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Results for “Substrate Cycling”

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PubMed1997

Glucose transport and glucose transporter GLUT4 are regulated by product(s) of intermediary metabolism in cardiomyocytes.

Alternative substrates of energy metabolism are thought to contribute to the impairment of heart and muscle glucose utilization in insulin-resistant states. We have investigated the acute effects of substrates in isolated rat cardiomyocytes. Exposure to lactate, pyruvate, propionate, acetate, palmitate, beta-hydroxybutyrate or alpha-oxoglutarate led to the depression of glucose transport by up to 50%, with lactate, pyruvate and propionate being the most potent agents. The percentage inhibition was greater in cardiomyocytes in which glucose transport was stimulated with the alpha-adrenergic agonist phenylephrine or with a submaximal insulin concentration than in basal or fully insulin-stimulated cells. Cardiomyocytes from fasted or diabetic rats displayed a similar sensitivity to substrates as did cells from control animals. On the other hand, the amination product of pyruvate (alanine), as well as valine and the aminotransferase inhibitors cycloserine and amino-oxyacetate, stimulated glucose transport about 2-fold. In addition, the effect of pyruvate was counteracted by cycloserine. Since reversible transamination reactions are known to affect the pool size of the citrate cycle, the influence of substrates, amino acids and aminotransferase inhibitors on citrate, malate and glutamate content was examined. A significant negative correlation was found between alterations in glucose transport and the levels of citrate (P < 0.01) or malate (P < 0.01), and there was a positive correlation between glucose transport and glutamate levels (P < 0.05). In contrast, there was no correlation with changes in [1-(14)C]pyruvate oxidation or in glucose-6-phosphate levels. Finally, pyruvate decreased the abundance of GLUT4 glucose transporters at the surface of phenylephrine- or insulin-stimulated cells by 34% and 27 % respectively, as determined by using the selective photoaffinity label [3H]ATB-BMPA [[3H]2-N-[4-(1-azi-2,2,2-trifluoroethyl)benzoyl]-1,3-bis-(D-man nos-4-yloxy)propyl-2-amine]. In conclusion, cardiomyocyte glucose transport is subject to counter-regulation by alternative substrates. The glucose transport system appears to be controlled by (a) compound(s) of intermediary metabolism (other than glucose 6-phosphate), but in a different way than pyruvate dehydrogenase. Transport inhibition eventually occurs via a decrease in the amount of glucose transporters in the plasma membrane.

3-Hydroxybutyric Acid↗

PubMed2004

Mutational analysis of ionizable residues proximal to the cytoplasmic interface of membrane spanning domain 3 of the multidrug resistance protein, MRP1 (ABCC1): glutamate 1204 is important for both the expression and catalytic activity of the transporter.

The multidrug resistance protein MRP1 is an ATP-dependent transporter of organic anions and chemotherapeutic agents. A significant number of ionizable amino acids are found in or proximal to the 17 transmembrane (TM) helices of MRP1, and we have investigated 6 of these at the cytoplasmic interface of TM13-17 for their role in MRP1 expression and transport activity. Opposite charge substitutions of TM13 Arg(1046) and TM15 Arg(1131) did not alter MRP1 expression nor did they substantially affect activity. In contrast, opposite charge substitutions of TM16 Arg(1202) and Glu(1204) reduced protein expression by >80%; however, MRP1 expression was not affected when Arg(1202) and Glu(1204) were replaced with neutral or same-charge residues. In addition, organic anion transport levels of the R1202L, R1202G, and R1202K mutants were comparable with wild-type MRP1. In contrast, organic anion transport by E1204L was substantially reduced, whereas transport by E1204D was comparable with wild-type MRP1, with the notable exception of GSH. Opposite charge substitutions of TM16 Arg(1197) and TM17 Arg(1249) did not affect MRP1 expression but substantially reduced transport. Mutants containing like-charge substitutions of Arg(1197) or Arg(1249) were also transport-inactive and no longer bound leukotriene C(4). In contrast, substrate binding by the transport-compromised E1204L mutant remained intact. Furthermore, vanadate-induced trapping of azido-ADP by E1204L was dramatically increased, indicating that this mutation may cause a partial uncoupling of the catalytic and transport activities of MRP1. Thus, Glu(1204) serves a dual role in membrane expression of MRP1 and a step in its catalytic cycle subsequent to initial substrate binding.

Adenosine Triphosphate↗

PubMed2000

Effect of light/dark cycle on bacterial hydrogen production by Rhodobacter sphaeroides RV. From hour to second range.

Hydrogen production by photosynthetic bacteria provides an efficient energy conversion method under low light intensity. However, under strong illumination, such as midday sunlight, the efficiency drops. This prevents the method from being applied industrially. To overcome this problem, we examined a method to thin out the excessive illumination. Light was given intermittently to reduce the total energy flux. The on/off ratio was set at 1/1 throughout the study, so that the time average of the light energy flux became half the continuous illumination. By keeping the time-average light flux constant (0.6 kW.m-2), the effects of the cycle period were examined in the range of hours to seconds. The hydrogen production rate was greatly affected by the cycle period, but cell growth and substrate consumption rates remained almost constant. The 30-min light/dark cycle (30 min on and 30 min off) provided the highest rate of hydrogen production (22 L.m-2.24 h-1). At the shorter cycles, the rate decreased except that there was a suboptimum at about 40 s. Under excessive light intensity (1.2 kW.m-2), the light-to-hydrogen conversion efficiency was greatly enhanced. The hydrogen production rate during the 30-min cycle was twice as high as during a 12-h cycle under the same conditions.

Activity Cycles↗

PubMed1995

Gluconeogenesis and effect of nutritional status on TCA cycle activity in the insect Manduca sexta.

Gluconeogenesis from isotopically substituted (3-13C)alanine (Ala) was demonstrated in the last larval instar of an insect, Manduca sexta, when maintained on low carbohydrate diets. 13C was incorporated into all carbons of the blood sugar trehalose (Tre), but enrichments of C1 and C6, and C2 and C5 were greatest. Relative to the amount of [3-13C]Ala metabolized, larvae maintained on a low carbohydrate diet supplemented with casein displayed the greatest enrichment of Tre. Very little de novo synthesis of Tre was observed in larvae maintained on a complete-balanced diet containing calorically equivalent amounts of sucrose and casein. Starvation failed to induce gluconeogenesis and 13C was not incorporated into Tre in starved insects. Activity of the TCA cycle contributed approximately 10% of the 13C incorporated into Tre in larvae on low carbohydrate diets, while the TCA cycle contribution in larvae on the complete diet approached 70%. The pattern of 13C enrichment of glucose in larvae on the low carbohydrate diets indicated that cytoplasmic carboxylation, possibly due to 'malic enzyme'-like activity, contributed significantly to the synthesis of Tre. The pentose phosphate pathway was evidenced in insects on all diets. Glucose labelling ratios indicated a pentose cycling flux of 10 to 20% in insects on the low carbohydrate diets and 50% in larvae on the complete diet. Glutamine together with lesser amounts of glutamate and glutathione were also products of the labelled Ala. The distribution of label in these products under different dietary conditions demonstrated shifts in the relative contribution of pyruvate carboxylase and pyruvate dehydrogenase activities for providing substrate to the TCA cycle. In the expected fashion starved insects and insects on the low carbohydrate diets incorporated a greater proportion of 13C into the TCA cycle via carboxylation while incorporation by the two pathways was similar in insects on the complete diet. The significance of these findings with regard to the regulation of gluconeogenesis in M. sexta and comparison of the present results with those obtained from studies of hepatic gluconeogenesis are discussed.

Alanine↗

PubMed2005

PTHrP signaling targets cyclin D1 and induces osteoblastic cell growth arrest.

UNLABELLED: PTHrP control of the MC3T3-E1 cell cycle machinery showed that, during differentiation, PTHrP induced G1 growth arrest. Cyclin D1 was a critical mediator as a downstream effector of cAMP, PKC, and MAPK signaling, and the process was PKA-independent. The involvement of JunB has been found critical for PTHrP effects. INTRODUCTION: PTH-related protein (PTHrP) has been implicated in the control of bone cell turnover, but the mechanisms underlying its effect on osteoblast proliferation and differentiation have not been clearly defined. The mechanisms by which PTHrP impacts cell cycle proteins and the role of signaling pathways in differentiated osteoblasts were studied. MATERIALS AND METHODS: To elucidate the role of PTHrP, flow cytometric analyses were performed using MC3T3-E1 and primary mouse calvarial cells. Relative protein abundance (Western blot), physical association of partners (immunoprecipitation), and kinase activities (in vitro kinase assays using either GST-Rb or H1-histone as substrates) of cell cycle-associated proteins in vehicle and PTHrP-treated 7-day differentiated cells were determined. ELISA and/or Northern blot analyses were done to evaluate JunB and cyclin D1 expression. SiRNA-mediated gene silencing experiments were performed to silence JunB protein. Finally, inhibitors of cAMP, protein kinase A (PKA), protein kinase C (PKC), and mitogen-activated protein kinase (MAPK) were used to determine involvement of different signaling pathways. RESULTS: PTHrP inhibited cyclin D1 protein expression 7-fold in a dose- and time-dependent manner and increased the level of p16 protein in differentiated osteoblasts. Additionally, PTHrP reduced cyclin D1-CDK4/CDK6 and CDK1 kinase activities. Forskolin, a cAMP agonist, mimicked PTHrP action, and the PKC inhibitor, GF109203X, slightly blocked downregulation of cyclin D1, implying involvement of both cAMP and PKC. U0126, a MAPK inhibitor, alone decreased cyclin D1 protein, suggesting that the basal cyclin D1 protein is MAPK dependent. H-89, a PKA inhibitor, did not alter the effect of PTHrP on cyclin D1, suggesting a PKA-independent mechanism. Finally, expression of JunB, an activating protein-1 transcription factor, was significantly upregulated, and silencing JunB (siRNA) partially reversed the cyclin D1 response, implying involvement of JunB in the PTHrP-mediated growth arrest of MC3T3-E1 cells. CONCLUSION: PTHrP upregulates JunB and reduces cyclin D1 expression while inducing G1 cell cycle arrest in differentiated osteoblasts. Such regulation could be an important determinant of the life span and bone-forming activity of osteoblasts.

3T3 Cells↗

PubMed2005

Substrate enhances the sensitivity of type I protein kinase a to cAMP.

The functional significance of the presence of two major (types I and II) isoforms of the cAMP-dependent protein kinase (PKA) is still enigmatic. The present study showed that peptide substrate enhanced the activation of PKA type I at low, physiologically relevant concentrations of cAMP through competitive displacement of the regulatory RI subunit. The effect was similar whether the substrate was a short peptide or the physiological 60-kDa protein tyrosine hydroxylase. In contrast, substrate failed to affect the cAMP-sensitivity of PKA type II. Size exclusion chromatography confirmed that substrate acted to physically enhance the dissociation of the RIalpha and Calpha subunits of PKA type I, but not the RIIalpha and Calpha subunits of PKA type II. Substrate availability can therefore fine-tune the activation of PKA type I by cAMP, but not PKA type II. The cAMP-dissociated RII and C subunits of PKA type II reassociated much faster than the PKA type I subunits in the presence of substrate peptide. This suggests that only PKA type II is able to rapidly reverse its activation after a burst of cAMP when exposed to high substrate concentration. We propose this as a possible reason why PKA type II is preferentially found in complexes with substrates undergoing rapid phosphorylation cycles.

Adenosine Diphosphate↗

PubMed1979

Regulation of neutral protease activity through the life cycles of Tetrahymena pyriformis.

Substantial fluctuations in the intracellular specific activity of neutral proteases, as assayed at pH 8 with azocasein as substrate, occur during the life cycle of the protozoan Tetrahymena pyriformis. Specific activity increases during growth in 2% proteose peptone, despite slow secretion into the medium. The most rapid increase occurs during late stationary phase and appears to be a response to one or more low molecular weight (less than 10 000), heat-stable, trypsin-insensitive, polar molecules secreted into the medium. In contrast, intracellular specific activity drops by a factor of 2--5 within the first 2--3 h after transfer to non-nutritive medium. The decrease in activity under these conditions results from an enhanced rate of secretion and the cessation of net synthesis. Its kinetics are unaffected by cycloheximide and concanavalin A.

Concanavalin A↗

PubMed2002

Effects of pentylenetetrazole and glutamate on metabolism of [U-(13)C]glucose in cultured cerebellar granule neurons.

This study was performed to analyze the effects of glutamate and the epileptogenic agent pentylenetetrazole (PTZ) on neuronal glucose metabolism. Cerebellar granule neurons were incubated for 2 h in medium containing 3 mM [U-(13)C]glucose, with and without 0.25 mM glutamate and/or 10 mM PTZ. In the presence of PTZ, decreased glucose consumption with unchanged lactate release was observed, indicating decreased glucose oxidation. PTZ also slowed down tricarboxylic acid (TCA) cycle activity as evidenced by the decreased amounts of labeled aspartate and [1,2-(13)C]glutamate. When glutamate was present, glucose consumption was also decreased. However, the amount of glutamate, derived from [U-(13)C]glucose via the first turn of the TCA cycle, was increased. The decreased amount of [1,2-(13)C]glutamate, derived from the second turn in the TCA cycle, and increased amount of aspartate indicated the dilution of label due to the entrance of unlabeled glutamate into TCA cycle. In the presence of glutamate plus PTZ, the effect of PTZ was enhanced by glutamate. Labeled alanine was detected only in the presence of glutamate plus PTZ, which indicated that oxaloacetate was a better amino acid acceptor than pyruvate. Furthermore, there was also evidence for intracellular compartmentation of oxaloacetate metabolism. Glutamate and PTZ caused similar metabolic changes, however, via different mechanisms. Glutamate substituted for glucose as energy substrate in the TCA cycle, whereas, PTZ appeared to decrease mitochondrial activity.

Alanine↗

PubMed2000

Kinetics and activation thermodynamics of methane monooxygenase compound Q formation and reaction with substrates.

The transient kinetics of formation and decay of the reaction cycle intermediates of the Methylosinus trichosporium OB3b methane monooxygenase (MMO) catalytic cycle are studied as a function of temperature and substrate type and deuteration. Kinetic evidence is presented for the existence of three intermediates termed compounds O, P, and P forming after the addition of O(2) to diferrous MMO hydroxylase (H(r)) and before the formation of the reactive intermediate compound Q. The Arrhenius plots for these reactions are linear and independent of substrate concentration and type, showing that substrate does not participate directly in the oxygen activation phase of the catalytic cycle. Analysis of the transient kinetic data revealed only small changes relative to the weak optical spectrum of H(r) for any of these intermediates. In contrast, large changes in the 430 nm spectral region are associated with the formation of Q. The decay reaction of Q exhibits an apparent first-order concentration dependence for all substrates tested, and the observed rate constant depends on the substrate type. The kinetics of the decay reaction of Q yield a nonlinear Arrhenius plot when methane is the substrate, and the rates in both segments of the plot increase linearly with methane concentration. Together these observations suggest that at least two reactions with a methane concentration dependence, and perhaps two methane molecules, are involved in the decay process. When CD(4) is used as the substrate, a large isotope effect and a linear Arrhenius plot are observed. Analogous plots for all other MMO substrates tested (e.g., ethane) are linear, and no isotope effect for deuterated analogues is observed. This demonstrates that a step other than C-H bond breaking is rate limiting for alternative MMO substrates. A two step Q decay mechanism is proposed that provides an explanation for the lack of an isotope effect for alternative MMO substrates and the fact that rate of oxidation of methane by Q exceeds that of many other hydrocarbons with weaker C-H bonds.

Catalysis↗

PubMed1997

Human Myt1 is a cell cycle-regulated kinase that inhibits Cdc2 but not Cdk2 activity.

Activation of the Cdc2.cyclin B kinase is a pivotal step of mitotic initiation. This step is mediated principally by the dephosphorylation of residues threonine 14 (Thr14) and tyrosine 15 (Tyr15) on the Cdc2 catalytic subunit. In several organisms homologs of the Wee1 kinase have been shown to be the major activity responsible for phosphorylating the Tyr15 inhibitory site. A membrane-bound kinase capable of phosphorylating residue Thr14, the Myt1 kinase, has been identified in the frog Xenopus laevis and more recently in human. In this study, we have examined the substrate specificity and cell cycle regulation of the human Myt1 kinase. We find that human Myt1 phosphorylates and inactivates Cdc2-containing cyclin complexes but not complexes containing Cdk2 or Cdk4. Analysis of endogenous Myt1 demonstrates that it remains membrane-bound throughout the cell cycle, but its kinase activity decreased during M phase arrest, when Myt1 became hyperphosphorylated. Further, Cdc2. cyclin B1 was capable of phosphorylating Myt1 in vitro, but this phosphorylation did not affect Myt1 kinase activity. These findings suggest that human Myt1 is negatively regulated by an M phase-activated kinase and that Myt1 inhibits mitosis due to its specificity for Cdc2.cyclin complexes.

Animals↗

PubMed1990

A hysteretic cycle in glucose 6-phosphate metabolism observed in a cell-free yeast extract.

The dynamics of a partial glycolytic reaction sequence which converts glucose 6-phosphate to triose phosphates is described. The study was performed with cell-free extracts from baker's yeast harvested in the logarithmic and stationary growth phases. The experiments are based on a flow-through reactor supplied with the desalted cell-free extract as well as glucose 6-phosphate, ATP and phosphoenolpyruvate. In the reaction system the quasi-irreversible reactions catalyzed by 6-phosphofructo-1-kinase, pyruvate kinase, and fructose-1,6-bisphosphatase are involved. When substrate is supplied continuously, only stable stationary states can be observed. With transient perturbations of the substrate supply, multiple stationary states appear. Cyclic transitions between unique stable stationary states were induced by appropriate changes of the rate of substrate supply. A hysteretic cycle could then be demonstrated when, during reverse transitions, a parameter region of multistability was passed. The presence (in resting yeast) or absence (in growing yeast) of fructose-1,6-bisphosphatase did not significantly influence the dynamic capabilities of the investigated reaction sequence. The kinetic properties of the cell-free extracts fit mathematical models developed for in vitro systems reconstituted from purified enzymes.

Adenosine Triphosphate↗

PubMed2005

Capacitive micromachined ultrasonic transducer design for high power transmission.

Capacitive micromachined ultrasonic transducers (cMUTs) were developed to meet the demands of the ultrasonic industry. To achieve maximum efficiency, the conventional operation of the cMUT requires a bias voltage close to the collapse voltage. Total acoustic output pressure is limited by the efficiency of the cMUT and the maximum-allowed pulse voltage on the membrane. In this paper, we propose the collapse-snapback operation of the cMUT: the membrane is collapsed onto the substrate in the collapsing cycle, and released in the snapback cycle. The collapse-snapback operation overcomes the above-mentioned limitations of the conventional operation. The collapse-snapback operation utilizes a larger range of membrane deflection profiles (both collapsed and released profiles) and generates higher acoustic output pressures. The static finite element calculations were performed to design cMUTs with specific collapse and snapback voltages by changing the electrode parameters (radius (re), position (de), and thickness (te)). These designs were refined for optimum average displacement per cycle. An electrode radius greater than 60% of the membrane radius significantly improved the displacement per volt. Moderately thick membranes (te approximately 0.2 microm) were preferred, as thicker membranes reduced the displacement per volt. Under proper bias conditions, the collapse-snapback operation, designed for high-power transmission, allowed the application of pulse voltages larger than the difference of collapse and snapback voltages. Dynamic finite element calculations of an infinite cMUT array on the substrate loaded with acoustic fluid medium were performed to determine the dynamic response of the cMUT. Commercially available FEM packages ANSYS and LS-DYNA were used for static and dynamic calculations, respectively. The cMUTs were fabricated for optimal performance in the collapse-snapback operation. The transmit experiments were performed on a 2-D cMUT array using a calibrated hydrophone. Taking into account the attenuation and diffraction losses, the pressure on the cMUT surface was extracted. The cMUT generated 0.47 MPa (6 kPa/V) and 1.04 MPa (11 kPa/V) in the conventional and collapse-snapback operations, respectively. Therefore, collapse-snapback operation of the cMUTs was superior for high-power transmission.

Journal Article↗

PubMed2002

Hypophosphorylation of poly(A) polymerase and increased polyadenylation activity are associated with human immunodeficiency virus type 1 Vpr expression.

The HIV-1 encoded accessory protein, viral protein R (Vpr) is responsible for several biological effects in HIV-1-infected cells including nuclear transport of the preintegration complex, activation of long terminal repeat (LTR)-mediated transcription, and the induction of cell-cycle arrest and apoptosis. Vpr's ability to arrest cells at the G2 phase of the cell cycle is due to the inactivation of p34(cdc2) cyclin B complex, resulting in hypophosphorylation of substrates involved in cell-cycle progression from G2 to mitosis (M). Poly(A) polymerase (PAP), the enzyme responsible for poly(A) addition to primary transcripts, contains multiple consensus phosphorylation sites for p34(cdc2) cyclin B kinase that regulates its catalytic activity. We investigated the effects of Vpr on the activity of PAP in Jurkat cells using a superinfection system. Superinfection of cells using Vpr+ vesicular stomatitis virus G protein (VSV-G)-pseudotyped virus caused a complete dephosphorylation of PAP. Cotransfection studies in 293T cells and Xenopus oocyte RNA injection experiments mirrored these effects. Vpr's dramatic effect on PAP dephosphorylation was reflected in enhanced polyadenylation activity in PAP activity assays. HIV-1 Vpr appears to enhance processes that are coupled to transcription such as polyadenylation and could ultimately prove to optimize HIV-1 replication and contribute to HIV-1 pathogenesis. (C)2002 Elsevier Science.

Cell Line↗

PubMed2000

Proteolysis and the cell cycle: with this RING I do thee destroy.

The ubiquitin system drives the cell division cycle by the timely destruction of numerous regulatory proteins. Remarkably, the two main activities that catalyze substrate ubiquitination in the cell cycle, the Skp1-Cdc53/cullin-F-box protein (SCF) complexes and the anaphase-promoting complex/cyclosome (APC/C), define a new superfamily of E3 ubiquitin ligases, all based on related cullin and RING-H2 finger protein subunits. The circuits that interconnect the SCF, APC/C and cyclin-dependent kinase activities form a master oscillator that coordinates the replication and segregation of the genome.

Anaphase-Promoting Complex-Cyclosome↗

PubMed2003

The increase in intramyocellular lipid content is a very early response to training.

The present study investigated the influences of a 2-wk training program on intramyocellular lipid (IMCL) content, IMCL decrease during exercise, fat oxidation, and insulin sensitivity. Nine untrained men (age, 23.3 +/- 3.2 yr; body mass index, 22.6 +/- 2.6 kg/m(2); maximal power output, 3.8 +/- 0.6 W/kg body weight) trained for 2 wk. Before and after training, subjects cycled for 3 h while substrate oxidation was measured. IMCL content in the vastus lateralis muscle was determined before and after cycling by proton magnetic resonance spectroscopy. Before and after training, insulin sensitivity was assessed by an insulin tolerance test. The training period resulted in a significant increase in IMCL content by 42 +/- 14%. IMCL content decreased significantly during cycling. However, 2 wk of training were not sufficient to achieve increases in fat oxidation and/or use of IMCL during exercise. All markers used to test insulin sensitivity point toward improved insulin sensitivity, albeit not significant. We conclude that the increase in IMCL content is a very early response to training, preceding significant changes in insulin sensitivity. The results suggest that the presence of triglycerides alone does not necessarily have detrimental effects on insulin sensitivity. We confirm earlier reports that IMCL contributes to the energy used during prolonged submaximal exercise.

Adipose Tissue↗

PubMed1998

The sequence, bacterial expression, and functional reconstitution of the rat mitochondrial dicarboxylate transporter cloned via distant homologs in yeast and Caenorhabditis elegans.

The dicarboxylate carrier (DIC) belongs to a family of transport proteins found in the inner mitochondrial membranes. The biochemical properties of the mammalian protein have been characterized, but the protein is not abundant. It is difficult to purify and had not been sequenced. We have used the sequence of the distantly related yeast DIC to identify a related protein encoded in the genome of Caenorhabditis elegans. Then, related murine expressed sequence tags were identified with the worm sequence, and the murine sequence was used to isolate the cDNA for the rat homolog. The sequences of the worm and rat proteins have features characteristic of the family of mitochondrial transport proteins. Both proteins were expressed in bacteria and reconstituted into phospholipid vesicles where their transport characteristics closely resembled those of whole rat mitochondria and of the rat DIC reconstituted into vesicles. As expected from the role of the DIC in gluconeogenesis and ureogenesis, its transcripts were detected in rat liver and kidney, but unexpectedly, they were also detected in rat heart and brain tissues where the protein may fulfill other roles, possibly in supplying substrates to the Krebs cycle.

Amino Acid Sequence↗

PubMed2000

Effect of gender, sex hormones, time variables and physiological urinary pH on apparent CYP2D6 activity as assessed by metabolic ratios of marker substrates.

The effects of gender, time variables, menstrual cycle phases, plasma sex hormone concentrations and physiologic urinary pH on CYP2D6 phenotyping were studied using two widely employed CYP2D6 probe drugs, namely dextromethorphan and metoprolol. Phenotyping on a single occasion of 150 young, healthy, drug-free women and men revealed that the dextromethorphan: dextrorphan metabolic ratio (MR) was significantly lower (P < 0.0001) in 56 female extensive metabolizers (0.008+/-0.021) compared to 86 male extensive metabolizers (0.020 +/-0.040). Urinary pH was a significant predictor of dextromethorphan: dextrorphan MRs in men and women (P < 0.001). Once-a-month phenotyping with dextromethorphan of 12 healthy young men (eight extensive metabolizers and four poor metabolizers) over a 1-year period, as well as every-other-day phenotyping with dextromethorphan of healthy, pre-menopausal women (10 extensive metabolizers and 2 poor metabolizers) during a complete menstrual cycle, did not follow a particular pattern and showed similar intrasubject variability ranging from 24.1% to 74.5% (mean 50.9%) in men and from 20.5% to 96.2% (mean 52.0%) in women, independent of the CYP2D6 phenotype (P = 0.342). Using metoprolol as a probe drug, considerable intrasubject variability (38.6+/- 12.0%) but no correlation between metoprolol: alpha-hydroxymetoprolol MRs and pre-ovulatory, ovulatory and luteal phases (mean +/- SD metoprolol: a-hydroxymetoprolol MRs: 1.086+/- 1.137 pre-ovulatory; 1.159+/-1.158 ovulatory and 1.002+/-1.405 luteal phase; P> 0.9) or 17beta-oestradiol, progesterone or testosterone plasma concentrations was observed. There was a significant inverse relationship between physiologic urinary pH and sequential dextromethorphan: dextrorphan MRs as well as metoprolol: alpha-hydroxymetoprolol MRs in men and women, with metabolic ratios varying up to six-fold with metoprolol and up to 20-fold with dextromethorphan (ANCOVA P < 0.001). We conclude that apparent CYP2D6 activity is highly variable, independent of menstrual cycle phases, sex hormones, time variables or phenotype. Up to 80% of the observed variability can be explained by variations of urinary pH within the physiological range. An apparent phenotype shift as a result of variations in urinary pH may be observed in individuals who have metabolic ratios close to the population antimode.

Administration, Oral↗

PubMed2003

Metabolic flux analysis of hepatocyte function in hormone- and amino acid-supplemented plasma.

Understanding the metabolic and regulatory pathways of hepatocytes is important for biotechnological applications involving liver cells. Previous attempts to culture hepatocytes in plasma yielded poor functional results. Recently we reported that hormone (insulin and hydrocortisone) and amino acid supplementation reduces intracellular lipid accumulation and restores liver-specific function in hepatocytes exposed to heparinized human plasma. In the current study, we performed metabolic flux analysis (MFA) using a simplified metabolic network model of cultured hepatocytes to quantitively estimate the changes in lipid metabolism and relevant intracellular pathways in response to hormone and amino acid supplementation. The model accounts for the majority of central carbon and nitrogen metabolism, and assumes pseudo-steady-state with no metabolic futile cycles. We found that beta-oxidation and tricarboxylic acid (TCA) cycle fluxes were upregulated by both hormone and amino acid supplementation, thus enhancing the rate of lipid oxidation. Concomitantly, hormone and amino acid supplementation increased gluconeogenic fluxes. This, together with an increased rate of glucose clearance, caused an increase in predicted glycogen synthesis. Urea synthesis was primarily derived from ammonia and aspartate generated through transamination reactions, while exogenous ammonia removal accounted for only 3-6% of the urea nitrogen. Amino acid supplementation increased the endogenous synthesis of oxaloacetate, and in turn that of aspartate, a necessary substrate for the urea cycle. These findings from MFA provide cues as to which genes/pathways relevant to fatty acid oxidation, urea production, and gluconeogenesis may be upregulated by plasma supplementation, and are consistent with current knowledge of hepatic amino acid metabolism, which provides further credence to this approach for evaluating the metabolic state of hepatocytes under various environmental conditions.

Albumins↗