Jorgete Constantin

Metabolic effects of silibinin in the rat liver.

The flavonolignan silibinin, which is a mixture of two diastereoisomers, silybin A and silybin B, is a component of the extract obtained from the fruit and seeds of the variegated milk thistle (Silybum marianum (L.) Gaertn. (Asteraceae)), known as silymarin. Among the therapeutic properties credited to silibinin, its antihyperglycaemic action has been extensively explored. Silibinin is structurally related to the flavonoids quercetin and fisetin, which have been previously demonstrated to be very active on liver metabolic processes related to glycaemic regulation. The aim of the present work was to investigate the effects of silibinin on metabolic pathways responsible for the maintenance of glycaemia, particularly glycogenolysis and gluconeogenesis, in the perfused rat liver. The activities of some key enzymes in these pathways and on parameters of energy metabolism in isolated mitochondria were also examined. At a concentration range of 50-300μM, silibinin inhibited gluconeogenesis in the fasted condition and inhibited glycogenolysis and glycolysis in the fed condition. The mechanisms by which silibinin exerted these actions were multiple and complex. It inhibited the activity of glucose 6-phosphatase, inhibited the pyruvate carrier, and reduced the efficiency of mitochondrial energy transduction. It can also act by reducing the supply of NADH for gluconeogenesis and mitochondria through its pro-oxidative actions. In general, the effects and the potency of silibinin were similar to those of quercetin and fisetin. However, silibinin exerted some distinct effects such as the inhibitory effect on oxygen consumption in the fed condition and a change in the energy status of the perfused livers. It can be concluded that the effects of silibinin on liver glucose metabolism may explain its antihyperglycaemic property. However, this effect was, in part, secondary to impairment in cellular energy metabolism, a finding that should be considered in its therapeutic usage.

Hepatic zonation of carbon and nitrogen fluxes derived from glutamine and ammonia transformations

BackgroundGlutaminase predominates in periportal hepatocytes and it has been proposed that it determines the glutamine-derived nitrogen flow through the urea cycle. Glutamine-derived urea production should, thus, be considerably faster in periportal hepatocytes. This postulate, based on indirect observations, has not yet been unequivocally demonstrated, making a direct investigation of ureogenesis from glutamine highly desirable.MethodsZonation of glutamine metabolism was investigated in the bivascularly perfused rat liver with [U-14C]glutamine infusion (0.6 mM) into the portal vein (antegrade perfusion) or into the hepatic vein (retrograde perfusion).ResultsAmmonia infusion into the hepatic artery in retrograde and antegrade perfusion allowed to promote glutamine metabolism in the periportal region and in the whole liver parenchyma, respectively. The results revealed that the space-normalized glutamine uptake, indicated by 14CO2 production, gluconeogenesis, lactate production and the associated oxygen uptake, predominates in the periportal region. Periportal predominance was especially pronounced for gluconeogenesis. Ureogenesis, however, tended to be uniformly distributed over the whole liver parenchyma at low ammonia concentrations (up to 1.0 mM); periportal predominance was found only at ammonia concentrations above 1 mM. The proportions between the carbon and nitrogen fluxes in periportal cells are not the same along the liver acinus.ConclusionsIn conclusion, the results of the present work indicate that the glutaminase activity in periportal hepatocytes is not the rate-controlling step of the glutamine-derived nitrogen flow through the urea cycle. The findings corroborate recent work indicating that ureogenesis is also an important ammonia-detoxifying mechanism in cells situated downstream to the periportal region.

Zonation of gluconeogenesis from lactate and pyruvate in the rat liver studied by means of anterograde and retrograde bivascular perfusion

Gluconeogenesis from lactate and pyruvate and associated parameters were investigated in the bivascularly and hemoglobin-free perfused rat liver. The substrates were infused either via the portal vein (anterograde perfusion mode), via the hepatic vein (retrograde mode) or via the hepatic artery (anterograde and retrograde modes). The rates of lactate and pyruvate infusion were 10.3 and 3.5 mumol min-1 g-1, respectively. The metabolic rates measured when the substrates were infused into the hepatic artery were referred to the cellular spaces accessible in each perfusion mode. The following results were obtained when the substrates were infused into the hepatic artery: (1) gluconeogenesis from lactate was equal to 2.08 +/- 0.2 mumol min-1 ml-1 in the retrograde mode and 1.33 +/- 0.08 mumol min-1 ml-1 in the anterograde mode (P = 0.019); (2) gluconeogenesis from pyruvate was equal to 0.66 +/- 0.11 mumol min-1 ml-1 in the retrograde mode and 0.7 +/- 0.11 mumol min-1 ml-1 in the anterograde mode (P = 0.78); (3) oxygen uptake increase with lactate was 1.75 +/- 0.14 mumol min-1 ml-1 in the retrograde mode and 1.05 +/- 0.07 mumol min-1 ml-1 in the anterograde mode (P = 0.002); (4) oxygen uptake increase with pyruvate was equal to 0.59 mumol min-1 ml-1 in the retrograde mode and 0.57 +/- 0.05 mumol min-1 ml-1 in the anterograde mode (P = 0.73); (5) pyruvate production from lactate was 0.28 +/- 0.06 mumol min-1 ml-1 in the retrograde mode and 0.39 +/- 0.05 mumol min-1 ml-1 in the anterograde mode (P = 0.28); (6) lactate production from pyruvate was equal to 0.52 +/- 0.05 mumol min-1 ml-1 in the retrograde mode and 0.99 +/- 0.08 mumol min-1 ml-1 in the anterograde mode (P < 0.001). Since only periportal cells are supplied with substrates when they are infused via the hepatic artery in retrograde perfusion, these results allow the conclusion that gluconeogenesis from lactate predominates in periportal hepatocytes. When pyruvate is the sole substrate, however, gluconeogenesis in periportal and perivenous cells presents no difference.

Glucose and glycogen catabolism in perfused livers of Walker-256 tumor-bearing rats and the response to hormones

The alterations in hepatic glucose and glycogen catabolism were evaluated in rats bearing the Walker-256 tumor. Food intake was monitored concomitantly with measurements of the in vivo hepatic glycogen levels. Glycogenolysis, glycolysis and oxygen uptake were measured in the isolated perfused liver. The hepatic glucose phosphorylating capacity was measured in the high-speed supernatant fraction of liver homogenates. Food intake was 21.4% reduced in tumor-bearing rats; the glycogen levels were decreased by 63.6%. Initial basal rates of glucose release (glycogenolysis) and lactate+pyruvate production from endogenous glycogen (glycolysis) in the perfused liver were not changed by the tumor-bearing state, resulting in a higher relative rate of glycogen breakdown (% of glycogen degradation per unit time). In absolute terms stimulation of glycogen mobilization by glucagon or norepinephrine was smaller in the tumor-bearing state. The percentage of extra glycogen degradation per unit time caused by both hormones, however, was practically the same in the control and in the tumor-bearing state. The hepatic glucose phosphorylating capacity was reduced from 3.92+/-0.39 nmolmin(-1)(mgprotein)(-1) in normal rats to 2.61+/-0.23 nmolmin(-1)(mgprotein)(-1) in livers from tumor-bearing rats. Glycolysis from exogenous glucose (20 mM) in perfused livers was diminished from 0.136+/-0.023 &mgr;molmin(-1)(gliver)(-1) in normal rats to 0.046+/-0.008 &mgr;molmin(-1)(gliver)(-1) in tumor-bearing rats. It can be concluded that livers from rats bearing the Walker-256 tumor are less able to transform glucose and accumulate glycogen while possessing a greater tendency of releasing glucose from the glycogen stores.

The actions of fisetin on glucose metabolism in the rat liver.

Fisetin is a flavonoid dietary ingredient found in the smoke tree (Cotinus coggyria) and in several fruits and vegetables. The effects of fisetin on glucose metabolism in the isolated perfused rat liver and some glucose‐regulating enzymatic activities were investigated. Fisetin inhibited glucose, lactate, and pyruvate release from endogenous glycogen. Maximal inhibitions of glycogenolysis (49%) and glycolysis (59%) were obtained with the concentration of 200 µM. The glycogenolytic effects of glucagon and dinitrophenol were suppressed by fisetin 300 µM. No significant changes in the cellular contents of AMP, ADP, and ATP were found. Fisetin increased the cellular content of glucose 6‐phosphate and inhibited the glucose 6‐phosphatase activity. Gluconeogenesis from lactate and pyruvate or fructose was inhibited by fisetin 300 µM. Pyruvate carboxylation in isolated intact mitochondria was inhibited (IC50 = 163.10 ± 12.28 µM); no such effect was observed in freeze‐thawing disrupted mitochondria. It was concluded that fisetin inhibits glucose release from the livers in both fed and fasted conditions. The inhibition of pyruvate transport into the mitochondria and the reduction of the cytosolic NADH‐NAD+ potential redox could be the causes of the gluconeogenesis inhibition. Fisetin could also prevent hyperglycemia by decreasing glycogen breakdown or blocking the glycogenolytic action of hormones. Copyright

Molecular mechanisms of citrus flavanones on hepatic gluconeogenesis.

It is well known that hyperglycaemia is the initiating cause of tissue damage associated with type 2 diabetes mellitus and that enhanced hepatic gluconeogenesis may account for the increase in blood glucose levels. The purpose of this work was to investigate the possible actions and mechanisms of three related citrus flavanones, namely hesperidin, hesperetin and naringenin, on hepatic gluconeogenesis and related parameters using isolated perfused rat liver. Hesperetin and naringenin (but not hesperidin) inhibited gluconeogenesis from lactate plus pyruvate, alanine and dihydroxyacetone. The inhibitory effects of these flavanones on gluconeogenesis from lactate and pyruvate (hesperetin IC50 75.6 μM; naringenin IC50 85.5 μM) as well as from alanine were considerably more pronounced than those from dihydroxyacetone. The main cause of gluconeogenesis inhibition is the reduction of pyruvate carboxylation by hesperetin (IC50 134.2 μM) and naringenin (IC50 143.5 μM) via inhibition of pyruvate transport into the mitochondria. Secondary causes are likely inhibition of energy metabolism, diversion of glucose 6-phosphate for glucuronidation reactions and oxidation of NADH by flavanone phenoxyl radicals. The influence of the structural differences between hesperetin and naringenin on their metabolic effects was negligible. Analytical evidence indicated that the presence of a rutinoside moiety in hesperidin noticeably decreases its metabolic effects, confirming that hesperetin and naringenin interact with intracellular enzymes and mitochondrial or cellular membranes better than hesperidin. Thus, the inhibition of the gluconeogenic pathway by citrus flavanones, which was similar to that of the drug metformin, may represent an attractive novel treatment strategy for type 2 diabetes.

Prooxidant activity of fisetin: effects on energy metabolism in the rat liver.

Flavonols, which possess the B‐catechol ring, as quercetin, are capable of producing o‐hemi‐ quinones 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 14CO2 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.

Zonation of the action of glucagon on gluconeogenesis studied in the bivascularly perfused rat liver

We have measured the action of glucagon, infused into the hepatic artery, on gluconeogenesis from lactate in the rat liver, bivascularly perfused in both the anterograde and retrograde modes. Concerning glucose production and oxygen uptake per unit cell space, the response of the periportal cells reached via the hepatic artery in retrograde perfusion to glucagon is superior to the response of the cells reached via the same vessel in anterograde perfusion. This phenomenon, however, most probably reflects zonation of gluconeogenesis rather than zonation of the hormonal action. The latter conclusion is based on the observation that the fractional change caused by the hormone is the same for all liver cells.

Heterogenic response of the liver parenchyma to ethanol studied in the bivascularly perfused rat liver.

Zonation of ethanol oxidation and metabolic effects along the hepatic acini were investigated in the bivascularly perfused liver of fed rats. Ethanol was infused into the hepatic artery in antegrade and retrograde perfusion. Inhibition of glycolysis by ethanol, expressed as μmol min−1 (ml accessible cell space)−1, was more pronounced in the retrograde mode; the retrograde/antegrade ratio was equal to 1.63 for an ethanol infusion rate of 37.5 μmol min−1 g−1. Stimulation of oxygen uptake by ethanol was more pronounced in the retrograde mode; the retrograde/antegrade ratio was equal to 1.77. Diminution of the citrate cycle caused by ethanol was more pronounced in the retrograde mode; the retrograde/antegrade ratio was equal to 1.46. Transformation of arterially infused ethanol into acetate was more pronounced in retrograde perfusion; the retrograde/antegrade ratio was equal to 1.63. The increments in glucose release (glycogenolysis) caused by ethanol in the antegrade and retrograde modes were similar. It was assumed that the changes caused by arterially infused ethanol in retrograde and antegrade perfusion closely reflect a significant part of the periportal parenchyma and an average over the whole liver parenchyma, respectively. Under such assumptions it can be concluded that, in the perfused liver from fed rats, four related parameters predominate in the periportal region: ethanol oxidation, glycolysis inhibition, oxygen uptake stimulation and citrate cycle inhibition. One of the main causes for this predominance could be the malate/aspartate shuttle, which operates more rapidly in the periportal area and is essential for NADH oxidation.

Catabolism of amino acids in livers from cafeteria-fed rats

Most studies using a hypercaloric diet to induce obesity have focused on the metabolism of fat and carbohydrates. Less concern has been given to the metabolism of amino acids, despite evidence of modifications in nitrogen metabolism during obesity. The aim of this study was to evaluate amino acid metabolism in livers from cafeteria diet-induced obese rats. Blood parameters were analysed, and histological sections of livers were stained with Sudan III. The enzymatic activities of some enzymes were determined in liver homogenates. Gluconeogenesis, ureagenesis, and oxygen consumption were evaluated in rat livers perfused with glutamine, alanine, or ammonium chloride. Compared to control rats, cafeteria-fed rats demonstrated higher levels of triacylglycerol and glucose in the blood and greater accumulation of fat in livers. Gluconeogenesis and urea production in livers perfused with glutamine and alanine at higher concentrations showed a substantial reduction in cafeteria-fed rats. However, no significant difference was observed among groups perfused with ammonium chloride. The activities of the enzymes alanine aminotransferase, glutaminase, and aspartate aminotransferase in the livers were reduced in cafeteria-fed rats. Taken together, these data are consistent with the hypothesis that livers from cafeteria diet-induced obese rats exhibit a limitation in their maximal capacity to metabolise glutamine and alanine to glucose, ammonia, and urea, not because of an impairment in gluconeogenesis and/or ureagenesis, but rather due to a depression in the activities of enzymes that catalyse the initial steps of amino acid metabolism.