The alpha beta epimerization of reduced nicotinamide adenine dinucleotide.

The proton magnetic resonance spectra of the dihydronicotinamide ring of αNADH³ and the nicotinamide ring of αNAD⁺ are reported and the proton absorptions assigned. The absolute assignment of the C4 methylene protons of αNADH is based on the generation of specifically deuterium-labeled (pro-S) B-deuterio-αNADH from enzymatically prepared B-deuterio-βNADH. The C4 proton absorption of αNAD⁺ is assigned by oxidation of B-deuterio-αNADH by the A specific, yeast alcohol dehydrogenase to yield 4-deuterio-αNAD⁺.The epimerization of either αNADH or βNADH yields an equilibrium ratio of approximately 9:1 βNADH to αNADH. The rate of epimerization of αNADH to βNADH at 38 °C in 0.05, pH 7.5, phosphate buffer is 3.1 × 10^?3 min^?1, corresponding to a half-life of 4 hr. Four related dehydrogenases, yeast and horse liver alcohol dehydrogenase and chicken M₄ and H₄ lactate dehydrogenase, are shown to oxidize αNADH to αNAD⁺ at rates three to four orders of magnitude slower than for βNADH. By using specifically labeled B-deuterio-αNADH the enzymatic oxidation by yeast alcohol dehydrogenase has been shown to occur with the identical stereospecificity as the oxidation of βNADH. The nonenzymatic epimerization of αNADH to βNADH and the enzymatic oxidation αNADH are discussed as a possible source of αNAD⁺in vivo.     

The effect of diabetes, nutritional factors, and sex on rat liver and kidney mevalonate kinase, mevalonate-5-phosphate kinase, and mevalonate-5-pyrophosphate decarboxylase

Isopycnic sucrose gradient separation of rat liver organelles revealed the presence of two distinct branched-chain α-keto acid decarboxylase activities; a mitochondrial activity, which decarboxylates the three branched-chain α-keto acids and requires CoA and NAD⁺ and a cytosolic activity, which decarboxylates α-ketoisocaproate, but not α-ketoisovalerate, or α-keto-β-methylvalerate. The latter enzyme does not require added CoA or NAD⁺. Assay conditions for the cytosolic α-ketoisocaproate decarboxylase activity were optimized and this activity was partially characterized. In rat liver cytosol preparations this activity has a pH optimum of 6.5 and is activated by 1.5 m ammonium sulfate. The decarboxylase activity has an apparent K_m of 0.03 mm for α-ketoisocaproate when optimized assay conditions are employed. Phenylpyruvate is a very potent inhibitor. α-Ketoisovalerate, α-keto-β-methylvalerate, α-ketobutyrate, and α-ketononanoate also inhibit the α-ketoisocaproate decarboxylase activity. The data indicate that the soluble α-ketoisocaproate decarboxylase is an oxidase. Rat liver cytosol preparations consumed oxygen when either α-ketoisocaproate or α-keto-γ-methiolbutyrate were added. None of the other α-keto acids tested stimulated oxygen consumption. 1-¹⁴C-Labeled α-keto-γ-methiolbutyrate is also decarboxylated by cytosol preparations. The α-ketoisocaproate oxidase was purified 20-fold from a 70,000g supernatant fraction of a rat liver homogenate. In these preparations the activity was increased 4-fold by the addition of dithiothreitol, ferrous iron, and ascorbate. The major product of this enzyme activity is β-hydroxyisovalerate. Isovalerate is not a free intermediate in the reaction. The data indicate an alternative pathway for metabolism of α-ketoisocaproate which produces β-hydroxyisovalerate.     

LYSINE METABOLISM IN THE RAT BRAIN: THE PIPECOLIC ACID-FORMING PATHWAY

Employing both the intraventricular and intraperitoneal injection techniques, ¹⁴C-l -lysine at non-overloading concentrations was found to be metabolized to l -¹⁴C-pipecolic acid at significantly high levels in the rat. Labeled pipecolic acid in the brain and liver was only found at rather low levels 24 h after intraperitoneal administration of ¹⁴C-l -lysine regardless of non-labeled lysine metabolite overload. A marked enhancement of pipecolic acid labeling was only found in the brain when ¹⁴C-l -lysine was intraventricularly administered to animals under various lysine metabolite overloads. While overloading doses of non-labeled saccharopine or α-aminoadipate did not significantly alter the labeling patterns of pipecolic acid in the brain, liver or urine when ¹⁴C-l -lysine was intraperitoneally administered, pipecolate overloading markedly reduced labeled pipecolic acid levels in the brain, liver and urine. These results indicate: pipecolic acid formation is subject to product inhibition, and saccharopine is not in the pathway of pipecolic acid synthesis from l -lysine. The labeling pattern of lysine metabolites was not significantly affected by the overloading injection of pipecolic acid when ¹⁴C-l -lysine was intraventricularly administered suggesting a blood-brain barrier for pipecolate. Besides ¹⁴C-pipecolic acid, labeled α-aminoadipic acid was also found at significant levels mostly in the brain. Labeled saccharopine was not detected in any tissues or urine samples analyzed. The ¹⁴C-l -lysine metabolic pattern of the newborn rats did not seem to be any different from the adult rats, i.e. labeled pipecolic acid was also detected in substantial quantities in the brain, liver and urine 5 h after injection. ¹⁴C-d -Lysine was mainly metabolized to l -¹⁴C-pipecolic acid through either route of administration. These experimental evidences indicate that the pipecolic acid-forming pathway is a significant route for lysine metabolism in the rat, and that the rat brain probably utilizes this pathway mainly for lysine metabolism. The present study also discusses the potential neurological significance of the pipecolic acid pathway in relation to the major lysine metabolic pathway (the saccharopine pathway).     

OXPHOS-Mediated Induction of NAD+ Promotes Complete Oxidation of Fatty Acids and Interdicts Non-Alcoholic Fatty Liver Disease

OXPHOS is believed to play an important role in non-alcoholic fatty liver disease (NAFLD), however, precise mechanisms whereby OXPHOS influences lipid homeostasis are incompletely understood. We previously reported that ectopic expression of LRPPRC, a protein that increases cristae density and OXPHOS, promoted fatty acid oxidation in cultured primary hepatocytes. To determine the biological significance of that observation and define underlying mechanisms, we have ectopically expressed LRPPRC in mouse liver in the setting of NAFLD. Interestingly, ectopic expression of LRPPRC in mouse liver completely interdicted NAFLD, including inflammation. Consistent with mitigation of NAFLD, two markers of hepatic insulin resistance—ROS and PKCε activity—were both modestly reduced. As reported by others, improvement of NAFLD was associated with improved whole-body insulin sensitivity. Regarding hepatic lipid homeostasis, the ratio of NAD⁺ to NADH was dramatically increased in mouse liver replete with LRPPRC. Pharmacological activators and inhibitors of the cellular respiration respectively increased and decreased the [NAD⁺]/[NADH] ratio, indicating respiration-mediated control of the [NAD⁺]/[NADH] ratio. Supporting a prominent role for NAD⁺, increasing the concentration of NAD⁺ stimulated complete oxidation of fatty acids. Importantly, NAD⁺ rescued impaired fatty acid oxidation in hepatocytes deficient for either OXPHOS or SIRT3. These data are consistent with a model whereby augmented hepatic OXPHOS increases NAD⁺, which in turn promotes complete oxidation of fatty acids and protects against NAFLD.     

The use of steady-state treatment in the rapid kinetics of horse liver alcohol dehydrogenase. The effect of addition of product on the "burst" reaction.

The effect of addition of product on the amplitude of the “burst” reaction of horse liver alcohol dehydrogenase was studied using a stopped-flow spectrophotofluorimeter. The amplitude of the “burst” formation of enzyme-bound NADH fluorescence was completely diminished by the addition of excess acetaldehyde or benzaldehyde in the reaction with NAD⁺ and ethanol or NAD⁺ and benzylalcohol, respectively. The results indicate that a significant concentration of the ternary enzyme-coenzyme-substrate complex was formed during the steady-state in the presence of product, and this ternary complex did not exhibit NADH fluorescence. The dissociation constants for the ternary complex were determined from the amplitudes of the “burst” reactions. The “active site” titration of the enzyme with NAD⁺ in the presence of ethanol and iso-butyramide is also described.     

Activity of liver mitochondrial Krebs cycle NAD<Superscript>+</Superscript>-dependent dehydrogenases in rats with hepatitis induced by acetaminophen under conditions of alimentary protein deficiency

Activity of isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, malate dehydrogenase, and the NAD⁺/NADН ratio were studied in the liver mitochondrial fraction of rats with toxic hepatitis induced by acetaminophen under conditions of alimentary protein deficiency. Acetaminophen-induced hepatitis was characterized by a decrease of isocitrate dehydrogenase, α-ketoglutarate dehydrogenase and malate dehydrogenase activities, while the mitochondrial NAD⁺/NADН ratio remained at the control level. Modeling of acetaminophen-induced hepatitis in rats with alimentary protein deficiency caused a more pronounced decrease in the activity of studied Krebs cycle NAD⁺-dependent dehydrogenases and a 2.2-fold increase of the mitochondrial NAD⁺/NADН ratio.     

Preferential uptake of d-α-aminoadipate from a racemic mixture by an Alcaligenes denitrificans

During growth on racemic α-aminoadipate as a sole source of carbon and nitrogen an Alcaligenes denitrificans displayed a two-phase (diauxic) growth not shown by a Pseudomonas putida. Oxidation studies in the presence of chloramphenicol showed that in both growth phases whole cells of A. denitrificans had the ability to oxidize either isomer of α-aminoadipate. These data negated as the physiological basis for diauxic growth a proposal that one isomer was repressing the synthesis of enzymes which transport or degrade the other enantiomer. Growth studies with various ratios of the d- and l-isomers of α-aminoadipate, differentiating one isomer from the other by radioactivity, indicated that diauxic growth resulted from a preferential uptake of d-α-aminoadipate over the l-isomer.     

Studies on the metabolism of galactose-6-phosphate in rat heart and brain.

The subcellular distribution of NADP⁺ and NAD⁺-dependent glucose-6-phosphate and galactose-6-phosphate dehydrogenases were studied in rat liver, heart, brain, and chick brain. Only liver particulate fractions oxidized glucose-6-phosphate and galactose-6-phosphate with either NADP⁺ or NAD⁺ as cofactor. While all of the tissues examined had NADP⁺-dependent glucose-6-phosphate dehydrogenase activity, only rat liver and rat brain soluble fractions had NADP⁺-dependent galactose-6-phosphate dehydrogenase activity. Rat liver microsomal and rat brain soluble galactose-6-phosphate dehydrogenase activities were kinetically different (K_m's 0.5 mm and 10 mm, respectively, for galactose-6-phosphate), although their reaction products were both 6-phosphogalactonate. Rat brain subcellular fractions did not oxidize 6-phosphogalactonate with either NADP⁺ or NAD⁺ cofactors but phosphatase activities hydrolyzing 6-phosphogalactonate, galactose-6-phosphate and galactose-1-phosphate were found in crude brain homogenates. In addition, galactose-6-phosphate and 6-phosphogalactonate were tested as inhibitors of various enzymes, with largely negative results, except that 6-phosphogalactonate was a competitive inhibitor (K_i = 0.5 mM) of rat brain 6-phosphogluconate dehydrogenase.     

Sulfhydryl groups in urocanase: Relationship to nicotinamide adenine dinucleotide binding

This study concerned the role of the sulfhydryl groups in urocanase of Pseudomonas putida. When p-chloromercuribenzoate was added to the enzyme, two sulfhydryl groups reacted at once with little inhibition; the enzyme slowly became inhibited while further sulfhydryls reacted. After the p-chloromercuribenzoate inhibition occurred, if a thiol was subsequently added, most of the original activity was recovered. As the incubation time with p-chloromercuribenzoate was increased, the thiol became less effective in reversing the inhibition. However, if NAD⁺ (10 μm) was added with the thiol, 60–90% of the initial activity was restored even after long p-chloromercuribenzoate incubations. Restoration of activity by NAD⁺ was concentration dependent and specific for NAD⁺. Radioactive NAD⁺ could be bound to urocanase. These results confirm the coenzyme role for NAD⁺ in urocanase. In urea, p-chloromercuribenzoate titration of urocanase measured 11.9 -SH groups per molecule. Sulfite-modified enzyme treated with p-chloromercuribenzoate and dialyzed was substantially photoactivated in the presence of a thiol; that is, NAD⁺ was not required to restore activity. From these results, it is proposed that this enzyme contains two reactive —SH groups and that an essential —SH group is involved in NAD⁺ binding. Forces present in the sulfite-modified enzyme prevent the release of the NAD⁺ in the presence of mercurials.     

Identification of activation of tryptophan–NAD+ pathway as a prominent metabolic response to thermally oxidized oil through metabolomics-guided biochemical analysis

Consumption of thermally oxidized oil is associated with metabolic disorders, but oxidized oil-elicited changes in the metabolome are not well defined. In this study, C57BL/6 mice were fed the diets containing either control soybean oil or heated soybean oil (HSO) for 4 weeks. HSO-responsive metabolic events were examined through untargeted metabolomics-guided biochemical analysis. HSO directly contributed to the presence of new HSO-derived metabolites in urine and the decrease of polyunsaturated fatty acid-containing phospholipids in serum and the liver. HSO disrupted redox balance by decreasing hepatic glutathione and ascorbic acid. HSO also activated peroxisome proliferator-activated receptors, leading to the decrease of serum triacylglycerols and the changes of cofactors and products in fatty acid oxidation pathways. Most importantly, multiple metabolic changes, including the decrease of tryptophan in serum; the increase of NAD⁺ in the liver; the increases of kynurenic acid, nicotinamide and nicotinamide N-oxide in urine; and the decreases of the metabolites from pyridine nucleotide degradation in the liver indicated that HSO activated tryptophan–NAD⁺ metabolic pathway, which was further confirmed by the upregulation of gene expression in this pathway. Because NAD⁺ and its metabolites are essential cofactors in many HSO-induced metabolic events, the activation of tryptophan–NAD⁺ pathway should be considered as a central metabolic response to the exposure of HSO.     

Exogenous NAD Effects on Plant Mitochondria: A Reinvestigation of the Transhydrogenase Hypothesis

Addition of NAD⁺ to purified potato (Solanum tuberosum L.) mitochondria respiring α-ketoglutarate and malate in the presence of the electron transport inhibitor rotenone, stimulated O₂ uptake. This stimulation was prevented by incubating mitochondria with N-4-azido-2-nitrophenyl-aminobutyryl-NAD⁺ (NAP₄-NAD⁺), an inhibitor of NAD⁺ uptake, but not by 1 mm EGTA, an inhibitor of external NADH oxidation. NAD⁺-stimulated malate-cytochrome c reductase activity, and reduction of added NAD⁺ by intact mitochondria, could be duplicated by rupturing the mitochondria and adding a small quantity to the cuvette. The extent of external NAD⁺ reduction was correlated with the amount of extra mitochondrial malate dehydrogenase present. Malate oxidation by potato mitochondria depleted of endogenous NAD⁺ by storing on ice for 72 hours, was completely dependent on added NAD⁺, and the effect of NAD⁺ on these mitochondria was prevented by incubating them with NAP₄-NAD⁺. External NAD⁺ reduction by these mitochondria was not affected by NAP₄-NAD⁺. We conclude that all effects of exogenous NAD⁺ on plant mitochondrial respiration can be attributed to net uptake of the NAD⁺ into the matrix space.     

Purification and properties of a mitochondrial NAD+ glycohydrolase

A 60- to 70-fold purification of an NAD⁺ glycohydrolase from the inner membrane of rat liver mitochondria to apparent homogeneity on sodium dodecyl sulfate (SDS)-polyacrylamide slab gel is described. The minimum molecular weight of the enzyme on polyacrylamide gels in the presence of SDS is around 62,000. The enzyme splits NAD⁺ to ADP-ribose and, presumably, nicotinamide. No phosphatase or phosphodiesterase activity is detected in the purified enzyme preparation. The enzyme shows high activity with NAD⁺ and moderate activity with NADP⁺ as substrates NAD(P)Hs are poor substrates. ATP and nicotinamide inhibit the enzyme. A possible participation of the enzyme in the mechanism of calcium release from rat liver mitochondria is discussed.     

Transport of NAD in Percoll-Purified Potato Tuber Mitochondria: Inhibition of NAD Influx and Efflux by N-4-Azido-2-nitrophenyl-4-aminobutyryl-3'-NAD

A mechanism by which intact potato (Solanum tuberosum) mitochondria may regulate the matrix NAD content was studied in vitro. If mitochondria were incubated with NAD⁺ at 25°C in 0.3 molar mannitol, 10 millimolar phosphate buffer (pH 7.4), 5 millimolar MgCl₂, and 5 millimolar α-ketoglutarate, the NAD pool size increased with time. In the presence of uncouplers, net uptake was not only inhibited, but NAD⁺ efflux was observed instead. Furthermore, the rate of NAD⁺ accumulation in the matrix space was strongly inhibited by the analog N-4-azido-2-nitrophenyl-4-aminobutyryl-3′-NAD⁺. When suspended in a medium that avoided rupture of the outer membrane, intact purified mitochondria progressively lost their NAD⁺ content. This led to a slow decrease of NAD⁺-linked substrates oxidation by isolated mitochondria The rate of NAD⁺ efflux from the matrix space was strongly temperature dependent and was inhibited by the analog inhibitor of NAD⁺ transport indicating that a carrier was required for net flux in either direction. It is proposed that uptake and efflux operate to regulate the total matrix NAD pool size.     

Subcellular Distribution of NAD+ between Cytosol and Mitochondria Determines the Metabolic Profile of Human Cells

The mitochondrial NAD pool is particularly important for the maintenance of vital cellular functions. Although at least in some fungi and plants, mitochondrial NAD is imported from the cytosol by carrier proteins, in mammals, the mechanism of how this organellar pool is generated has remained obscure. A transporter mediating NAD import into mammalian mitochondria has not been identified. In contrast, human recombinant NMNAT3 localizes to the mitochondrial matrix and is able to catalyze NAD⁺ biosynthesis in vitro. However, whether the endogenous NMNAT3 protein is functionally effective at generating NAD⁺ in mitochondria of intact human cells still remains to be demonstrated. To modulate mitochondrial NAD⁺ content, we have expressed plant and yeast mitochondrial NAD⁺ carriers in human cells and observed a profound increase in mitochondrial NAD⁺. None of the closest human homologs of these carriers had any detectable effect on mitochondrial NAD⁺ content. Surprisingly, constitutive redistribution of NAD⁺ from the cytosol to the mitochondria by stable expression of the Arabidopsis thaliana mitochondrial NAD⁺ transporter NDT2 in HEK293 cells resulted in dramatic growth retardation and a metabolic shift from oxidative phosphorylation to glycolysis, despite the elevated mitochondrial NAD⁺ levels. These results suggest that a mitochondrial NAD⁺ transporter, similar to the known one from A. thaliana, is likely absent and could even be harmful in human cells. We provide further support for the alternative possibility, namely intramitochondrial NAD⁺ synthesis, by demonstrating the presence of endogenous NMNAT3 in the mitochondria of human cells.     

New enzyme cycling method for determination of oxidized and reduced nicotinamide adenine dinucleotide

A highly sensitive enzyme cycling method for the measurement of NAD⁺(H) concentration in tissue extracts has been described. The assay measured propanediol formed during the coupled oxidation-reduction reactions between ethanol and lactaldehyde as catalyzed by liver alcohol dehydrogenase in the presence of catalytic concentrations of NAD⁺(H). The assay can be used to determine NAD⁺(H) concentrations in the range of 0.4 to 4 pmoles of coenzyme in a 0.1 ml reaction volume.     

Vitamin E deficiency in the rat. IV. Alteration in mitochondrial membrane and its relation to respiratory decline

The respiratory control and rate of oxidation of exogenous NADH in vitro by liver mitochondria from vitamin E deficient rats were studied as a means of providing information concerning possible mitochondrial membrane alterations due to the deficiency.When mitochondria were aged at different temperatures for various periods of time, half-maximal inhibition of respiratory control occurred at lower temperatures and shorter aging periods in deficient mitochondria than in normal ones. Also, respiratory control was lost more rapidly in deficient mitochondria than in normal ones in the presence of either digitonin or low (hypotonic) concentrations of mannitol.Microsomes, both freshly prepared and boiled, dramatically lowered respiratory control and the effect was greater in the deficient mitochondria. Bovine serum albumin overcame the suppressed respiratory control, and exogenously added fatty acids mimiced the action of the microsomes.NADH oxidation by normal mitochondria proceeded slowly in isotonic media, while mitochondria of vitamin E deficient rats oxidized NADH much more rapidly. When mitochondria were subjected to ultrasonic disruption or incubated in hypotonic media, the rates of NADH oxidation by both types of mitochondria were similar.Respiratory decline associated with oxidation of β-hydroxybutyrate by the deficient mitochondria was decreased by including in the medium either a high concentration of NAD⁺, 0.5 mm oxalacetate, or 2 mm aspartate plus 1 mm α-ketoglutarate. This observation, plus the finding of similar activities of malate dehydrogenase and glutamic-oxalacetic transminase in normal and deficient livers, suggests that the action of each was due to an elevation of the mitochondrial NAD⁺/NADH ratio via a malate shuttle and cytoplasmic and mitochondrial glutamic-oxalacetate transaminase. It is postulated that the marked mitochondrial respiratory decline in the deficient rats is attributed to a limiting availability of NAD⁺ and a low ratio of NAD⁺ to NADH.     

A new ternary complex between horse liver alcohol dehydrogenase, NAD+, and acetone

Acetone was found to form a dead-end ternary complex with horse liver alcohol dehydrogenase and oxidized nicotinamide adenine dinucleotide (NAD⁺) when the reactants were incubated for a long time at relatively high concentrations. The complex formation was demonstrated by measuring the increase in absorbance at 320 nm, the quenching of protein fluorescence, and the loss of enzyme activity. Since acetone is a substrate of liver alcohol dehydrogenase, and the presence of acetaldehyde or pyrazole prevents acetone from forming the dead-end complex with liver alcohol dehydrogenase and NAD⁺, the acetone molecule in the complex may be bound to the substrate binding site of liver alcohol dehydrogenase. The dissociation of the complex was demonstrated by prolonged dialysis or by addition of reduced nicotinamide adenine dinucleotide (NADH) and iso-butyramide. A modified nicotinamide adenine dinucleotide was obtained as a main product from the dead-end complex after dissociation of the complex or denaturation of the apoenzyme. The modified nicotinamide adenine dinucleotide was found to exhibit an absorption spectrum similar to that of NADH; however, it was not oxidizable by liver alcohol dehydrogenase in the presence of acetaldehyde and exhibited no fluorescence.     

Nutritional Energy Stimulates NAD+ Production to Promote Tankyrase-Mediated PARsylation in Insulinoma Cells

The poly-ADP-ribosylation (PARsylation) activity of tankyrase (TNKS) regulates diverse physiological processes including energy metabolism and wnt/β-catenin signaling. This TNKS activity uses NAD⁺ as a co-substrate to post-translationally modify various acceptor proteins including TNKS itself. PARsylation by TNKS often tags the acceptors for ubiquitination and proteasomal degradation. Whether this TNKS activity is regulated by physiological changes in NAD⁺ levels or, more broadly, in cellular energy charge has not been investigated. Because the NAD⁺ biosynthetic enzyme nicotinamide phosphoribosyltransferase (NAMPT) in vitro is robustly potentiated by ATP, we hypothesized that nutritional energy might stimulate cellular NAMPT to produce NAD⁺ and thereby augment TNKS catalysis. Using insulin-secreting cells as a model, we showed that glucose indeed stimulates the autoPARsylation of TNKS and consequently its turnover by the ubiquitin-proteasomal system. This glucose effect on TNKS is mediated primarily by NAD⁺ since it is mirrored by the NAD⁺ precursor nicotinamide mononucleotide (NMN), and is blunted by the NAMPT inhibitor FK866. The TNKS-destabilizing effect of glucose is shared by other metabolic fuels including pyruvate and amino acids. NAD⁺ flux analysis showed that glucose and nutrients, by increasing ATP, stimulate NAMPT-mediated NAD⁺ production to expand NAD⁺ stores. Collectively our data uncover a metabolic pathway whereby nutritional energy augments NAD⁺ production to drive the PARsylating activity of TNKS, leading to autoPARsylation-dependent degradation of the TNKS protein. The modulation of TNKS catalytic activity and protein abundance by cellular energy charge could potentially impose a nutritional control on the many processes that TNKS regulates through PARsylation. More broadly, the stimulation of NAD⁺ production by ATP suggests that nutritional energy may enhance the functions of other NAD⁺-driven enzymes including sirtuins.