Source Of NADPH Research Articles

The ratio of ursodeoxycholyltaurine to 7-oxolithocholyltaurine serves as a biomarker of decreased 11β-hydroxysteroid dehydrogenase 1 activity in mouse.

Background and Purpose11β‐Hydroxysteroid dehydrogenase 1 (11β‐HSD1) regulates tissue‐specific glucocorticoid metabolism and its impaired expression and activity are associated with major diseases. Pharmacological inhibition of 11β‐HSD1 is considered a promising therapeutic strategy. This study investigated whether alternative 7‐oxo bile acid substrates of 11β‐HSD1 or the ratios to their 7‐hydroxy products can serve as biomarkers for decreased enzymatic activity.Experimental ApproachBile acid profiles were measured by ultra‐HPLC tandem‐MS in plasma and liver tissue samples of four different mouse models with decreased 11β‐HSD1 activity: global (11KO) and liver‐specific 11β‐HSD1 knockout mice (11LKO), mice lacking hexose‐6‐phosphate dehydrogenase (H6pdKO) that provides cofactor NADPH for 11β‐HSD1 and mice treated with the pharmacological inhibitor carbenoxolone. Additionally, 11β‐HSD1 expression and activity were assessed in H6pdKO‐ and carbenoxolone‐treated mice.Key ResultsThe enzyme product to substrate ratios were more reliable markers of 11β‐HSD1 activity than absolute levels due to large inter‐individual variations in bile acid concentrations. The ratio of the 7β‐hydroxylated ursodeoxycholyltaurine (UDC‐Tau) to 7‐oxolithocholyltaurine (7oxoLC‐Tau) was diminished in plasma and liver tissue of all four mouse models and decreased in H6pdKO‐ and carbenoxolone‐treated mice with moderately reduced 11β‐HSD1 activity. The persistence of 11β‐HSD1 oxoreduction activity in the face of H6PD loss indicates the existence of an alternative NADPH source in the endoplasmic reticulum.Conclusions and ImplicationsThe plasma UDC‐Tau/7oxo‐LC‐Tau ratio detects decreased 11β‐HSD1 oxoreduction activity in different mouse models. This ratio may be a useful biomarker of decreased 11β‐HSD1 activity in pathophysiological situations or upon pharmacological inhibition.LINKED ARTICLESThis article is part of a themed issue on Oxysterols, Lifelong Health and Therapeutics. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.16/issuetoc

Abstract 17224: Metabolic Switch and Redox Imbalance Contributes to Pulmonary Hypertension in Glucose-6-Phosphate Dehydrogenase Deficiency

Introduction: Pulmonary hypertension (PH) is a fatal disorder with inadequate therapeutic choices and diminished survival rate with later prognosis. We previously reported that several patients with idiopathic pulmonary arterial hypertension had different types of glucose-6 phosphate dehydrogenase (G6PD) deficiency. G6PD is the key regulator enzyme in the pentose phosphate pathway (PPP) and the only source of NADPH in erythrocytes. However, the pathogenic mechanism of how G6PD deficiency contributes to PH development remains elusive. Hypothesis: We hypothesize that G6PD deficiency-induced PH is mediated through a multifactorial mechanism by increased red blood cell fragility, oxidative stress, and a metabolic switch. Methods: To delineate the contribution of G6PD in PH pathogenesis, we utilized a G6PD knockdown mouse line (11-13 week old) with decreased expression of G6PD (10% from wild-type level). Results: Hemodynamic and histological studies confirmed that G6PD deficient mice developed PH phenotype by an increase in right ventricular systolic pressure (30.08±0.91mmHg; p≤0.001), Fulton index (0.358±0.03; p≤0.01) and pulmonary vascular remodeling. G6PD deficiency resulted in increased free hemoglobin and activation of the p38/MAPK pathway, which we recently reported, induces the development of PH in the sugen/hypoxia model via endothelial barrier dysfunction. Metabolomics analysis of G6PD-deficient mice indicates the switch to alternative metabolic fluxes that feed into PPP, resulting in the upregulation of oxidative stress, fatty acid pathway, and reduction in pyruvate production. Thus, G6PD deficiency did not reduce PPP flux that is important for proliferation but activated collateral pathways at the cost of increased oxidative stress. Indeed, we found upregulation of Myo-inositol oxidase (p≤0.05), reduction in GSH/GSSG ratio (p≤0.01), and increased nitration (p≤0.05) in the lungs of G6PD deficient mice. Increased oxidative stress also results in the activation of PI3K, ERK1/2 and AMPK that contributes to the proliferation of pulmonary vasculature. Conclusions: Based on these results we infer that G6PD deficiency has a multi-modal effect, including hemolysis, metabolic reprogramming, and oxidative stress leading to PH.

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

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‐13C3]glycerol to measure the PPP activity by analysis of 13C 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.

Genome-scale metabolic modeling of Acetobacter pasteurianus 386B reveals its metabolic adaptation to cocoa fermentation conditions

Acetobacter pasteurianus 386B has been selected as a candidate functional starter culture to better control the cocoa fermentation process. Previously, its genome has been sequenced and a genome-scale metabolic model (GEM) has been reconstructed. To understand its metabolic adaptation to cocoa fermentation conditions, different flux balance analysis (FBA) simulations were performed and compared with experimental data. In particular, metabolic flux distributions were simulated for two phases that characterize the growth of A. pasteurianus 386B under cocoa fermentation conditions, predicting a switch in respiratory chain usage in between these phases. The possible influence on the resulting energy production was shown using a reduced version of the GEM. FBA simulations revealed the importance of the compartmentalization of the ethanol oxidation reactions, namely in the periplasm or in the cytoplasm, and highlighted the potential role of ethanol as a source of carbon, energy, and NADPH. Regarding the latter, the physiological function of a proton-translocating NAD(P)+ transhydrogenase was further investigated in silico. This study revealed the potential of using a GEM to simulate the metabolism of A. pasteurianus 386B, and may provide a general framework toward a better physiological understanding of functional starter cultures in food fermentation processes.

In Vivo Selection for Formate Dehydrogenases with High Efficiency and Specificity toward NADP.

The efficient regeneration of cofactors is vital for the establishment of biocatalytic processes. Formate is an ideal electron donor for cofactor regeneration due to its general availability, low reduction potential, and benign byproduct (CO2). However, formate dehydrogenases (FDHs) are usually specific to NAD+, such that NADPH regeneration with formate is challenging. Previous studies reported naturally occurring FDHs or engineered FDHs that accept NADP+, but these enzymes show low kinetic efficiencies and specificities. Here, we harness the power of natural selection to engineer FDH variants to simultaneously optimize three properties: kinetic efficiency with NADP+, specificity toward NADP+, and affinity toward formate. By simultaneously mutating multiple residues of FDH from Pseudomonas sp. 101, which exhibits practically no activity toward NADP+, we generate a library of >106 variants. We introduce this library into an E. coli strain that cannot produce NADPH. By selecting for growth with formate as the sole NADPH source, we isolate several enzyme variants that support efficient NADPH regeneration. We find that the kinetically superior enzyme variant, harboring five mutations, has 5-fold higher efficiency and 14-fold higher specificity in comparison to the best enzyme previously engineered, while retaining high affinity toward formate. By using molecular dynamics simulations, we reveal the contribution of each mutation to the superior kinetics of this variant. We further determine how nonadditive epistatic effects improve multiple parameters simultaneously. Our work demonstrates the capacity of in vivo selection to identify highly proficient enzyme variants carrying multiple mutations which would be almost impossible to find using conventional screening methods.

Investigation of Heterologously Expressed Glucose-6-Phosphate Dehydrogenase Genes in a Yeast zwf1 Deletion.

Glucose-6-phosphate dehydrogenase (G6PD) is a key enzyme of the oxidative part of the pentose phosphate pathway and serves as the major source of NADPH for metabolic reactions and oxidative stress response in pro- and eukaryotic cells. We here report on a strain of the model yeast Saccharomyces cerevisiae which lacks the G6PD-encoding ZWF1 gene and displays distinct growth retardation on rich and synthetic media, as well as a strongly reduced chronological lifespan. This strain was used as a recipient to introduce plasmid-encoded heterologous G6PD genes, synthesized in the yeast codon usage and expressed under the control of the native PFK2 promotor. Complementation of the hypersensitivity of the zwf1 mutant towards hydrogen peroxide to different degrees was observed for the genes from humans (HsG6PD1), the milk yeast Kluyveromyces lactis (KlZWF1), the bacteria Escherichia coli (EcZWF1) and Leuconostoc mesenteroides (LmZWF1), as well as the genes encoding three different plant G6PD isoforms from Arabidopsis thaliana (AtG6PD1, AtG6PD5, AtG6PD6). The plastidic AtG6PD1 isoform retained its redox-sensitive activity when produced in the yeast as a cytosolic enzyme, demonstrating the suitability of this host for determination of its physiological properties. Mutations precluding the formation of a disulfide bridge in AtG6PD1 abolished its redox-sensitivity but improved its capacity to complement the yeast zwf1 deletion. Given the importance of G6PD in human diseases and plant growth, this heterologous expression system offers a broad range of applications.

Structural defect leads to human severe (Class I) loss of function in glucose‐6‐phosphate dehydrogenase

Oxidative stress caused by infection, medication, food, and imbalance of metabolic cycles damages DNA and organelles, which may lead to cancer, blood disorders, and other serious diseases. Glucose‐6‐phosphate dehydrogenase (G6PD) is the rate‐limiting enzyme in the pentose phosphate pathway, which is essential for nucleotide, fatty acid, cholesterol, and hormone synthesis. In addition to those roles, G6PD reduces NADP+ to NADPH, which is crucial for reducing reactive oxygen species. Dysfunction of G6PD increases susceptibility to oxidative stress, especially in erythrocyte due to the lack of mitochondria, which is another source of NADPH. Interestingly, 400 million people worldwide have mutations on the g6pd gene, and the World Health Organization (WHO) has classified more than 160 missense mutations into five classes. The Class I G6PD deficiency is the most severe form; patients have less than 10% enzymatic activity of wild‐type G6PD and suffer from chronic non‐spherocytic hemolytic anemia. Here, we provide a new insight into the loss of activity in G6PD deficiency, based on the structure of the Class I pathogenic mutants of G6PD. We will discuss the structural basis of the Class I mutants of G6PD and its implications to various symptoms in G6PD deficiency.Support or Funding InformationNational Institutes of Health, R01 grant, HD084422; Japan Society for the Promotion of Science KAKENHI, Grant‐in Aid for Research Activity Start‐up, JP19K23713; University of Tsukuba; Stanford University; SLAC National Accelerator Laboratory

Identification and Characterization of NADH Kinase-3 from a Stress-Tolerant Wild Mung Bean Species (Vigna luteola (Jacq.) Benth.) with a Possible Role in Waterlogging Tolerance

In plants, reactive oxygen species accumulate to a toxic level under various abiotic stresses. Many antioxidant defense systems require NADPH as a principal reducing energy equivalent. However, the source of NADPH and the molecular mechanisms associated with the maintenance of cytoplasmic redox balance are still unknown. The present study describes Vigna NADH kinase (VlNADHK), an enzyme involved in NADPH synthesis and prefers NADH as a diphospho-nicotinamide nucleotide donor. We analyzed the enzymatic activity of a putative cytoplasmic NADH kinase during waterlogging in contrasting mung bean genotypes Vigna luteola (tolerant) and Vigna radiata cv. T44 (susceptible) under pot-culture condition. The tolerant cultivar showed higher enzymatic activity under waterlogging as well as after recovery. Similarly, the transcript level of waterlogging-induced NADHK expression was also studied and found to be upregulated in response to waterlogging in the roots of V. luteola and T44. PCR amplicons of partial and full-length sequences were cloned and sequenced from V. luteola. To the best of our knowledge, this is the first time an ATP-dependent NADH kinase gene has been recognized as a component of waterlogging stress tolerance in legumes. Our study indicated that this cytoplasmic NADH kinase is a primary source of the cytosolic NADPH and might have a role in waterlogging tolerance in legumes.

Pharmacological HIF1 Inhibition Eliminates Downregulation of the Pentose Phosphate Pathway and Prevents Neuronal Apoptosis in Rat Hippocampus Caused by Severe Hypoxia.

The pentose phosphate pathway (PPP) of glucose metabolism in the brain serves as a primary source of NADPH which in turn plays a crucial role in multiple cellular processes, including maintenance of redox homeostasis and antioxidant defense. In our model of protective mild hypobaric hypoxia in rats (3MHH), an inverse correlation between hypoxia-inducible factor-1 (HIF1) activity and mRNA levels of glucose-6-phosphate dehydrogenase (G6PD), the key enzyme of PPP, was observed. In the present study, it was demonstrated that severe hypobaric hypoxia (SH) induced short-term upregulation of HIF1 alpha-subunit (HIF1α) in the hippocampal CA1 subfield and decreased the activity of G6PD. The levels of NADPH were also reduced, promoting oxidative stress, triggering apoptosis, and neuronal loss. Injection of a HIF1 inhibitor (HIF1i), topotecan hydrochloride (5mg/kg, i.p.), before SH prevented the upregulation of HIF1α and normalized G6PD activity. In addition, HIF1i injection caused an increase in NADPH levels, normalization of total glutathione levels and of the cellular redox status as well as suppression of free-radical and apoptotic processes. These results demonstrate a new molecular mechanism of post-hypoxic cerebral pathology development which involves HIF1-dependent PPP depletion and support a recently suggested injurious role of HIF1 activation in the acute phase of cerebral hypoxia/ischemia. Application of PPP stimulators in early post-hypoxic/ischemic period might represent a promising neuroprotective strategy. Graphical abstract HIF1-dependent down-regulation of the pentose phosphate pathway contributes to the hypoxia-induced oxidative stress and neuronal apoptosis in the rat hippocampus.

Cellular Pharmacodynamics of a Novel Pyrrolo[3,2-d]pyrimidine Inhibitor Targeting Mitochondrial and Cytosolic One-Carbon Metabolism

Folate-dependent one-carbon (C1) metabolism is compartmentalized in the mitochondria and cytosol and is a source of critical metabolites for proliferating tumors. Mitochondrial C1 metabolism including serine hydroxymethyltransferase 2 (SHMT2) generates glycine for de novo purine nucleotide and glutathione biosynthesis and is an important source of NADPH, ATP, and formate, which affords C1 units as 10-formyl-tetrahydrofolate and 5,10-methylene-tetrahydrofolate for nucleotide biosynthesis in the cytosol. We previously discovered novel first-in-class multitargeted pyrrolo[3,2-d]pyrimidine inhibitors of SHMT2 and de novo purine biosynthesis at glycinamide ribonucleotide formyltransferase and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase with potent in vitro and in vivo antitumor efficacy toward pancreatic adenocarcinoma cells. In this report, we extend our findings to an expanded panel of pancreatic cancer models. We used our lead analog AGF347 [(4-(4-(2-amino-4-oxo-3,4-dihydro-5H-pyrrolo[3,2-d]pyrimidin-5-yl)butyl)-2-fluorobenzoyl)-l-glutamic acid] to characterize pharmacodynamic determinants of antitumor efficacy for this series and demonstrated plasma membrane transport into the cytosol, uptake from cytosol into mitochondria, and metabolism to AGF347 polyglutamates in both cytosol and mitochondria. Antitumor effects of AGF347 downstream of SHMT2 and purine biosynthesis included suppression of mammalian target of rapamycin signaling, and glutathione depletion with increased levels of reactive oxygen species. Our results provide important insights into the cellular pharmacology of novel pyrrolo[3,2-d]pyrimidine inhibitors as antitumor compounds and establish AGF347 as a unique agent for potential clinical application for pancreatic cancer, as well as other malignancies. SIGNIFICANCE STATEMENT: This study establishes the antitumor efficacies of novel inhibitors of serine hydroxymethyltransferase 2 and of cytosolic targets toward a panel of clinically relevant pancreatic cancer cells and demonstrates the important roles of plasma membrane transport, mitochondrial accumulation, and metabolism to polyglutamates of the lead compound AGF347 to drug activity. We also establish that loss of serine catabolism and purine biosynthesis resulting from AGF347 treatment impacts mammalian target of rapamycin signaling, glutathione pools, and reactive oxygen species, contributing to antitumor efficacy.

Inhibition effects of some antidepressant drugs on pentose phosphate pathway enzymes

The glucose metabolism in the pentose cycle is essential to the source of NADPH. Deficiency of these enzymes have been linked to depression and psychotic disorders. Depression is an increasingly prevalent mental disorder which may cause loss of labor. Antidepressant drugs are commonly employed in treatments of mood disorders and anxiety treatment. The purpose of this study is to investigate the effects of aripiprazole, mirtazapine, risperidone, escitalopram and haloperidol on the activity of 6-phosphogluconate dehydrogenase (6PGD) and glucose-6-phosphate dehydrogenase (G6PD) enzymes purified from human erythrocytes. It was found that aripiprazole, mirtazapine, risperidone, escitalopram and haloperidol show effective inhibitor properties on purified G6PD and 6PGD enzymes. The IC50 values of these drugs were found in the range of 26.34 μM-5.78 mM for 6PGD and 16.26 μM-3.85 mM for G6PD. The Ki values of the drugs were found in the range of 30.21 ± 4.31 μM-4.51 ± 1.83 mM for 6PGD and 14.12 ± 3.48 μM-4.98 ± 1.14 mM for G6PD. Usage of drugs with significant biological effects may be a hazard in some conditions.

Targeting the H3K4 Demethylase KDM5B Reprograms the Metabolome and Phenotype of Melanoma Cells

Melanoma cells shift between epigenetic-metabolic states to adapt to stress and, particularly, to drugs. Here, we unraveled the metabolome of an H3K4 demethylase (KDM5B/JARID1B)-driven melanoma cell phenotype that is known to be multidrug resistant. We set up a fast protocol for standardized, highly sensitive liquid chromatography-high resolution mass spectrometry analyzing stably controlled KDM5B expression by RNAi or doxycycline-induced overexpression. Within the KDM5B-dependent metabolome, we found significant and highly specific regulation of 11 intracellular metabolites. Functionally, overexpression of KDM5B in melanoma cells led to broadening of their oxidative metabolism from mainly glutamine-dependent to additionally glucose- and fatty acid-utilizing, upregulation of the pentose phosphate pathway as a source of antioxidant NADPH, and maintenance of a high ratio of reduced to oxidized glutathione. Histone lysine demethylase inhibition (GSK-J1, 2,4-PDCA) decreased colony formation and invasion in three-dimensional models. Thus, targeting KDM5B could represent an alternative way of modulating the metabolome and malignant cell behavior in melanoma.

Nicotinamide nucleotide transhydrogenase is required for brain mitochondrial redox balance under hampered energy substrate metabolism and high-fat diet.

Among mitochondrial NADP-reducing enzymes, nicotinamide nucleotide transhydrogenase (NNT) establishes an elevated matrix NADPH/NADP+ by catalyzing the reduction of NADP+ at the expense of NADH oxidation coupled to inward proton translocation across the inner mitochondrial membrane. Here, we characterize NNT activity and mitochondrial redox balance in the brain using a congenic mouse model carrying the mutated Nnt gene from the C57BL/6J strain. The absence of NNT activity resulted in lower total NADPH sources activity in the brain mitochondria of young mice, an effect that was partially compensated in aged mice. Nonsynaptic mitochondria showed higher NNT activity than synaptic mitochondria. In the absence of NNT, an increased release of H2 O2 from mitochondria was observed when the metabolism of respiratory substrates occurred with restricted flux through relevant mitochondrial NADPH sources or when respiratory complex I was inhibited. In accordance, mitochondria from Nnt-/- brains were unable to sustain NADP in its reduced state when energized in the absence of carbon substrates, an effect aggravated after H2 O2 bolus metabolism. These data indicate that the lack of NNT in brain mitochondria impairs peroxide detoxification, but peroxide detoxification can be partially counterbalanced by concurrent NADPH sources depending on substrate availability. Notably, only brain mitochondria from Nnt-/- mice chronically fed a high-fat diet exhibited lower activity of the redox-sensitive aconitase, suggesting that brain mitochondrial redox balance requires NNT under the metabolic stress of a high-fat diet. Overall, the role of NNT in the brain mitochondria redox balance especially comes into play under mitochondrial respiratory defects or high-fat diet.

NADPH-Auxotrophic E. coli: A Sensor Strain for Testing in Vivo Regeneration of NADPH.

Insufficient rate of NADPH regeneration often limits the activity of biosynthetic pathways. Expression of NADPH-regenerating enzymes is commonly used to address this problem and increase cofactor availability. Here, we construct an Escherichia coli NADPH-auxotroph strain, which is deleted in all reactions that produce NADPH with the exception of 6-phosphogluconate dehydrogenase. This strain grows on a minimal medium only if gluconate is added as NADPH source. When gluconate is omitted, the strain serves as a "biosensor" for the capability of enzymes to regenerate NADPH in vivo. We show that the NADPH-auxotroph strain can be used to quantitatively assess different NADPH-regenerating enzymes and provide essential information on expression levels and concentrations of reduced substrates required to support optimal NADPH production rate. The NADPH-auxotroph strain thus serves as an effective metabolic platform for evaluating NADPH regeneration within the cellular context.

Absence of hexose-6-phosphate dehydrogenase results in reduced overall glucose consumption but does not prevent 11β-hydroxysteroid dehydrogenase-1-dependent glucocorticoid activation.

Hexose-6-phosphate dehydrogenase (H6PD) is thought to be the major source of NADPH within the endoplasmic reticulum (ER), determining 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) reaction direction to convert inert 11-oxo- to potent 11β-hydroxyglucocorticoids. Here, we tested the hypothesis whether H6pd knock-out (KO) in primary murine bone marrow-derived macrophages results in a switch from 11β-HSD1 oxoreduction to dehydrogenation, thereby inactivating glucocorticoids (GC) and affecting macrophage phenotypic activation as well as causing a more aggressive M1 macrophage phenotype. H6pdKO did not lead to major disturbances of macrophage activation state, although a slightly more pronounced M1 phenotype was observed with enhanced proinflammatory cytokine release, an effect explained by the decreased 11β-HSD1-dependent GC activation. Unexpectedly, ablation of H6pd did not switch 11β-HSD1 reaction direction. A moderately decreased 11β-HSD1 oxoreduction activity by 40-50% was observed in H6pdKO M1 macrophages but dehydrogenation activity was undetectable, providing strong evidence for the existence of an alternative source of NADPH in the ER. H6pdKO M1 activated macrophages showed decreased phagocytic activity, most likely a result of the reduced 11β-HSD1-dependent GC activation. Other general macrophage functions reported to be influenced by GC, such as nitrite production and cholesterol efflux, were altered negligibly or not at all. Importantly, assessment of energy metabolism using an extracellular flux analyzer and lactate measurements revealed reduced overall glucose consumption in H6pdKO M1 activated macrophages, an effect that was GC independent. The GC-independent influence of H6PD on energy metabolism and the characterization of the alternative source of NADPH in the ER warrant further investigations. ENZYMES: 11β-HSD1, EC 1.1.1.146; H6PD, EC 1.1.1.47.

Case 3: Early Severe Jaundice in a Term Infant

An appropriate-for–gestational age (AGA) African American female infant is born at term via elective, repeat cesarean section at 39 weeks’ gestation to a gravida 2, para 2 woman. The mother’s pregnancy had been complicated only by gestational hypertension, and maternal antenatal testing results were unremarkable. Maternal blood type is O positive with a negative antibody screen during pregnancy. Meconium fluid is noted at delivery; however, the infant is vigorous with a strong cry and requires only routine resuscitation with Apgar scores of 8 and 9 at 1 and 5 minutes, respectively. After resuscitation, the infant makes a transition to routine couplet care with an anticipated 48-hour stay. A cord blood sample is sent for blood type and direct antiglobulin test (DAT) per hospital protocol secondary to infant blood type finding of B-negative type with a negative DAT result. Her first 24 hours are uneventful with direct breastfeeding every 2 to 3 hours, 2 documented voids, 3 stools, and minimal weight loss. All documented vital signs and examination results are normal for a term newborn. At 24 hours after birth, a transcutaneous bilirubin test is attempted per standard newborn care plan, with the resulting value too high to detect. Serum total and direct bilirubin concentrations are found to be 21.7 mg/dL (360 μmol/L) and 0.6 mg/dL (10 μmol/L), respectively. The infant is transported directly to the NICU for continued management and preparation for possible exchange transfusion. With any case of hyperbilirubinemia, especially with early-onset or rapidly increasing levels, it is important to keep a broad differential to avoid missing benign versus more severe causes of jaundice. Categorizing by time of onset into 3 categories can be useful in establishing a differential diagnosis. These categories are based on newborn age: less than 24 hours after birth, between 24 and 72 hours after …

Novel Modes of Inhibition of Wild-Type Isocitrate Dehydrogenase 1 (IDH1): Direct Covalent Modification of His315

IDH1 plays a critical role in a number of metabolic processes and serves as a key source of cytosolic NADPH under conditions of cellular stress. However, few inhibitors of wild-type IDH1 have been reported. Here we present the discovery and biochemical characterization of two novel inhibitors of wild-type IDH1. In addition, we present the first ligand-bound crystallographic characterization of these novel small molecule IDH1 binding pockets. Importantly, the NADPH competitive α,β-unsaturated enone 1 makes a unique covalent linkage through active site H315. As few small molecules have been shown to covalently react with histidine residues, these data support the potential utility of an underutilized strategy for reversible covalent small molecule design.

13C Metabolic Flux Analysis of Enhanced Lipid Accumulation Modulated by Ethanolamine in Crypthecodinium cohnii.

The heterotrophic microalga Crypthecodinium cohnii has attracted considerable attention due to its capability of accumulating lipids with a high fraction of docosahexaenoic acid (DHA). In our previous study, ethanolamine (ETA) was identified as an effective chemical modulator for lipid accumulation in C. cohnii. In this study, to gain a better understanding of the lipid metabolism and mechanism for the positive effects of modulator ETA, metabolic flux analysis was performed using 13C-labeled glucose with and without 1 mM ETA modulator. The analysis of flux distribution showed that with the addition of ETA, flux in glycolysis pathway and citrate pyruvate cycle was strengthened while flux in pentose phosphate pathway was decreased. In addition, flux in TCA cycle was slightly decreased compared with the control without ETA. The enzyme activity of malic enzyme (ME) was significantly increased, suggesting that NADP+-dependent ME might be the major source of NADPH for lipid accumulation. The flux information obtained by this study could be valuable for the further efforts in improving lipid accumulation and DHA production in C. cohnii.

Understanding the impact of the cofactor swapping of isocitrate dehydrogenase over the growth phenotype of Escherichia coli on acetate by using constraint-based modeling

It has been proposed that NADP+-specificity of isocitrate dehydrogenase (ICDH) evolved as an adaptation of microorganisms to grow on acetate as the sole source of carbon and energy. In Escherichia coli, changing the cofactor specificity of ICDH from NADP+ to NAD+ (cofactor swapping) decreases the growth rate on acetate. However, the metabolic basis of this phenotype has not been analyzed. In this work, we used constraint-based modeling to investigate the effect of the cofactor swapping of ICDH in terms of energy production, response of alternative sources of NADPH, and partitioning of fluxes between ICDH and isocitrate lyase (ICL) -a crucial bifurcation when the bacterium grows on acetate-. We generated E. coli strains expressing NAD+-specific ICDH instead of the native enzyme, and bearing the deletion of the NADPH-producing transhydrogenase PntAB. We measured their growth rate and acetate uptake rate, modeled the distribution of metabolic fluxes by Flux Balance Analysis (FBA), and quantified the specific activities of NADPH-producing dehydrogenases in central pathways. The cofactor swapping of ICDH led to one-third decrease in biomass yield, irrespective of the presence of PntAB. According to our simulations, the diminution in growth rate observed upon cofactor swapping could be explained by one-half decrease in the total production of NADPH and a lower availability of carbon for biosynthesis because of a change in the partition at the isocitrate bifurcation. Together with an increased total ATP production, this scenario resulted in a 10-fold increment in the flux of ATP not used for growing purposes. PntAB was identified as the primary NADPH balancing response, with the dehydrogenases of the oxidative branch of the Pentose Phosphate Pathway and the malic enzyme playing a role in its absence. We propose that in the context of E. coli growing on acetate, the NADP+-specificity of ICDH is a trait that impacts not only NADPH production, but also the efficient allocation of carbon and energy.

Loss of IDH2 Accelerates Age-related Hearing Loss in Male Mice

Isocitrate dehydrogenase (IDH) 2 participates in the TCA cycle and catalyzes the conversion of isocitrate to α-ketoglutarate and NADP+ to NADPH. In the mitochondria, IDH2 also plays a key role in protecting mitochondrial components from oxidative stress by supplying NADPH to both glutathione reductase (GSR) and thioredoxin reductase 2 (TXNRD2). Here, we report that loss of Idh2 accelerates age-related hearing loss, the most common form of hearing impairment, in male mice. This was accompanied by increased oxidative DNA damage, increased apoptotic cell death, and profound loss of spiral ganglion neurons and hair cells in the cochlea of 24-month-old Idh2−/− mice. In young male mice, loss of Idh2 resulted in decreased NADPH redox state and decreased activity of TXNRD2 in the mitochondria of the inner ear. In HEI-OC1 mouse inner ear cell lines, knockdown of Idh2 resulted in a decline in cell viability and mitochondrial oxygen consumption. This was accompanied by decreased NADPH redox state and decreased activity of TXNRD2 in the mitochondria of the HEI-OC1 cells. Therefore, IDH2 functions as the principal source of NADPH for the mitochondrial thioredoxin antioxidant defense and plays an essential role in protecting hair cells and neurons against oxidative stress in the cochlea of male mice.