Fabrício Bracht

The action of extracellular NAD+ on gluconeogenesis in the perfused rat liver

In the rat liver NAD+ infusion produces increases in portal perfusion pressure and glycogenolysis and transient inhibition of oxygen consumption. The aim of the present work was to investigate the possible action of this agent on gluconeogenesis using lactate as a gluconeogenic precursor. Hemoglobin-free rat liver perfusion in antegrade and retrograde modes was used with enzymatic determination of glucose production and polarographic assay of oxygen uptake. NAD+ infusion into the portal vein (antegrade perfusion) produced a concentration-dependent (25–100 μM) transient inhibition of oxygen uptake and gluconeogenesis. For both parameters inhibition was followed by stimulation. NAD+ infusion into the hepatic vein (retrograde perfusion) produced only transient stimulations. During Ca2+-free perfusion the action of NAD+ was restricted to small transient stimulations. Inhibitors of eicosanoid synthesis with different specificities (indo-methacin, nordihydroguaiaretic acid, bromophenacyl bromide) either inhibited or changed the action of NAD+. The action of NAD+ on gluconeogenesis is probably mediated by eicosanoids synthesized in non-parenchymal cells. As in the fed state, in the fasted condition extracellular NAD+ is also able to exert two opposite effects, inhibition and stimulation. Since inhibition did not manifest significantly in retrograde perfusion it is likely that the generating signal is located in pre-sinusoidal regions.

Distribution, lipid-bilayer affinity and kinetics of the metabolic effects of dinoseb in the liver

&NA; Dinoseb is a highly toxic pesticide of the dinitrophenol group. Its use has been restricted, but it can still be found in soils and waters in addition to being a component of related pesticides that, after ingestion by humans or animals, can originate the compound by enzymatic hydrolysis. As most dinitrophenols, dinoseb uncouples oxidative phosphorylation. In this study, distribution, lipid bilayer affinity and kinetics of the metabolic effects of dinoseb were investigated, using mainly the isolated perfused rat liver, but also isolated mitochondria and molecular dynamics simulations. Dinoseb presented high affinity for the hydrophobic region of the lipid bilayers, with a partition coefficient of 3.75 × 104 between the hydrophobic and hydrophilic phases. Due to this high affinity for the cellular membranes dinoseb underwent flow‐limited distribution in the liver. Transformation was slow but uptake into the liver space was very pronounced. For an extracellular concentration of 10 &mgr;M, the equilibrium intracellular concentration was equal to 438.7 &mgr;M. In general dinoseb stimulated catabolism and inhibited anabolism. Half‐maximal stimulation of oxygen uptake in the whole liver occurred at concentrations (2.8–5.8 &mgr;M) at least ten times above those in isolated mitochondria (0.28 &mgr;M). Gluconeogenesis and ureagenesis were half‐maximally inhibited at concentrations between 3.04 and 5.97 &mgr;M. The ATP levels were diminished, but differently in livers from fed and fasted rats. Dinoseb disrupts metabolism in a complex way at concentrations well above its uncoupling action in isolated mitochondria, but still at concentrations that are low enough to be dangerous to animals and humans even at sub‐lethal doses. Graphical abstract Figure. No caption available. HighlightsDinoseb presents high affinity for the hydrophobic region of the lipid bilayers.Its cellular concentration may exceed the extracellular one by a factor of 43.9.One order of magnitude separates the active concentrations in mitochondria and liver.Hepatic metabolism is disrupted in the concentration range up to 10 &mgr;M.

Transformation and action of extracellular NAD+ in perfused rat and mouse livers.

AbstractAim:Transformation and possible metabolic effects of extracellular NAD+ were investigated in the livers of mice (Mus musculus; Swiss strain) and rats (Rattus novergicus; Holtzman and Wistar strains).Methods:The livers were perfused in an open system using oxygen-saturated Krebs/Henseleit-bicarbonate buffer (pH 7.4) as the perfusion fluid. The transformation of NAD+ was monitored using high-performance liquid chromatography.Results:In the mouse liver, the single-pass metabolism of 100 μmol/L NAD+ was almost complete; ADP-ribose and nicotinamide were the main products in the outflowing perfusate. In the livers of both Holtzman and Wistar rats, the main transformation products were ADP-ribose, uric acid and nicotinamide; significant amounts of inosine and AMP were also identified. On a weight basis, the transformation of NAD+ was more efficient in the mouse liver. In the rat liver, 100 μmol/L NAD+ transiently inhibited gluconeogenesis and oxygen uptake. Inhibition was followed by a transient stimulation. Inhibition was more pronounced in the Wistar strain and stimulation was more pronounced in the Holtzman strain. In the mouse liver, no clear effects on gluconeogenesis and oxygen uptake were found even at 500 μmol/L NAD+.Conclusion:It can be concluded that the functions of extracellular NAD+ are species-dependent and that observations in one species are strictly valid for that species. Interspecies extrapolations should thus be made very carefully. Actually, even variants of the same species can demonstrate considerably different responses.

The action of extracellular NAD+ in the liver of healthy and tumor-bearing rats: model analysis of the tumor-induced modified response.

The chronic inflammatory state induced by cancer is expected to affect the actions of extracellular NAD(+) in the liver because these are largely mediated by eicosanoids. Under this assumption the present work was planned to investigate the influence of the Walker-256 tumor on the action of extracellular NAD(+) on metabolism and hemodynamics in the perfused rat liver. The experiments were done with livers from healthy and tumor-bearing rats with measurements of gluconeogenesis from lactate, pyruvate production, oxygen consumption and portal pressure. A model describing the biphasic effects of NAD(+) was proposed as an auxiliary worktool for interpretation. The Walker-256 tumor modified the responses of metabolism to extracellular NAD(+) by delaying the peak of maximal responses and by prolonging the inhibitory effects. The transient increase in portal perfusion pressure caused by NAD(+) was enhanced and delayed. The model was constructed assuming the mediation of a down-regulator (inhibition), an up-regulator (stimulation) and receptor dessensitization. Analysis suggested that the productions of both the down- and up-regulators were substantially increased and delayed in time in the tumor-bearing condition. Since the regulators are probably eicosanoids, this analysis is consistent with the increased capacity of producing these agents in the chronic inflammatory state induced by cancer.

Structural and functional characterization of a highly secreted α-l-arabinofuranosidase (GH62) from Aspergillus nidulans grown on sugarcane bagasse

Carbohydrate-Active Enzymes are key enzymes for biomass-to-bioproducts conversion. α-l-Arabinofuranosidases that belong to the Glycoside Hydrolase family 62 (GH62) have important applications in biofuel production from plant biomass by hydrolyzing arabinoxylans, found in both the primary and secondary cell walls of plants. In this work, we identified a GH62 α-l-arabinofuranosidase (AnAbf62Awt) that was highly secreted when Aspergillus nidulans was cultivated on sugarcane bagasse. The gene AN7908 was cloned and transformed in A. nidulans for homologous production of AnAbf62Awt, and we confirmed that the enzyme is N-glycosylated at asparagine 83 by mass spectrometry analysis. The enzyme was also expressed in Escherichia coli and the studies of circular dichroism showed that the melting temperature and structural profile of AnAbf62Awt and the non-glycosylated enzyme from E. coli (AnAbf62Adeglyc) were highly similar. In addition, the designed glycomutant AnAbf62AN83Q presented similar patterns of secretion and activity to the AnAbf62Awt, indicating that the N-glycan does not influence the properties of this enzyme. The crystallographic structure of AnAbf62Adeglyc was obtained and the 1.7Å resolution model showed a five-bladed β-propeller fold, which is conserved in family GH62. Mutants AnAbf62AY312F and AnAbf62AY312S showed that Y312 was an important substrate-binding residue. Molecular dynamics simulations indicated that the loop containing Y312 could access different conformations separated by moderately low energy barriers. One of these conformations, comprising a local minimum, is responsible for placing Y312 in the vicinity of the arabinose glycosidic bond, and thus, may be important for catalytic efficiency.

Influence of adjuvant-induced arthritis on the action of extracellular NAD+ on hepatic gluconeogenesis and related parameters

The influence of arthritis on some metabolic and hemodynamic effects of extracellular NAD+ was investigated in the perfused liver of Holtzman rats with the additional purpose of comparing the present results with previous results obtained with rats from the Wistar strain. Fasted rats were used and glucose plus pyruvate productions from lactate and the associated oxygen uptake increment were measured as well as the portal perfusion pressure. Livers from arthritic rats presented higher transient increments in perfusion pressure upon NAD+ infusion. The metabolic effects of NAD+ consisted in initial inhibitions of glucose and pyruvate production and oxygen uptake, followed by recovery and stimulation. In the arthritis condition, the inhibitions were more accentuated in relative terms and also more persistent in time, but the subsequent stimulations were smaller or even absent. The inhibitory phase was almost abolished by indomethacin, inhibitor of eicosanoid synthesis, but not by nordihydroguaiaretic acid, an inhibitor of leukotriene synthesis. In comparison with Wistar rats, the response to NAD+ of the liver from Holtzman rats in antegrade perfusion was characterized by a shorter and less intense inhibitory phase and by a more vigorous stimulation. In retrograde perfusion, however, where stimulation was the only effect, there was no difference between both strains. This observation suggests that the more accentuated stimulation in Holtzman rats could be the consequence of a short and less intense inhibitory phase. The stronger and more persistent inhibitions of gluconeogenesis and oxygen uptake as well as the enhanced portal pressure increase in the arthritis condition are consistent with an accentuated capacity of producing eicosanoids by animals under the influence of the disease.