevention of DCM.

Iron Overload Is Highly Prevalent in All Disease Severity States in Pyruvate Kinase Deficiency (PKD)

▪Background: PKD causes a defect in glycolysis resulting in a hereditary non-spherocytic hemolytic anemia. The prevalence of iron overload is not well described for PKD.Aim: We aim to describe the demographic features and prevalence of iron overload in transfusion dependent and transfusion independent patients with PKD.Methods: Between March 2014 and April 2016, 203 patients enrolled on the PKD Natural History Study at 29 IRB approved sites. All patients were confirmed to have two compound heterozygous or homozygous mutations in the PKLR gene. Children < 1 year of age (n=9) were excluded from this analysis, because elevated ferritin levels are less reliably related to iron overload. Patients were designated with iron overload at the time of enrollment if the plasma ferritin was >1000 ng/mL or the patient was on chelation therapy at any time during the prior 12 months. Patients were designated with having had iron overload if a MRI ever showed liver iron content (LIC) >3 mg/g dry weight or if they had ever received chelation therapy. Tests of association were performed using Fisher’s exact test (categorical) and Wilcoxon rank sum test (continuous). Linear associations between variables were measured by Pearson correlation coefficient. P-values <0.05 were considered statistically significant.Results: Of the 194 patients, 111 (57%) were adults ≥18 years and 83 (43%) were children. The median age of enrollment was 20.6 y (range: 1.3-69.9). Splenectomy had been performed in 65% (126/194). Screening ferritin levels were available for 72% (140/194) and LIC for 32% (62/194). At enrollment, 48% (70/147) had iron overload as defined by ferritin and/or current chelation. Using the LIC criterion, iron overload had been present at some point in 86% (95/110) of patients. Ferritin positively correlated with LIC (n=45); r=0.62, p<0.0001. However, of 29 patients with an LIC measurement and a mean ferritin <1000 ng/mL, 15 (52%) had a LIC >3 mg/g DW.Baseline characteristics in patients with and without iron overload are shown in the Table. Notably, even in patients that were never regularly transfused and had a hemoglobin (Hb) >8.7 g/dl, the prevalence of iron overload was 26% (8/31). The frequency of iron overload was significantly higher in patients who had a prior splenectomy (p<0.0001), even after controlling for transfusion history (p<0.0001). Age was associated with iron overload (p=0.046); although, the age range of patients with iron overload was broad (1.3-69.9 years). The frequency of iron overload was significantly higher in those with a lower baselineHb(p=0.004) and higher bilirubin (p=0.03).Data on cardiac iron status was available for 66 patients.Only 2 had cardiac iron overload (defined as T2*<20ms); they were age 5 (T2* 17.8ms, LIC 5 mg/g) and 22 years (T2* 19.7ms, LIC 14 mg/g) at the time of the MRI.Of the 194 patients, 52 (27%) were from the Pennsylvania Amish community. These patients were managed differently than the non-Amish, in that only 2% of the Amish patients were on iron chelation therapy in the 12 months prior to enrollment compared with 43% among the non-Amish cohort. In addition, the Amish had a significant higher prevalence of splenectomy (96% vs 52%, p<0.0001) and proportion who had been historically transfused (79% vs 32%, p<0.0001). Despite these differences, the Amish patients had a lower prevalence of iron overload (34% vs. 51%). There was no significant difference inHbor enrollment age between the Amish and non-Amish cohorts.Conclusion: Iron overload is a common, serious complication in PKD, regardless of age, disease severity, or transfusion status. Although ferritin correlates with LIC for the PKD population overall, at the individual patient level, ferritin is not a good predictor of LIC and a ferritin <1000 ng/ml does not exclude hepatic iron overload. Therefore, we recommend that all patients with PKD starting at age 1 year should be screened annually for iron overload using ferritin and, at least once, using MRI. [Display omitted] DisclosuresBarcellini:Agios: Consultancy. Neufeld:Novartis: Consultancy. Morton:Agios Pharmaceuticals: Research Funding. Yaish:Octapharma: Other: Study investigator. Kuo:Agios Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Alexion: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Apotex: Other: unrestricted educational grant; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Thompson:bluebird bio: Consultancy, Research Funding; Eli Lily: Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mast: Research Funding; Baxalta (now part of Shire): Research Funding; ApoPharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Amgen: Research Funding. Grace:Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Poly(ADP-ribose) polymerase-dependent energy depletion occurs through inhibition of glycolysis.

Excessive poly(ADP-ribose) (PAR) polymerase-1 (PARP-1) activation kills cells via a cell-death process designated "parthanatos" in which PAR induces the mitochondrial release and nuclear translocation of apoptosis-inducing factor to initiate chromatinolysis and cell death. Accompanying the formation of PAR are the reduction of cellular NAD(+) and energetic collapse, which have been thought to be caused by the consumption of cellular NAD(+) by PARP-1. Here we show that the bioenergetic collapse following PARP-1 activation is not dependent on NAD(+) depletion. Instead PARP-1 activation initiates glycolytic defects via PAR-dependent inhibition of hexokinase, which precedes the NAD(+) depletion in N-methyl-N-nitroso-N-nitroguanidine (MNNG)-treated cortical neurons. Mitochondrial defects are observed shortly after PARP-1 activation and are mediated largely through defective glycolysis, because supplementation of the mitochondrial substrates pyruvate and glutamine reverse the PARP-1-mediated mitochondrial dysfunction. Depleting neurons of NAD(+) with FK866, a highly specific noncompetitive inhibitor of nicotinamide phosphoribosyltransferase, does not alter glycolysis or mitochondrial function. Hexokinase, the first regulatory enzyme to initiate glycolysis by converting glucose to glucose-6-phosphate, contains a strong PAR-binding motif. PAR binds to hexokinase and inhibits hexokinase activity in MNNG-treated cortical neurons. Preventing PAR formation with PAR glycohydrolase prevents the PAR-dependent inhibition of hexokinase. These results indicate that bioenergetic collapse induced by overactivation of PARP-1 is caused by PAR-dependent inhibition of glycolysis through inhibition of hexokinase.

Loss of positive allosteric interactions between neuronal nitric oxide synthase and phosphofructokinase contributes to defects in glycolysis and increased fatigability in muscular dystrophy

Duchenne muscular dystrophy (DMD) involves a complex pathophysiology that is not easily explained by the loss of the protein dystrophin, the primary defect in DMD. Instead, many features of the pathology are attributable to the secondary loss of neuronal nitric oxide synthase (nNOS) from dystrophin-deficient muscle. In this investigation, we tested whether the loss of nNOS contributes to the increased fatigability of mdx mice, a model of DMD. Our findings show that the expression of a muscle-specific, nNOS transgene increases the endurance of mdx mice and enhances glycogen metabolism during treadmill-running, but did not affect vascular perfusion of muscles. We also find that the specific activity of phosphofructokinase (PFK; the rate limiting enzyme in glycolysis) is positively affected by nNOS in muscle; PFK-specific activity is significantly reduced in mdx muscles and the muscles of nNOS null mutants, but significantly increased in nNOS transgenic muscles and muscles from mdx mice that express the nNOS transgene. PFK activity measured under allosteric conditions was significantly increased by nNOS, but unaffected by endothelial NOS or inducible NOS. The specific domain of nNOS that positively regulates PFK activity was assayed by cloning and expressing different domains of nNOS and assaying their effects on PFK activity. This approach yielded a polypeptide that included the flavin adenine dinucleotide (FAD)-binding domain of nNOS as the region of the molecule that promotes PFK activity. Smaller peptides in this domain were then synthesized and used in activity assays that showed a 36-amino acid peptide in the FAD-binding domain in which most of the positive allosteric activity of nNOS for PFK resides. Mapping this peptide onto the structure of nNOS shows that the peptide is exposed on the surface, readily available for binding. Collectively, these findings indicate that defects in glycolytic metabolism and increased fatigability in dystrophic muscle may be caused in part by the loss of positive allosteric interactions between nNOS and PFK.

Diagnostic value of in situ muscle fiber conduction velocity measurements in myopathies.

In situ studies on muscle fiber conduction velocity (MFCV) were performed in 54 patients with histologically and biochemically defined myopathies. MFCV was measured over a 10 cm segment of the rectus femoris muscle by intramuscular stimulation and recording. Muscle disorders included muscular dystrophies, myotonic dystrophy, inflammatory myopathies, metabolic myopathies, endocrine myopathies, and congenital myopathies with structural abnormalities. Ten healthy volunteers served as controls. MFCV was significantly reduced in all patients except those with a defect in glycolysis and those that had recovered from acute myositis. MFCV did not vary with either sex. age or the duration of the disease. This shows that MFCV slowing is an unspecific finding in most myopathies. However, in some patients with normal needle electromyography, MFCV provided additional information in diagnosing muscle disease.

Skeletal muscle glycolytic capacity and phosphofructokinase regulation in horses with polysaccharide storage myopathy

Abstract Objective To determine whether polysaccharide storage myopathy (PSSM) in Quarter Horses is attributable to a defect in glycolysis or in the allosteric regulation of phosphofructokinase (PFK) enzyme. Animals Muscle biopsy specimens were obtained from 6 Quarter Horses with PSSM and 8 Quarter Horse or Thoroughbred control horses. Procedures Maximal activity of glycogenolytic and glycolytic enzymes was determined spectrophotometrically. Maximal activity of PFK was determined for each horse at pH 8.0, and at pH 7.0 when variable concentrations of the activators, fructose 6 phosphate, fructose 2,6 bisphosphate, and adenosine monophosphate or inhibitors adenosine triphosphate and citrate were added to the reaction mixture. Relative activity was calculated as activity at pH 7/maximal PFK activity. Results Deficiencies in glycogenolytic or glycolytic enzyme activities were not evident in horses with PSSM. Differences between horses with PSSM and control horses in relative activity of PFK were not apparent for any of the activators or inhibitors used in the study. Conclusions In a group of horses with PSSM, we were unable to detect a glycogenolytic or glycolytic enzyme deficiency or abnormality in the allosteric regulation of PFK. Clinical Relevance Although PSSM is clinically and histologically similar to glycogenolytic/glycolytic enzyme deficiencies in human beings and other animal species, abnormalities in this metabolic pathway are not present in horses with PSSM. (Am J Vet Res 1998;59:782-785)

Pyruvate Metabolism in Lafora Disease

Lafora disease is an autosomal recessive and progressive degenerative disorder of the central nervous system (CNS). The pathogenic mechanism has been presumed to be an inborn error of carbohydrate metabolism, although this has never been proved. In a case of proven Lafora disease, pyruvate metabolism, which has a central position in carbohydrate metabolism, was studied in body fluids under various conditions and in brain biopsy material. No abnormalities in this metabolic pathway were found. This finding plus earlier reports in the literature exclude a defect in glycolysis; thus, a disturbance of carbohydrate metabolism as the pathogenic mechanism of Lafora disease is unlikely.

The ischemic exercise test in normal adults and in patients with weakness and cramps.

Data from 23 normal men and women were used to derive 95% confidence limits for maximum changes in ammonia and lactate values following ischemic forearm exercise. Most normal subjects raised serum lactate and ammonia concentrations more than 20 mg/dl and 100 micrograms/dl, respectively, over baseline values. No significant correlations were found among age, sex, duration of exercise, or estimate of work performed and the maximum ammonia and lactate values achieved. When 70 patients with complaints of weakness, fatigue, or cramps were evaluated, the ischemic exercise test identified 5 patients who proved to have defects in glycolysis or purine metabolism. The test also distinguished those patients with type III glycogen storage disease who lacked debrancher enzyme activity in muscle.

Isolation of Bacillus subtilis mutants pleiotropically insensitive to glucose catabolite repression.

A pleiotropic mutant of Bacillus subtilis was isolated which overproduced in the presence of glucose several enzymes whose synthesis is subject to glucose catabolite repression. Examination of intracellular metabolites suggested that the mutation may have resulted in a defect in glycolysis, increasing phosphoenolpyruvate and decreasing pyruvate, 2-ketoglutarate, and oxaloacetate.