Physiological Substrate Concentrations Research Articles

Fructose-1-kinase has pleiotropic roles in Escherichia coli

In Escherichia coli, the master transcription regulator Catabolite Repressor Activator (Cra) regulates >100 genes in central metabolism. Cra binding to DNA is allosterically regulated by binding to fructose-1-phosphate (F-1-P), but the only documented source of F-1-P is from the concurrent import and phosphorylation of exogenous fructose. Thus, many have proposed that fructose-1,6-bisphosphate (F-1,6-BP) is also a physiological regulatory ligand. However, the role of F-1,6-BP has been widely debated. Here, we report that the E. coli enzyme fructose-1-kinase (FruK) can carry out its “reverse” reaction under physiological substrate concentrations to generate F-1-P from F-1,6-BP. We further show that FruK directly binds Cra with nanomolar affinity and forms higher order, heterocomplexes. Growth assays with a ΔfruK strain and fruK complementation show that FruK has a broader role in metabolism than fructose catabolism. Since fruK itself is repressed by Cra, these newly-reported events add layers to the dynamic regulation of E. coli central metabolism that occur in response to changing nutrients. These findings might have wide-spread relevance to other γ-proteobacteria, which conserve both Cra and FruK.

Phosphoserine Aminotransferase Pathogenetic Variants in Serine Deficiency Disorders: A Functional Characterization.

In humans, the phosphorylated pathway (PP) converts the glycolytic intermediate D-3-phosphoglycerate (3-PG) into L-serine through the enzymes 3-phosphoglycerate dehydrogenase, phosphoserine aminotransferase (PSAT) and phosphoserine phosphatase. From the pathogenic point of view, the PP in the brain is particularly relevant, as genetic defects of any of the three enzymes are associated with a group of neurometabolic disorders known as serine deficiency disorders (SDDs). We recombinantly expressed and characterized eight variants of PSAT associated with SDDs and two non-SDD associated variants. We show that the pathogenetic mechanisms in SDDs are extremely diverse, including low affinity of the cofactor pyridoxal 5'-phosphate and thermal instability for S179L and G79W PSAT, loss of activity of the holo form for R342W PSAT, aggregation for D100A PSAT, increased Km for one of the substrates with invariant kcats for S43R PSAT, and a combination of increased Km and decreased kcat for C245R PSAT. Finally, we show that the flux through the in vitro reconstructed PP at physiological concentrations of substrates and enzymes is extremely sensitive to alterations of the functional properties of PSAT variants, confirming PSAT dysfunctions as a cause of SSDs.

3βHSD activity saturates at physiological substrate concentrations in intact cells.

Conversion of adrenally produced dehydroepiandrosterone (DHEA) to the potent androgen dihydrotestosterone (DHT) is an important mechanism by which prostate cancer reaches castration resistance. At the start of this pathway is a branch point at which DHEA can be converted to Δ4 -androstenedione by the enzyme 3β-hydroxysteroid dehydrogenase (3βHSD) or to Δ5 -androstenediol by 17βHSD. To better understand this process, we studied the kinetics of these reactions in cells. Prostate cancer cells (LNCaP cell line) were incubated with steroids (DHEA and Δ5 -androstenediol) over a range of concentrations and the steroid metabolism reaction products were measured by mass spectrometry or by high-performance liquid chromatography to determine reaction kinetics. To confirm the generalizability of results, experiments were also performed in JEG-3 placental choriocarcinoma cells. The two reactions displayed very different saturation profiles, with only the 3βHSD-catalyzed reaction beginning to saturate within a physiological substrate concentration range. Strikingly, incubating LNCaP cells with low (in the ~10 nM range) concentrations of DHEA resulted in a large majority of the DHEA undergoing 3βHSD-catalyzed conversion to Δ4 -androstenedione, whereas high concentrations of DHEA (in the 100s of nM range) resulted in most of the DHEA undergoing 17βHSD-catalyzed conversion to Δ5 -androstenediol. Contrary to expectations from previous studies that used purified enzyme, cellular metabolism of DHEA by 3βHSD begins to saturate in the physiological concentration range, suggesting that fluctuations in DHEA concentrations could be buffered at the downstream active androgen level.

The human phosphorylated pathway: a multienzyme metabolic assembly for l-serine biosynthesis.

De novo l-serine biosynthesis in the mammalian astrocytes proceeds via a linear, three-step pathway (the phosphorylated pathway) catalysed by 3-phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase (PSAT) and phosphoserine phosphatase (PSP). The first reaction, catalysed by PHGDH and using the glycolytic intermediate 3-phosphoglycerate, is strongly shifted towards the reagents, and coupling to the following step by PSAT is required to push the equilibrium towards l-serine formation; the last step, catalysed by PSP, is virtually irreversible and inhibited by the final product l-serine. Very little is known about the regulation of the human phosphorylated pathway and the ability of the three enzymes to organise in a complex with potential regulatory functions. Here, the complex formation was investigated in differentiated human astrocytes, by proximity ligation assay, and in vitro on the human recombinant enzymes. The results indicate that the three enzymes co-localise in cytoplasmic clusters that more stably engage PSAT and PSP. Although in vitro analyses based on native PAGE, size exclusion chromatography and cross-linking experiments do not show the formation of a stable complex, kinetic studies of the reconstituted pathway using physiological enzyme and substrate concentrations support cluster formation and indicate that PHGDH catalyses the rate-limiting step while PSP reaction is the driving force for the whole pathway. The enzyme agglomerate assembly of the phosphorylated pathway (the putative 'serinosome') delivers a relevant level of sophistication to the control of l-serine biosynthesis in human cells, a process strictly related to the modulation of the brain levels of d-serine and glycine, the main co-agonists of N-methyl-d-aspartate receptors and various pathological states.

Simulation modeling of glutamate cysteine ligase activity

L-Y-glutamyl-L-cysteinyl glycine, or glutathione, as one of the basic intracellular antioxidants, plays a vital role in cellular metabolism. In mammalian cells, glutathione is synthesized via two steps. The first step that is considered rate limiting is catalyzed by glutamate cysteine ligase. In this work, a stochastic algorithm based on continuous-time Markov chains was used to simulate the activity of glutamate-cysteine ligase. Several different mechanisms of enzymatic activity including reversible inhibition of glutathione, and an ATP binding motif have been considered. Based on physiological metabolite measurements made for human erythrocytes, the activity of glutamate cysteine ligase was determined. There are many possible ways for substrates to bind to an active site of the studied enzyme, but, only the mechanism by which primary binding to ATP can occur makes it possible to obtain the catalytic rate value similar to that of the experimentally measured glutamatecysteine ligase activity relative to physiological concentrations of substrates. In other cases, the values differ by more than one order of magnitude. The performed analysis allows the conclusion that when models for glutathione biosynthesis are constructed in vivo conditions, the ATP concentration and reversible inhibition of glutathione should be taken into account.

Genetic and biocatalytic basis of formate dependent growth of Escherichia coli strains evolved in continuous culture

The reductive glycine pathway was described as the most energetically favorable synthetic route of aerobic formate assimilation. Here we report the successful implementation of formatotrophy in Escherichia coli by means of a stepwise adaptive evolution strategy. Medium swap and turbidostat regimes of continuous culture were applied to force the channeling of carbon flux through the synthetic pathway to pyruvate establishing growth on formate and CO2 as sole carbon sources. Labeling with 13C-formate proved the assimilation of the C1 substrate via the pathway metabolites. Genetic analysis of intermediate isolates revealed a mutational path followed throughout the adaptation process. Mutations were detected affecting the copy number (gene ftfL) or the coding sequence (genes folD and lpd) of genes which specify enzymes implicated in the three steps forming glycine from formate and CO2, the central metabolite of the synthetic pathway. The mutation R191S present in methylene-tetrahydrofolate dehydrogenase/cyclohydrolase (FolD) abolishes the inhibition of cyclohydrolase activity by the substrate formyl-tetrahydrofolate. The mutation R273H in lipoamide dehydrogenase (Lpd) alters substrate affinities as well as kinetics at physiological substrate concentrations likely favoring a reactional shift towards lipoamide reduction. In addition, genetic reconstructions proved the necessity of all three mutations for formate assimilation by the adapted cells. The largely unpredictable nature of these changes demonstrates the usefulness of the evolutionary approach enabling the selection of adaptive mutations crucial for pathway engineering of biotechnological model organisms.

The advantages of describing covalent inhibitor in vitro potencies by IC50 at a fixed time point. IC50 determination of covalent inhibitors provides meaningful data to medicinal chemistry for SAR optimization

Recent years have seen a resurgence in drug discovery efforts aimed at the identification of covalent inhibitors which has led to an explosion of literature reports in this area and most importantly new approved therapies. These reports and breakthroughs highlight the significant investments made across the industry in SAR campaigns to optimize inhibitors. The potency of covalent inhibitors is generally considered to be more accurately described by the time-independent kinetic parameter kinact/Ki rather than a by a simple IC50 since the latter is a time-dependent parameter. Enzyme substrate concentrations are an additional important factor to consider when attempting to translate parameters derived from enzymology experiments to phenotypic behavior in a physiologically relevant cell-based system. Theoretical and experimental investigations into the relationship between IC50, time, substrate concentration and Kinact/Ki provided us with an effective approach to provide meaningful data for SAR optimization. The data we generated for our JAK3 irreversible covalent inhibitor program using IC50 values provided by enzyme assays with long incubations (>1h) coupled with physiological substrate concentration provided the medicinal chemist with optimal information in a rapid and efficient manner. We further document the wide applicability of this method by applying it to other enzymes systems where we have run covalent inhibitor programs.

Physiological Role of Glutamate Dehydrogenase in Cancer Cells.

increased growth rates and final densities of several human metastatic cancer cells. To assess whether glutamate dehydrogenase (GDH) in cancer cells may catalyze the reverse reaction of fixation, its covalent regulation and kinetic parameters were determined under near-physiological conditions. Increased total protein and phosphorylation were attained in -supplemented metastatic cells, but total cell GDH activity was unchanged. Higher Vmax values for the GDH reverse reaction vs. forward reaction in both isolated hepatoma (HepM) and liver mitochondria [rat liver mitochondria (RLM)] favored an -fixing role. GDH sigmoidal kinetics with , ADP, and leucine fitted to Hill equation showed nH values of 2 to 3. However, the K0.5 values for were over 20 mM, questioning the physiological relevance of the GDH reverse reaction, because intracellular in tumors is 1 to 5 mM. In contrast, data fitting to the Monod–Wyman–Changeux (MWC) model revealed lower Km values for , of 6 to 12 mM. In silico analysis made with MWC equation, and using physiological concentrations of substrates and modulators, predicted GDH N-fixing activity in cancer cells. Therefore, together with its thermodynamic feasibility, GDH may reach rates for its reverse, -fixing reaction that are compatible with an anabolic role for supporting growth of cancer cells.

Thermal sensitivity of white muscle lactate dehydrogenase isolated from a lake trout, (Salmo trutta), inhabiting lake Plav, Montenegro

To shed light on thermoadaptive properties of Salmo trutta from lake Plav (Montenegro), we undertook kinetic studies of pyruvate reduction rates and thermal stability analyses of white muscle LDH. We compared these with the data obtained for trout of the same, confirmed by us, Danubian lineage living in rivers and streams of Serbia and Montenegro. We also tested the effect of acclimation in captivity at 4 and 14 °C. The lake trout was of a typical smoltified phenotype (the size, the elongated silver colored body). At physiological substrate concentration, the breaks in the Arrhenius plots (critical temperature - Tc) correlated with acclimation temperatures or habitat water temperatures. Q10 values for temperatures above Tc were close to one, in all cases except 4 °C acclimated trout. At temperatures below Tc Q10 was close to two, except in the case of 14 °C acclimated trout. Lake trout had a highest Q10 values at temperatures below Tc. It was conspicuous that within the entire range of tested temperatures the differences in Q10 resulted from the effect of environmental temperature. Higher Q10 values were obtained with LDH isolated from trout acclimated to 4 °C compared with LDH acclimated to 14 °C. Ea values were much lower at a temperature below Tc compared with temperatures above Tc. Thermal stability of muscle LDH was lower after acclimation to 14 compared to 4 °C, while extremely high thermostability was obtained with the lake trout enzyme. Our data support the concept that Tc values have distinct physiological significance.

Protein disulfide isomerase and glutathione are alternative substrates in the one Cys catalytic cycle of glutathione peroxidase 7

BackgroundMammalian GPx7 is a monomeric glutathione peroxidase of the endoplasmic reticulum (ER), containing a Cys redox center (CysGPx). Although containing a peroxidatic Cys (CP) it lacks the resolving Cys (CR), that confers fast reactivity with thioredoxin (Trx) or related proteins to most other CysGPxs. MethodsReducing substrate specificity and mechanism were addressed by steady-state kinetic analysis of wild type or mutated mouse GPx7. The enzymes were heterologously expressed as a synuclein fusion to overcome limited expression. Phospholipid hydroperoxide was the oxidizing substrate. Enzyme–substrate and protein–protein interaction were analyzed by molecular docking and surface plasmon resonance analysis. ResultsOxidation of the CP is fast (k+1>103M−1s−1), however the rate of reduction by GSH is slow (k′+2=12.6M−1s−1) even though molecular docking indicates a strong GSH–GPx7 interaction. Instead, the oxidized CP can be reduced at a fast rate by human protein disulfide isomerase (HsPDI) (k+1>103M−1s−1), but not by Trx. By surface plasmon resonance analysis, a KD=5.2μM was calculated for PDI–GPx7 complex. Participation of an alternative non-canonical CR in the peroxidatic reaction was ruled out. Specific activity measurements in the presence of physiological reducing substrate concentration, suggest substrate competition in vivo. ConclusionsGPx7 is an unusual CysGPx catalyzing the peroxidatic cycle by a one Cys mechanism in which GSH and PDI are alternative substrates. General significanceIn the ER, the emerging physiological role of GPx7 is oxidation of PDI, modulated by the amount of GSH.

Regulation of succinate-fueled mitochondrial respiration in liver and skeletal muscle of hibernating thirteen-lined ground squirrels

Hibernating ground squirrels (Ictidomys tridecemlineatus) alternate between two distinct metabolic states throughout winter: torpor, during which metabolic rate (MR) and body temperature (Tb) are considerably suppressed, and interbout euthermia (IBE), during which MR and Tb briefly return to euthermic levels. Previous studies showed suppression of succinate-fuelled respiration during torpor in liver and skeletal muscle mitochondria; however, these studies used only a single, saturating succinate concentration. Therefore, they could not address whether mitochondrial metabolic suppression occurs under physiological substrate concentrations or whether differences in the kinetics of mitochondrial responses to changing substrate concentration might also contribute to mitochondrial metabolic regulation during torpor. The present study confirmed that succinate oxidation is reduced during torpor in liver and skeletal muscle at 37 and 10°C over a 100-fold range of succinate concentrations. At 37°C, this suppression resulted from inhibition of succinate dehydrogenase (SDH), which had a greater affinity for oxaloacetate (an SDH inhibitor) during torpor. At 10°C, SDH was not inhibited, suggesting that SDH inhibition initiates but does not maintain mitochondrial suppression during torpor. Moreover, in both liver and skeletal muscle, mitochondria from torpid animals maintained relatively higher respiration rates at low succinate concentrations, which reduces the extent of energy savings that can be achieved during torpor, but may also maintain mitochondrial oxidative capacity above some lower critical threshold, thereby preventing cellular and/or mitochondrial injury during torpor and facilitating rapid recruitment of oxidative capacity during arousal.

Complement C3 is a substrate for activated factor XIII that is cross‐linked to fibrin during clot formation

Complement C3 is a substrate for activated factor <scp>XIII</scp> that is cross‐linked to fibrin during clot formation

Biochemical Characterization of Enzyme Fidelity of Influenza A Virus RNA Polymerase Complex

BackgroundIt is widely accepted that the highly error prone replication process of influenza A virus (IAV), together with viral genome assortment, facilitates the efficient evolutionary capacity of IAV. Therefore, it has been logically assumed that the enzyme responsible for viral RNA replication process, influenza virus type A RNA polymerase (IAV Pol), is a highly error-prone polymerase which provides the genomic mutations necessary for viral evolution and host adaptation. Importantly, however, the actual enzyme fidelity of IAV RNA polymerase has never been characterized.Principal FindingsHere we established new biochemical assay conditions that enabled us to assess both polymerase activity with physiological NTP pools and enzyme fidelity of IAV Pol. We report that IAV Pol displays highly active RNA-dependent RNA polymerase activity at unbiased physiological NTP substrate concentrations. With this robust enzyme activity, for the first time, we were able to compare the enzyme fidelity of IAV Pol complex with that of bacterial phage T7 RNA polymerase and the reverse transcriptases (RT) of human immunodeficiency virus (HIV-1) and murine leukemia virus (MuLV), which are known to be low and high fidelity enzymes, respectively. We observed that IAV Pol displayed significantly higher fidelity than HIV-1 RT and T7 RNA polymerase and equivalent or higher fidelity than MuLV RT. In addition, the IAV Pol complex showed increased fidelity at lower temperatures. Moreover, upon replacement of Mg++ with Mn++, IAV Pol displayed increased polymerase activity, but with significantly reduced processivity, and misincorporation was slightly elevated in the presence of Mn++. Finally, when the IAV nucleoprotein (NP) was included in the reactions, the IAV Pol complex exhibited enhanced polymerase activity with increased fidelity.SignificanceOur study indicates that IAV Pol is a high fidelity enzyme. We envision that the high fidelity nature of IAV Pol may be important to counter-balance the multiple rounds of IAV genome amplification per infection cycle, which provides IAV Pol with ample opportunities to generate and amplify genomic founder mutations, and thus achieve optimal viral mutagenesis for its evolution.

Nomegestrol acetate is an anti-aromatase agent in human MCF-7aro breast cancer cells.

The progestogen nomegestrol acetate (NOMAC), a 17α-hydroxy-nor-progesterone derivative (LUTENYL®) is largely used as an oral contraceptive and to treat menopausal complaints. In previous studies, we demonstrated that NOMAC is an anti-sulfatase agent in MCF-7 and T-47D breast cancer cells. In this study, we explore the effect of NOMAC on aromatase activity in a stable aromatase-expressing human breast cancer cell line: MCF-7aro. Cells were incubated with physiological concentrations of androgen substrates [3H]-testosterone or [3H]-androstenedione (5×10-9mol/L) alone, or in the presence of NOMAC (5×10-5mol/L-5×10-8mol/L) for 24h at 37°C. [3H]-Estradiol (E2), [3H]-estrone (E1), [3H]-testosterone and [3H]-androstenedione were characterized by thin layer chromatography and quantified using the corresponding standard. Aromatase activity levels are high in MCF-7aro cells because the [3H]-E2 concentration after incubation of [3H]-testosterone was 5.8±0.31pmol/mg DNA in non-treated cells. At concentrations of 5×10-5mol/L, 5×10-6mol/L and 5×10-7mol/L NOMAC significantly inhibits this conversion by 49.7%, 29.9% and 18.1%, respectively. After [3H]-androstenedione incubation, similar inhibition levels were observed with NOMAC for [3H]-E1 formation; whereas, inhibition of [3H]-E2 production, which implicates 17β-hydroxysteroid dehydrogenase activity in this pathway, is greater because NOMAC also inhibits this enzyme. The MCF-7aro cell line shows high aromatase activity and NOMAC can act as an anti-aromatase agent by inhibiting this activity. This is an important new effect of this progestogen. Because NOMAC can also inhibit sulfatase activity in breast cancer cells, we suggest that this dual effect of NOMAC has attractive possibilities for clinical trials.

The metal ion requirements of Arabidopsis thaliana Glx2-2 for catalytic activity

In an effort to better understand the structure, metal content, the nature of the metal centers, and enzyme activity of Arabidopsis thaliana Glx2-2, the enzyme was overexpressed, purified, and characterized using metal analyses, kinetics, and UV-vis, EPR, and (1)H NMR spectroscopies. Glx2-2-containing fractions that were purple, yellow, or colorless were separated during purification, and the differently colored fractions were found to contain different amounts of Fe and Zn(II). Spectroscopic analyses of the discrete fractions provided evidence for Fe(II), Fe(III), Fe(III)-Zn(II), and antiferromagnetically coupled Fe(II)-Fe(III) centers distributed among the discrete Glx2-2-containing fractions. The individual steady-state kinetic constants varied among the fractionated species, depending on the number and type of metal ion present. Intriguingly, however, the catalytic efficiency constant, k(cat)/K(m), was invariant among the fractions. The value of k(cat)/K(m) governs the catalytic rate at low, physiological substrate concentrations. We suggest that the independence of k(cat)/K(m) on the precise makeup of the active-site metal center is evolutionarily related to the lack of selectivity for either Fe versus Zn(II) or Fe(II) versus Fe(III), in one or more metal binding sites.

Controlling the surface properties of nanostructures for studies of polymerases

We report the successful functionalization of optically accessible nanostructures, suitablefor single-molecule experiments at physiological substrate concentrations, with polyethyleneglycol. Characterization of the coating in terms of roughness, protein repellence, andspecific immobilization of DNA is described. We present an application of this technique inthe detection of polymerase activity within nanostructures, which demonstrates theopportunities made possible through the integration of nanofabricated structures withsurface functionalization.

Role of Induced Fit in Enzyme Specificity: A Molecular Forward/Reverse Switch

Enzyme structures solved with and without bound substrate often show that substrate-induced conformational changes bring catalytic residues into alignment, alter the local environment, and position the substrate for catalysis. Although the structural data are compelling, the role of conformational changes in enzyme specificity has been controversial in that specificity is a kinetic property that is not easy to predict based upon structure alone. Recent studies on DNA polymerization have illuminated the role of substrate-induced conformational changes in enzyme specificity by showing that the rate at which the enzyme opens to release the bound substrate is a key kinetic parameter. The slow release of a correct substrate commits it to the forward reaction so that specificity is determined solely by the rate of substrate binding, including the isomerization step, and not by the slower rate of the chemical reaction. In contrast, fast dissociation of an incorrect substrate favors release rather than reaction. Thus, the conformational change acts as a molecular switch to select the right substrate and to recognize and disfavor the reaction of an incorrect substrate. A conformational switch may also favor release rather than reverse reaction of the product.

Experimental validation of metabolic pathway modeling

In the search for new drug targets in the human parasite Entamoeba histolytica, metabolic control analysis was applied to determine, experimentally, flux control distribution of amebal glycolysis. The first (hexokinase, hexose-6-phosphate isomerase, pyrophosphate-dependent phosphofructokinase (PP(i)-PFK), aldolase and triose-phosphate isomerase) and final (3-phosphoglycerate mutase, enolase and pyruvate phosphate dikinase) glycolytic segments were reconstituted in vitro with recombinant enzymes under near-physiological conditions of pH, temperature and enzyme proportion. Flux control was determined by titrating flux with each enzyme component. In parallel, both glycolytic segments were also modeled by using the rate equations and kinetic parameters previously determined. Because the flux control distribution predicted by modeling and that determined by reconstitution were not similar, kinetic interactions among all the reconstituted components were experimentally revised to unravel the causes of the discrepancy. For the final segment, it was found that 3-phosphoglycerate was a weakly competitive inhibitor of enolase, whereas PP(i) was a moderate inhibitor of 3-phosphoglycerate mutase and enolase. For the first segment, PP(i) was both a strong inhibitor of aldolase and a nonessential mixed-type activator of amebal hexokinase; in addition, lower V(max) values for hexose-6-phosphate isomerase, PP(i)-PFK and aldolase were induced by PP(i) or ATP inhibition. It should be noted that PP(i) and other metabolites were absent from the 3-phosphoglycerate mutase and enolase or aldolase and hexokinase kinetics experiments, but present in reconstitution experiments. Only by incorporating these modifications in the rate equations, modeling predicted values of flux control distribution, flux rate and metabolite concentrations similar to those experimentally determined. The experimentally validated segment models allowed 'in silico experimentation' to be carried out, which is not easy to achieve in in vivo or in vitro systems. The results predicted a nonsignificant effect on flux rate and flux control distribution by adding parallel routes (pyruvate kinase for the final segment and ATP-dependent PFK for the first segment), because of the much lower activity of these enzymes in the ameba. Furthermore, modeling predicted full flux-control by 3-phosphoglycerate mutase and hexokinase, in the presence of low physiological substrate and product concentrations. It is concluded that the combination of in vitro pathway reconstitution with modeling and enzyme kinetics experimentation permits a more comprehensive understanding of the pathway behavior and control properties.

Metabolic fluxes in the liver of rats bearing the Walker‐256 tumour: influence of the circulating levels of substrates and fatty acids

Studies on fatty acid and amino acid metabolism in the liver of Walker-256 tumour-bearing rats have revealed several changes. Comparisons, however, have been based on experiments performed with non-physiological, frequently unrealistic, substrate concentrations. The aim of the present work was to examine the influence of physiological substrate concentrations on gluconeogenesis, ketogenesis and related parameters. Isolated livers were perfused and substrates were infused at concentrations that were reported to occur in healthy and tumour-bearing rats. Ketogenesis and the mitochondrial NADH/NAD+ ratio were smaller in the tumour-bearing condition at low (0.2 mM) and high (0.8 mM) oleate concentrations. In the absence of oleate, gluconeogenesis from alanine (0.7 mM) and gluconeogenesis plus the associated changes in oxygen uptake due to lactate/pyruvate (2/0.2 and 6/0.3 mM) were smaller in livers of tumour-bearing rats. However, the response of gluconeogenesis from lactate/pyruvate in livers of tumour-bearing rats to 0.8 mM oleate was more pronounced so that a trend towards normalization was apparent at high substrate and oleate concentrations. Gluconeogenesis from 0.7 mM alanine was not significantly changed by oleate in the tumour-bearing state; in the control condition, stimulation occurred at 0.2 mM oleate and inhibition at 0.8 mM oleate. This diminution almost equalized the hepatic alanine-dependent gluconeogenesis of both control and tumour-bearing rats. Ureogenesis was smaller in the tumour-bearing state and was not affected by oleate. It was concluded that the high concentrations of fatty acids and lactate/pyruvate, which predominate in rats bearing the Walker-256 tumour, could be effective in normalizing the gluconeogenic response of livers from tumour-bearing rats.

Genetic Variants of PXR (NR1I2) and CAR (NR1I3) and Their Implications in Drug Metabolism and Pharmacogenetics

The defense mechanisms responsible for protecting the body from endogenous toxins are also involved in the metabolism of drugs and are composed of phase I and phase II drug metabolizing enzymes, as well as drug transporters. Numerous drugs and chemicals have been shown to modulate the expression of the genes involved in these three drug-detoxifying processes. Induction of these genes contributes to both auto-induction of drug clearance and to drug-drug interactions in combination therapies. The orphan nuclear receptors PXR (pregnane X receptor) and CAR (Constitutive androstane receptor) are xenosensors that mediate drug-induced changes by increasing transcription of genes that are involved in drug clearance and disposition. Co-administration of drugs, one of which is a nuclear receptor agonist or antagonist, can either lead to altered clearance of the second drug and severe toxicity, or a loss of therapeutic efficacy or an imbalance in physiological substrate concentrations, providing a novel molecular mechanism for drug-drug interactions. Thus, genetic variability in these nuclear receptors will contribute to human variation in the magnitude of clinically significant drug-drug interactions. This review describes common PXR and CAR genetic variants that have been identified to date in the human population and the functional consequence of these variant alleles. In addition, alternatively spliced variants of PXR and CAR that may also contribute to individual variability as well as tissue specific expression of these receptors are also described. Identification of PXR and CAR genetic variants and alternatively spliced mRNAs may ultimately allow predictions of interindividual differences in target gene induction and drug-drug interactions.