Comparative Biochemistry and Physiology - Part C: Pharmacology, Toxicology and Endocrinology, 1996
1. The actions of piroxicam, a nonsteroidal and noncarboxylic anti-inflammatory drug, on the meta... more 1. The actions of piroxicam, a nonsteroidal and noncarboxylic anti-inflammatory drug, on the metabolism of the isolated perfused rat liver were investigated. The main purpose was to verify if piroxicam is also active on glycogenolysis and energy metabolism, as demonstrated for several carboxylic nonsteroidal anti-inflammatories. 2. Piroxicam increased oxygen consumption in livers from both fed and fasted rats. 3. Piroxicam increased glucose release and glycolysis from endogenous glycogen (glycogenolysis). 4. Gluconeogenesis from lactate plus pyruvate was inhibited. 5. The action of piroxicam on oxygen consumption was blocked by antimycin A, but not by atractyloside. 6. The action of piroxicam in the perfused rat liver metabolism seems to be a consequence of its action on mitochondria. 7. It can be concluded that inhibition of energy metabolism and stimulation of glycogenolysis are not specific properties of carboxylic nonsteroidal anti-inflammatory drugs.
The crucial difference between the biochemical education in Brazil and more developed countries r... more The crucial difference between the biochemical education in Brazil and more developed countries resides not in the highest degrees but in the lowest ones: the bachelor degree in Biochemistry is still a rarity in Brazil. Whereas more than 600 institutions in the US offer the bachelor degree in biochemistry/molecular biology, presently only 3 are offered in Brazil. This situation may be surprising, but it has historical reasons among others. The field of Biochemistry in Brazil played a pioneering role in institutionalizing master's and doctoral programs, thus reinforcing also research activities. Teachers and researchers involved in these programs, however, had in general little if any interest in implementing undergraduate biochemistry studies, except as a fundamental one year discipline to be taken by future biologists, pharmacists, medical students, etc. The second reason for the virtual absence of the biochemistry bachelor degree before 2005 in Brazil are the so called baccalaureates in "Pharmacy and Biochemistry" a strange denomination whose more correct name should be "Pharmacy and Clinical Analyses". This situation generated the notion, within and outside academic circles, that there is a very especial relationship between Pharmacy and Biochemistry or even that they are one and the same thing. These and other erroneous notions have proven to be adverse for the brazilian biochemistry in both professional and academic spheres, especially in more recent times when the master's and doctoral programs ceased to offer the more basic disciplines. Many graduates with a master's or even doctoral degree have a highly defficient background on physical and organic chemistry, or even on metabolic biochemistry and enzymology if the program is centered on molecular biology.
1. The influence of Ca2+ on the effects of glucagon on glycolysis was investigated in the isolate... more 1. The influence of Ca2+ on the effects of glucagon on glycolysis was investigated in the isolated perfused rat liver. Livers from fed rats were perfused in an open system with Krebs/Henseleit-bicarbonate buffer (pH 7.4). Glucose release, lactate plus pyruvate production (glycolysis) and oxygen uptake were measured. The following results were obtained: 2. In livers perfused with Ca(2+)-free Krebs/Henseleit-bicarbonate buffer and after depletion of the intracellular pools, the initial and transient stimulation of glycolysis, which is normally observed shortly after the onset of glucagon infusion, was more pronounced when compared to livers perfused with normal perfusion fluid (2.5 mM Ca2+) and without previous depletion of the intracellular pools (controls); the subsequent inhibition of glycolysis was delayed in Ca(2+)-free perfused livers and was less pronounced in comparison with the controls at the end of the glucagon infusion period (20 min). 3. Perfusion with a Ca(2+)-free medium supplemented with EDTA, without previous depletion of the intracellular pools, also produced a substantial reduction in the effects of glucagon on glycolysis. 4. Ca(2+)-free perfusion did not affect the stimulative action of glucagon on glucose release (glycogenolysis) and oxygen uptake. 5. Glycolysis inhibition by cAMP also was abolished in Ca(2+)-free perfused livers, and the initial stimulation was enhanced. 6. Mn2+, a metal ion known as a competitor of Ca2+, considerably reduced the action of glucagon on glycolysis; Mn2+ did not affect the basal rates of glycolysis. 7. Sr2+, a metal ion that is often recognized as Ca2+ by several biological structures and processes, increased the inhibitory action of glucagon on glycolysis. 8. Several organic compounds, which directly or indirectly take part in Ca2+ fluxes, were also able to diminish (e.g., verapamil) or even to abolish (carbenoxolone) the inhibitory action of glucagon on glycolysis. 9. It was concluded that, under the conditions of the living cell, Ca2+ is important for glycolysis inhibition by glucagon. In principle at least, the results can be explained in terms of the known Ca2+ dependencies of several protein kinases and protein phosphatases.
Brazilian journal of medical and biological research = Revista brasileira de pesquisas médicas e biológicas / Sociedade Brasileira de Biofísica ... [et al.], 1986
The effect of aqueous extracts of Stevia rebaudiana leaves on a glucose tolerance test was invest... more The effect of aqueous extracts of Stevia rebaudiana leaves on a glucose tolerance test was investigated in 16 normal volunteers. Aqueous extracts of 5 grams of leaves were administered to volunteers at regular 6-h intervals for 3 days. Glucose tolerance tests were performed before and after extract administration. A second group of 6 normal volunteers who ingested an aqueous arabinose solution was also studied to eliminate possible stress effects. The extract of Stevia rebaudiana increased glucose tolerance. The extract significantly decreased plasma glucose levels during the test and after overnight fasting in all volunteers.
Research communications in molecular pathology and pharmacology, 2000
A kinetic model describing the behavior of extracellularly supplied cAMP in the perfused rat live... more A kinetic model describing the behavior of extracellularly supplied cAMP in the perfused rat liver was derived and compared with experimental data. The model was based on the following conditions and assumptions: a) labeled cAMP is being constantly infused (step input); b) permeation of the cell membrane is an essentially irreversible step (k(in) as transfer coefficient); c) the adenine moiety of cAMP incorporates into a nucleotide pool (km1 as transformation coefficient), which cannot permeate the cell membrane; d) the adenine moiety of cAMP can be transferred from the nucleotide pool to a nucleoside + free base pool (km2 as transformation coefficient), which is able to permeate the cell membrane (k(ef) as transfer coefficient). These events were described by a series of differential equations for which an analytical solution was obtained. Total cellular incorporation of label derived from [3H]cAMP was measured in the isolated perfused rat liver. The equations of the model were fit...
Research communications in molecular pathology and pharmacology, 1998
The hemodynamic effects of diltiazem in the liver are strictly Ca2+ -dependent Consequently, Ca2+... more The hemodynamic effects of diltiazem in the liver are strictly Ca2+ -dependent Consequently, Ca2+ -free perfusion can be used for investigating the metabolic effects of diltiazem without interference by hemodynamics. Livers were perfused with Krebs/Henseleit-bicarbonate buffer (pH 7.4). For performing Ca2+ -free perfusion the cation was omitted from the perfusion fluid and the cellular pools were exhausted by repeated phenylephrine infusions. Three conditions were investigated with and without Ca2+ : (1) substrate-free perfusion fluid; (2) 0.3 mM [1-(14)C]octanoate infusion; (3) 0.3 mM [1-(14)C]palmitate infusion. The following results were obtained: 1. Oxygen uptake stimulation caused by octanoate and palmitate was abolished by 500 microM diltiazem in the presence of Ca2+; in the absence of Ca2+ there was no inhibition (octanoate) or it was much smaller (palmitate); 2. The 14CO2 production was inhibited in the presence of Ca2+; in the absence of Ca2+ there was no inhibition (palmit...
Research communications in chemical pathology and pharmacology, 1989
The action of glucagon, epinephrine and cyanide on hepatic glycogen catabolism in meal-fed rats a... more The action of glucagon, epinephrine and cyanide on hepatic glycogen catabolism in meal-fed rats and the corresponding controls was investigated using the isolated perfused liver. The amounts of glycogen catabolites (glucose, L-lactate and pyruvate) released were correlated with the glycogen content of the livers at the different times after feeding. Irrespective of the metabolic condition, the sensitivity of glycogenolysis to glucagon was roughly proportional to the glycogen content of the livers. However, glycogenolysis in livers from meal-fed rats was less sensitive to epinephrine and cyanide. The difference between meal-fed and controls (ad libitum-fed) was particularly pronounced at 22 hours after feeding. It was concluded that resistance to hepatic glycogen depletion in meal-fed rats during the fasting period may be, partly at least, the consequence of a reduced sensitivity of glycogenolysis to epinephrine and to situations of reduced rates of oxidative phosphorylation.
Brazilian journal of medical and biological research = Revista brasileira de pesquisas médicas e biológicas / Sociedade Brasileira de Biofísica ... [et al.], 1989
The multiple indicator dilution technique was used to investigate octanoate transport in the isol... more The multiple indicator dilution technique was used to investigate octanoate transport in the isolated, perfused rat liver. The form of the octanoate outflow profiles depends on albumin concentration. The steady-state intracellular octanoate concentration and the rate of uptake increase when the albumin concentration is decreased. At physiological albumin concentrations, the intracellular concentration of octanoate is only 7% of the extracellular concentration. Exchange between the intra- and extracellular space, however, is very rapid, irrespective of the albumin concentration and consequently exchange is not rate limiting for metabolism.
Diltiazem causes vasoconstriction in the liver when present at high concentrations, an action tha... more Diltiazem causes vasoconstriction in the liver when present at high concentrations, an action that is strictly Ca 2+-dependent. Diltiazem is also active on energy metabolism. This toxic action could be partly a consequence of hemodynamic e¡ects. In the absence of Ca 2+ , the hemodynamic e¡ects are no longer present and, consequently, Ca 2+-free experiments are useful for distinguishing between hemodynamics-dependent and hemodynamics-independent e¡ects. The experimental system used was the hemoglobin-free perfused rat liver from fed and fasted rats. Diltiazem was infused at various concentrations in the presence and absence of Ca 2+. Several metabolic parameters were measured: lactate and pyruvate production (glycolysis), glycogenolysis, oxygen uptake, gluconeogenesis, and the cellular levels of lactate, pyruvate, glucose, AMP, ADP, and ATP. The e¡ects of diltiazem can be divided into three groups: (1) E¡ects that are strictly dependent on the Ca 2+mediated hemodynamic action. This group comprises inhibition of oxygen uptake at all concentrations (50^500 mmol/L) inhibition of lactate, pyruvate, and glucose release at high concentrations; the decrease in cellular ATP; the increase in cellular AMP; and the cellular accumulation of glucose and lactate. (2) E¡ects that are independent of the hemodynamic action. The most relevant e¡ect of this type is inhibition of gluconeogenesis. (3) E¡ects that are in£uenced by Ca 2+ but are independent of the hemodynamic e¡ects. This is the typical case of lactate and glucose release from endogenous glycogen, whose stimulation by low diltiazem concentrations is more pronounced in the presence of Ca 2+ than in its absence.
The heterogeneity of the liver parenchyma in relation to uric acid production from adenosine was ... more The heterogeneity of the liver parenchyma in relation to uric acid production from adenosine was investigated using the bivascularly perfused rat liver in the anterograde and retrograde modes. Adenosine was infused in livers from fed rats during 20 min at four different concentrations (20, 50, 100 and 200 µM) according to four experimental protocols as follows: (A) anterograde perfusion, with adenosine infusion into the portal vein; (B) anterograde perfusion, with adenosine in the hepatic artery, (C) retrograde perfusion, with adenosine in the hepatic vein; (D) retrograde perfusion, with adenosine in the hepatic artery. With protocols A, B, and D uric acid production from adenosine was always characterized by initial bursts followed by progressive decreases toward smaller steady-states. With protocol C the initial burst was present only when 200 µM adenosine was infused. The initial bursts in uric acid production were accompanied by simultaneous increases in the ratio of uric acid production/adenosine uptake rate. These initial bursts are thus representing increments in the production of uric acid that are not corresponded by similar increments in the metabolic uptake rates of adenosine. Global analysis of uric acid production revealed that the final steady-state rates were approximately equal for all infusion rates with protocols A, B and C, but smaller with protocol D. This difference, however, can be explained in terms of the differences in accessible cellular spaces, which are much smaller when protocol D is employed. When the analysis was performed in terms of the extra amounts of uric acid produced during the infusion of adenosine, where the initial bursts are also taken into account, different dose-response curves were found for each experimental protocol. These differences cannot be explained in terms of the accessible cell spaces and they are likely to reflect regional heterogeneities. From the various dose-response curves and from the known characteristics of the microcirculation of the rat liver it can be concluded that the initial bursts in uric acid production are generated in periportal hepatocytes. The reason for this heterogeneity could be related to the metabolic effects of adenosine, especially to oxygen uptake inhibition, which is likely to produce changes in the ATP/AMP ratios.
1. The influence of quercetin on glycogen catabolism and related parameters was investigated in t... more 1. The influence of quercetin on glycogen catabolism and related parameters was investigated in the isolated perfused rat liver and subcellular systems. 2. Quercetin stimulated glycogenolysis (glucose release). This effect was already evident at a concentration of 50 microM maximal at 300 microM and declined at higher concentrations. Quercetin also stimulated oxygen consumption, with a similar concentration dependence. 3. Lactate production from endogenous glycogen (glycolysis) was diminished by quercetin without significant changes in pyruvate production. 4. Quercetin did not inhibit glucose transport into cells but decreased intracellular sequestration of [5-(3)H]glucose under conditions of net glucose release. 5. In isolated mitochondria, quercetin diminished the energy transduction efficiency. It also inhibited several enzymatic activities, e.g. the K(+)-ATPase/Na(+)-ATPase of plasma membrane vesicles and the glucose 6-phosphatase of isolated microsomes. 6. No significant changes of the cellular contents of AMP, ADP and ATP were found. The cellular content of glucose 6-phosphate, however, was increased (3.12-fold). 7. Some of the effects of quercetin (glycogenolysis stimulation) can be attributed to its action on mitochondrial energy metabolism, as, for example, uncoupling of oxidative phosphorylation. However, the multiplicity of the effects on several enzymatic systems certainly produces an intricate interplay that also generates complex and apparently contradictory effects.
Hepatic metabolic fluxes (glycolysis, glucose release, glycogenolysis, oxygen consumption, ketoge... more Hepatic metabolic fluxes (glycolysis, glucose release, glycogenolysis, oxygen consumption, ketogenesis and gluconeogenesis), hepatic glycogen and food ingestion in meal-fed rats were measured and compared to appropriate controls. The following results were obtained: 1) in livers from meal-fed rats a higher fraction of glucosyl units derived from glycogen is used in glycolysis instead of being released in the form of glucose; 2) the rate of glycogen catabolism in livers from meal-fed rats is less than expected when one compares their glycogen levels with those of the appropriate controls; 3) the livers from meal-fed rats become much less ketotic than the livers from rats which were not trained to eat a single meal daily. It was concluded that the liver of meal-fed rats is well adapted to the main characteristics of those animals, e.g., increased lipogenesis from glycolysis products and a reduced need for carbon units from the liver (glucose and ketone bodies) as a consequence of enhanced food intake.
In the rat liver NAD+ infusion produces increases in portal perfusion pressure and glycogenolysis... more 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 microM) 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.
Journal of Biochemical and Molecular Toxicology, 2010
Flavonols, which possess the B-catechol ring, as quercetin, are capable of producing o-hemiquinon... more Flavonols, which possess the B-catechol ring, as quercetin, are capable of producing o-hemiquinones and to oxidize NADH in a variety of mammalian cells. The purpose of this study was to investigate whether fisetin affects the liver energy metabolism and the mitochondrial NADH to NAD+ ratio. The action of fisetin on hepatic energy metabolism was investigated in the perfused rat liver and isolated mitochondria. In isolated mitochondria, fisetin decreased the respiratory control and ADP/O ratios with the substrates α-ketoglutarate and succinate. In the presence of ADP, respiration of isolated mitochondria was inhibited with both substrates, indicating an inhibitory action on the ATP-synthase. The stimulation of the ATPase activity of coupled mitochondria and the inhibition of NADH-oxidase activity pointed toward a possible uncoupling action and the interference of fisetin with mitochondrial energy transduction mechanisms. In livers from fasted rats, fisetin inhibited ketogenesis from endogenous sources. The β-hydroxybutyrate/ acetoacetate ratio, which reflects the mitochondrial NADH/NAD+ redox ratio, was also decreased. In addition, fisetin (200 μM) increased the production of (14)CO2 from exogenous oleate. The results of this investigation suggest that fisetin causes a shift in the mitochondrial redox potential toward a more oxidized state with a clear predominance of its prooxidant activity.
The zonation of the purinergic action of AMP in the hepatic parenchyma was investigated in the bi... more The zonation of the purinergic action of AMP in the hepatic parenchyma was investigated in the bivascularly perfused rat liver by means of anterograde and retrograde perfusion. Livers from fed rats were used and AMP (100 microM) was infused according to four different experimental protocols: (A) anterograde perfusion and AMP infusion via the portal vein; (B) anterograde perfusion and AMP infusion via the hepatic artery; (C) retrograde perfusion and AMP via the hepatic vein; (D) retrograde perfusion and AMP via the hepatic artery. The response of the liver cells was heterogeneous. Oxygen uptake inhibition by AMP predominates in cells situated shortly after the intrasinusoidal confluence of the portal vein and hepatic artery. Oxygen consumption in all other cells seems to be increased by AMP. Glycogenolysis stimulation by AMP (glucose release) was more pronounced in the periportal cells situated in the region of the intrasinusoidal confluence of the portal vein and the hepatic artery. It can be concluded that the heterogenic response of the liver to AMP is similar to the heterogenic response to ATP.
The purpose of the present work was to investigate the possible inhibitory effect of a pinhão coa... more The purpose of the present work was to investigate the possible inhibitory effect of a pinhão coat extract rich in condensed tannin on the activity of α-amylases (human salivary and porcine pancreatic). Experiments with the classic α-amylase inhibitor acarbose and a condensed tannin from Acacia mearnsii were done for comparative purposes. Fourier transform-infrared spectroscopy analysis of the pinhão coat tannin revealed a higher proportion of procyanidins to prodelphinidins when compared to the A. mearnsii tannin. The pinhão coat extract rich in condensed tannin was an effective inhibitor of both human salivary and porcine pancreatic α-amylase. Inhibition was of the mixed S-parabolic I-parabolic type. For the human salivary α-amylase the inhibition constants were 56.88 ± 5.74 and 103.27 ± 11.85 μg/L; for the porcine pancreatic α-amylase the inhibition constants were smaller, namely, 20.25 ± 1.97 and 46.79 ± 4.57 μg/L. The decreasing potency sequence was: acarbose N pinhão coat extract rich in tannin N A. mearnsii tannin. Similarly to acarbose and the A. mearnsii tannin, the pinhão coat extract was also effective in diminishing the post-prandial glycemic levels in rats after starch administration. The inhibitory properties of the pinhão coat extract indicate that it can be used to suppress postprandial hyperglycemia in diabetic patients.
Page 1. Research Article Effects of a New 1,3,4-Thiadiazolium Mesoionic Compound, MI-D, on the Ac... more Page 1. Research Article Effects of a New 1,3,4-Thiadiazolium Mesoionic Compound, MI-D, on the Acute Inflammatory Response Júlio C. Cardoso,1 Sılvia MSC Cadena,1 Aleksander Zampronio,2 Ana Maria S. Arruda,2w Eva ...
Usnic acid is a naturally occurring dibenzofuran derivative found in several lichen species. The ... more Usnic acid is a naturally occurring dibenzofuran derivative found in several lichen species. The compound has been marketed as an ingredient of food supplements for weight reduction. There is evidence that the comp ound acts as an uncouple r of mitochondrial oxidati ve phosphorylation and it is also clear that consumption of the drug can lead to severe hepatotoxicity depending on the doses. Based on these and other ideas the objective of the present work was to investigate the possibl e effects of usnic acid on liver metaboli sm. Livers of male Wistar rats were perfused in a non-recirc ulating system. Usnic acid stimulated oxygen consumption at low concentrations, diminished the cellular ATP levels, increased the cytosolic but diminished the mitochondrial NADH/NAD + ratio, strongly inhibite d gluconeoge nesis from three different substrates (IC 50 between 1.33 and 3.61 lM), stimulated glycolysis, fructolysis, glycogenolysis and ammoniagenesis and inhibited ureogenesis. The 14 CO 2 production from [1-14 C]octanoate and [1-14 C]oleate was increased by usnic acid, but ketogenesis from octanoate was diminished and that from oleate was not affected. It may be concluded that the effects of usnic acid up to 2.5 lM reflect predominantly its activity as an uncouple r. At higher concentrations, however, several other effects may become significant, including inhibition of mitochondrial electron flow and inhibition of medium-chain fatty acid oxidatio n. In metabolic terms, toxicity of usnic acid can be predicted to be espec ially dangerous in the fasted state due to the combination of several deleterius events such as diminished hepatic glucose and ketone bodies output to the brain and increased ammonia produc tion.
Comparative Biochemistry and Physiology - Part C: Pharmacology, Toxicology and Endocrinology, 1996
1. The actions of piroxicam, a nonsteroidal and noncarboxylic anti-inflammatory drug, on the meta... more 1. The actions of piroxicam, a nonsteroidal and noncarboxylic anti-inflammatory drug, on the metabolism of the isolated perfused rat liver were investigated. The main purpose was to verify if piroxicam is also active on glycogenolysis and energy metabolism, as demonstrated for several carboxylic nonsteroidal anti-inflammatories. 2. Piroxicam increased oxygen consumption in livers from both fed and fasted rats. 3. Piroxicam increased glucose release and glycolysis from endogenous glycogen (glycogenolysis). 4. Gluconeogenesis from lactate plus pyruvate was inhibited. 5. The action of piroxicam on oxygen consumption was blocked by antimycin A, but not by atractyloside. 6. The action of piroxicam in the perfused rat liver metabolism seems to be a consequence of its action on mitochondria. 7. It can be concluded that inhibition of energy metabolism and stimulation of glycogenolysis are not specific properties of carboxylic nonsteroidal anti-inflammatory drugs.
The crucial difference between the biochemical education in Brazil and more developed countries r... more The crucial difference between the biochemical education in Brazil and more developed countries resides not in the highest degrees but in the lowest ones: the bachelor degree in Biochemistry is still a rarity in Brazil. Whereas more than 600 institutions in the US offer the bachelor degree in biochemistry/molecular biology, presently only 3 are offered in Brazil. This situation may be surprising, but it has historical reasons among others. The field of Biochemistry in Brazil played a pioneering role in institutionalizing master's and doctoral programs, thus reinforcing also research activities. Teachers and researchers involved in these programs, however, had in general little if any interest in implementing undergraduate biochemistry studies, except as a fundamental one year discipline to be taken by future biologists, pharmacists, medical students, etc. The second reason for the virtual absence of the biochemistry bachelor degree before 2005 in Brazil are the so called baccalaureates in "Pharmacy and Biochemistry" a strange denomination whose more correct name should be "Pharmacy and Clinical Analyses". This situation generated the notion, within and outside academic circles, that there is a very especial relationship between Pharmacy and Biochemistry or even that they are one and the same thing. These and other erroneous notions have proven to be adverse for the brazilian biochemistry in both professional and academic spheres, especially in more recent times when the master's and doctoral programs ceased to offer the more basic disciplines. Many graduates with a master's or even doctoral degree have a highly defficient background on physical and organic chemistry, or even on metabolic biochemistry and enzymology if the program is centered on molecular biology.
1. The influence of Ca2+ on the effects of glucagon on glycolysis was investigated in the isolate... more 1. The influence of Ca2+ on the effects of glucagon on glycolysis was investigated in the isolated perfused rat liver. Livers from fed rats were perfused in an open system with Krebs/Henseleit-bicarbonate buffer (pH 7.4). Glucose release, lactate plus pyruvate production (glycolysis) and oxygen uptake were measured. The following results were obtained: 2. In livers perfused with Ca(2+)-free Krebs/Henseleit-bicarbonate buffer and after depletion of the intracellular pools, the initial and transient stimulation of glycolysis, which is normally observed shortly after the onset of glucagon infusion, was more pronounced when compared to livers perfused with normal perfusion fluid (2.5 mM Ca2+) and without previous depletion of the intracellular pools (controls); the subsequent inhibition of glycolysis was delayed in Ca(2+)-free perfused livers and was less pronounced in comparison with the controls at the end of the glucagon infusion period (20 min). 3. Perfusion with a Ca(2+)-free medium supplemented with EDTA, without previous depletion of the intracellular pools, also produced a substantial reduction in the effects of glucagon on glycolysis. 4. Ca(2+)-free perfusion did not affect the stimulative action of glucagon on glucose release (glycogenolysis) and oxygen uptake. 5. Glycolysis inhibition by cAMP also was abolished in Ca(2+)-free perfused livers, and the initial stimulation was enhanced. 6. Mn2+, a metal ion known as a competitor of Ca2+, considerably reduced the action of glucagon on glycolysis; Mn2+ did not affect the basal rates of glycolysis. 7. Sr2+, a metal ion that is often recognized as Ca2+ by several biological structures and processes, increased the inhibitory action of glucagon on glycolysis. 8. Several organic compounds, which directly or indirectly take part in Ca2+ fluxes, were also able to diminish (e.g., verapamil) or even to abolish (carbenoxolone) the inhibitory action of glucagon on glycolysis. 9. It was concluded that, under the conditions of the living cell, Ca2+ is important for glycolysis inhibition by glucagon. In principle at least, the results can be explained in terms of the known Ca2+ dependencies of several protein kinases and protein phosphatases.
Brazilian journal of medical and biological research = Revista brasileira de pesquisas médicas e biológicas / Sociedade Brasileira de Biofísica ... [et al.], 1986
The effect of aqueous extracts of Stevia rebaudiana leaves on a glucose tolerance test was invest... more The effect of aqueous extracts of Stevia rebaudiana leaves on a glucose tolerance test was investigated in 16 normal volunteers. Aqueous extracts of 5 grams of leaves were administered to volunteers at regular 6-h intervals for 3 days. Glucose tolerance tests were performed before and after extract administration. A second group of 6 normal volunteers who ingested an aqueous arabinose solution was also studied to eliminate possible stress effects. The extract of Stevia rebaudiana increased glucose tolerance. The extract significantly decreased plasma glucose levels during the test and after overnight fasting in all volunteers.
Research communications in molecular pathology and pharmacology, 2000
A kinetic model describing the behavior of extracellularly supplied cAMP in the perfused rat live... more A kinetic model describing the behavior of extracellularly supplied cAMP in the perfused rat liver was derived and compared with experimental data. The model was based on the following conditions and assumptions: a) labeled cAMP is being constantly infused (step input); b) permeation of the cell membrane is an essentially irreversible step (k(in) as transfer coefficient); c) the adenine moiety of cAMP incorporates into a nucleotide pool (km1 as transformation coefficient), which cannot permeate the cell membrane; d) the adenine moiety of cAMP can be transferred from the nucleotide pool to a nucleoside + free base pool (km2 as transformation coefficient), which is able to permeate the cell membrane (k(ef) as transfer coefficient). These events were described by a series of differential equations for which an analytical solution was obtained. Total cellular incorporation of label derived from [3H]cAMP was measured in the isolated perfused rat liver. The equations of the model were fit...
Research communications in molecular pathology and pharmacology, 1998
The hemodynamic effects of diltiazem in the liver are strictly Ca2+ -dependent Consequently, Ca2+... more The hemodynamic effects of diltiazem in the liver are strictly Ca2+ -dependent Consequently, Ca2+ -free perfusion can be used for investigating the metabolic effects of diltiazem without interference by hemodynamics. Livers were perfused with Krebs/Henseleit-bicarbonate buffer (pH 7.4). For performing Ca2+ -free perfusion the cation was omitted from the perfusion fluid and the cellular pools were exhausted by repeated phenylephrine infusions. Three conditions were investigated with and without Ca2+ : (1) substrate-free perfusion fluid; (2) 0.3 mM [1-(14)C]octanoate infusion; (3) 0.3 mM [1-(14)C]palmitate infusion. The following results were obtained: 1. Oxygen uptake stimulation caused by octanoate and palmitate was abolished by 500 microM diltiazem in the presence of Ca2+; in the absence of Ca2+ there was no inhibition (octanoate) or it was much smaller (palmitate); 2. The 14CO2 production was inhibited in the presence of Ca2+; in the absence of Ca2+ there was no inhibition (palmit...
Research communications in chemical pathology and pharmacology, 1989
The action of glucagon, epinephrine and cyanide on hepatic glycogen catabolism in meal-fed rats a... more The action of glucagon, epinephrine and cyanide on hepatic glycogen catabolism in meal-fed rats and the corresponding controls was investigated using the isolated perfused liver. The amounts of glycogen catabolites (glucose, L-lactate and pyruvate) released were correlated with the glycogen content of the livers at the different times after feeding. Irrespective of the metabolic condition, the sensitivity of glycogenolysis to glucagon was roughly proportional to the glycogen content of the livers. However, glycogenolysis in livers from meal-fed rats was less sensitive to epinephrine and cyanide. The difference between meal-fed and controls (ad libitum-fed) was particularly pronounced at 22 hours after feeding. It was concluded that resistance to hepatic glycogen depletion in meal-fed rats during the fasting period may be, partly at least, the consequence of a reduced sensitivity of glycogenolysis to epinephrine and to situations of reduced rates of oxidative phosphorylation.
Brazilian journal of medical and biological research = Revista brasileira de pesquisas médicas e biológicas / Sociedade Brasileira de Biofísica ... [et al.], 1989
The multiple indicator dilution technique was used to investigate octanoate transport in the isol... more The multiple indicator dilution technique was used to investigate octanoate transport in the isolated, perfused rat liver. The form of the octanoate outflow profiles depends on albumin concentration. The steady-state intracellular octanoate concentration and the rate of uptake increase when the albumin concentration is decreased. At physiological albumin concentrations, the intracellular concentration of octanoate is only 7% of the extracellular concentration. Exchange between the intra- and extracellular space, however, is very rapid, irrespective of the albumin concentration and consequently exchange is not rate limiting for metabolism.
Diltiazem causes vasoconstriction in the liver when present at high concentrations, an action tha... more Diltiazem causes vasoconstriction in the liver when present at high concentrations, an action that is strictly Ca 2+-dependent. Diltiazem is also active on energy metabolism. This toxic action could be partly a consequence of hemodynamic e¡ects. In the absence of Ca 2+ , the hemodynamic e¡ects are no longer present and, consequently, Ca 2+-free experiments are useful for distinguishing between hemodynamics-dependent and hemodynamics-independent e¡ects. The experimental system used was the hemoglobin-free perfused rat liver from fed and fasted rats. Diltiazem was infused at various concentrations in the presence and absence of Ca 2+. Several metabolic parameters were measured: lactate and pyruvate production (glycolysis), glycogenolysis, oxygen uptake, gluconeogenesis, and the cellular levels of lactate, pyruvate, glucose, AMP, ADP, and ATP. The e¡ects of diltiazem can be divided into three groups: (1) E¡ects that are strictly dependent on the Ca 2+mediated hemodynamic action. This group comprises inhibition of oxygen uptake at all concentrations (50^500 mmol/L) inhibition of lactate, pyruvate, and glucose release at high concentrations; the decrease in cellular ATP; the increase in cellular AMP; and the cellular accumulation of glucose and lactate. (2) E¡ects that are independent of the hemodynamic action. The most relevant e¡ect of this type is inhibition of gluconeogenesis. (3) E¡ects that are in£uenced by Ca 2+ but are independent of the hemodynamic e¡ects. This is the typical case of lactate and glucose release from endogenous glycogen, whose stimulation by low diltiazem concentrations is more pronounced in the presence of Ca 2+ than in its absence.
The heterogeneity of the liver parenchyma in relation to uric acid production from adenosine was ... more The heterogeneity of the liver parenchyma in relation to uric acid production from adenosine was investigated using the bivascularly perfused rat liver in the anterograde and retrograde modes. Adenosine was infused in livers from fed rats during 20 min at four different concentrations (20, 50, 100 and 200 µM) according to four experimental protocols as follows: (A) anterograde perfusion, with adenosine infusion into the portal vein; (B) anterograde perfusion, with adenosine in the hepatic artery, (C) retrograde perfusion, with adenosine in the hepatic vein; (D) retrograde perfusion, with adenosine in the hepatic artery. With protocols A, B, and D uric acid production from adenosine was always characterized by initial bursts followed by progressive decreases toward smaller steady-states. With protocol C the initial burst was present only when 200 µM adenosine was infused. The initial bursts in uric acid production were accompanied by simultaneous increases in the ratio of uric acid production/adenosine uptake rate. These initial bursts are thus representing increments in the production of uric acid that are not corresponded by similar increments in the metabolic uptake rates of adenosine. Global analysis of uric acid production revealed that the final steady-state rates were approximately equal for all infusion rates with protocols A, B and C, but smaller with protocol D. This difference, however, can be explained in terms of the differences in accessible cellular spaces, which are much smaller when protocol D is employed. When the analysis was performed in terms of the extra amounts of uric acid produced during the infusion of adenosine, where the initial bursts are also taken into account, different dose-response curves were found for each experimental protocol. These differences cannot be explained in terms of the accessible cell spaces and they are likely to reflect regional heterogeneities. From the various dose-response curves and from the known characteristics of the microcirculation of the rat liver it can be concluded that the initial bursts in uric acid production are generated in periportal hepatocytes. The reason for this heterogeneity could be related to the metabolic effects of adenosine, especially to oxygen uptake inhibition, which is likely to produce changes in the ATP/AMP ratios.
1. The influence of quercetin on glycogen catabolism and related parameters was investigated in t... more 1. The influence of quercetin on glycogen catabolism and related parameters was investigated in the isolated perfused rat liver and subcellular systems. 2. Quercetin stimulated glycogenolysis (glucose release). This effect was already evident at a concentration of 50 microM maximal at 300 microM and declined at higher concentrations. Quercetin also stimulated oxygen consumption, with a similar concentration dependence. 3. Lactate production from endogenous glycogen (glycolysis) was diminished by quercetin without significant changes in pyruvate production. 4. Quercetin did not inhibit glucose transport into cells but decreased intracellular sequestration of [5-(3)H]glucose under conditions of net glucose release. 5. In isolated mitochondria, quercetin diminished the energy transduction efficiency. It also inhibited several enzymatic activities, e.g. the K(+)-ATPase/Na(+)-ATPase of plasma membrane vesicles and the glucose 6-phosphatase of isolated microsomes. 6. No significant changes of the cellular contents of AMP, ADP and ATP were found. The cellular content of glucose 6-phosphate, however, was increased (3.12-fold). 7. Some of the effects of quercetin (glycogenolysis stimulation) can be attributed to its action on mitochondrial energy metabolism, as, for example, uncoupling of oxidative phosphorylation. However, the multiplicity of the effects on several enzymatic systems certainly produces an intricate interplay that also generates complex and apparently contradictory effects.
Hepatic metabolic fluxes (glycolysis, glucose release, glycogenolysis, oxygen consumption, ketoge... more Hepatic metabolic fluxes (glycolysis, glucose release, glycogenolysis, oxygen consumption, ketogenesis and gluconeogenesis), hepatic glycogen and food ingestion in meal-fed rats were measured and compared to appropriate controls. The following results were obtained: 1) in livers from meal-fed rats a higher fraction of glucosyl units derived from glycogen is used in glycolysis instead of being released in the form of glucose; 2) the rate of glycogen catabolism in livers from meal-fed rats is less than expected when one compares their glycogen levels with those of the appropriate controls; 3) the livers from meal-fed rats become much less ketotic than the livers from rats which were not trained to eat a single meal daily. It was concluded that the liver of meal-fed rats is well adapted to the main characteristics of those animals, e.g., increased lipogenesis from glycolysis products and a reduced need for carbon units from the liver (glucose and ketone bodies) as a consequence of enhanced food intake.
In the rat liver NAD+ infusion produces increases in portal perfusion pressure and glycogenolysis... more 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 microM) 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.
Journal of Biochemical and Molecular Toxicology, 2010
Flavonols, which possess the B-catechol ring, as quercetin, are capable of producing o-hemiquinon... more Flavonols, which possess the B-catechol ring, as quercetin, are capable of producing o-hemiquinones and to oxidize NADH in a variety of mammalian cells. The purpose of this study was to investigate whether fisetin affects the liver energy metabolism and the mitochondrial NADH to NAD+ ratio. The action of fisetin on hepatic energy metabolism was investigated in the perfused rat liver and isolated mitochondria. In isolated mitochondria, fisetin decreased the respiratory control and ADP/O ratios with the substrates α-ketoglutarate and succinate. In the presence of ADP, respiration of isolated mitochondria was inhibited with both substrates, indicating an inhibitory action on the ATP-synthase. The stimulation of the ATPase activity of coupled mitochondria and the inhibition of NADH-oxidase activity pointed toward a possible uncoupling action and the interference of fisetin with mitochondrial energy transduction mechanisms. In livers from fasted rats, fisetin inhibited ketogenesis from endogenous sources. The β-hydroxybutyrate/ acetoacetate ratio, which reflects the mitochondrial NADH/NAD+ redox ratio, was also decreased. In addition, fisetin (200 μM) increased the production of (14)CO2 from exogenous oleate. The results of this investigation suggest that fisetin causes a shift in the mitochondrial redox potential toward a more oxidized state with a clear predominance of its prooxidant activity.
The zonation of the purinergic action of AMP in the hepatic parenchyma was investigated in the bi... more The zonation of the purinergic action of AMP in the hepatic parenchyma was investigated in the bivascularly perfused rat liver by means of anterograde and retrograde perfusion. Livers from fed rats were used and AMP (100 microM) was infused according to four different experimental protocols: (A) anterograde perfusion and AMP infusion via the portal vein; (B) anterograde perfusion and AMP infusion via the hepatic artery; (C) retrograde perfusion and AMP via the hepatic vein; (D) retrograde perfusion and AMP via the hepatic artery. The response of the liver cells was heterogeneous. Oxygen uptake inhibition by AMP predominates in cells situated shortly after the intrasinusoidal confluence of the portal vein and hepatic artery. Oxygen consumption in all other cells seems to be increased by AMP. Glycogenolysis stimulation by AMP (glucose release) was more pronounced in the periportal cells situated in the region of the intrasinusoidal confluence of the portal vein and the hepatic artery. It can be concluded that the heterogenic response of the liver to AMP is similar to the heterogenic response to ATP.
The purpose of the present work was to investigate the possible inhibitory effect of a pinhão coa... more The purpose of the present work was to investigate the possible inhibitory effect of a pinhão coat extract rich in condensed tannin on the activity of α-amylases (human salivary and porcine pancreatic). Experiments with the classic α-amylase inhibitor acarbose and a condensed tannin from Acacia mearnsii were done for comparative purposes. Fourier transform-infrared spectroscopy analysis of the pinhão coat tannin revealed a higher proportion of procyanidins to prodelphinidins when compared to the A. mearnsii tannin. The pinhão coat extract rich in condensed tannin was an effective inhibitor of both human salivary and porcine pancreatic α-amylase. Inhibition was of the mixed S-parabolic I-parabolic type. For the human salivary α-amylase the inhibition constants were 56.88 ± 5.74 and 103.27 ± 11.85 μg/L; for the porcine pancreatic α-amylase the inhibition constants were smaller, namely, 20.25 ± 1.97 and 46.79 ± 4.57 μg/L. The decreasing potency sequence was: acarbose N pinhão coat extract rich in tannin N A. mearnsii tannin. Similarly to acarbose and the A. mearnsii tannin, the pinhão coat extract was also effective in diminishing the post-prandial glycemic levels in rats after starch administration. The inhibitory properties of the pinhão coat extract indicate that it can be used to suppress postprandial hyperglycemia in diabetic patients.
Page 1. Research Article Effects of a New 1,3,4-Thiadiazolium Mesoionic Compound, MI-D, on the Ac... more Page 1. Research Article Effects of a New 1,3,4-Thiadiazolium Mesoionic Compound, MI-D, on the Acute Inflammatory Response Júlio C. Cardoso,1 Sılvia MSC Cadena,1 Aleksander Zampronio,2 Ana Maria S. Arruda,2w Eva ...
Usnic acid is a naturally occurring dibenzofuran derivative found in several lichen species. The ... more Usnic acid is a naturally occurring dibenzofuran derivative found in several lichen species. The compound has been marketed as an ingredient of food supplements for weight reduction. There is evidence that the comp ound acts as an uncouple r of mitochondrial oxidati ve phosphorylation and it is also clear that consumption of the drug can lead to severe hepatotoxicity depending on the doses. Based on these and other ideas the objective of the present work was to investigate the possibl e effects of usnic acid on liver metaboli sm. Livers of male Wistar rats were perfused in a non-recirc ulating system. Usnic acid stimulated oxygen consumption at low concentrations, diminished the cellular ATP levels, increased the cytosolic but diminished the mitochondrial NADH/NAD + ratio, strongly inhibite d gluconeoge nesis from three different substrates (IC 50 between 1.33 and 3.61 lM), stimulated glycolysis, fructolysis, glycogenolysis and ammoniagenesis and inhibited ureogenesis. The 14 CO 2 production from [1-14 C]octanoate and [1-14 C]oleate was increased by usnic acid, but ketogenesis from octanoate was diminished and that from oleate was not affected. It may be concluded that the effects of usnic acid up to 2.5 lM reflect predominantly its activity as an uncouple r. At higher concentrations, however, several other effects may become significant, including inhibition of mitochondrial electron flow and inhibition of medium-chain fatty acid oxidatio n. In metabolic terms, toxicity of usnic acid can be predicted to be espec ially dangerous in the fasted state due to the combination of several deleterius events such as diminished hepatic glucose and ketone bodies output to the brain and increased ammonia produc tion.
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