Zonation of the action of ethanol on gluconeogenesis and ketogenesis studied in the bivascularly perfused rat liver

Zonation of the actions of ethanol on gluconeogenesis and ketogenesis from lactate were investigated in the bivascularly perfused rat liver. Livers from fasted rats were perfused bivascularly in the antegrade and retrograde modes. Ethanol and lactate were infused into the hepatic artery (antegrade and retrograde) and portal vein. A previously described quantitative analysis that takes into account the microcirculatory characteristics of the rat liver was extended to the analysis of zone-specific effects of inhibitors. Confirming previous reports, gluconeogenesis and the corresponding oxygen uptake increment due to saturable lactate infusions were more pronounced in the periportal region. Arterially infused ethanol inhibited gluconeogenesis more strongly in the periportal region (inhibition constant = 3.99 ± 0.22 mM) when compared to downstream localized regions (inhibition constant = 8.64 ± 2.73 mM). The decrease in oxygen uptake caused by ethanol was also more pronounced in the periportal zone. Lactate decreased ketogenesis dependent on endogenous substrates in both regions, periportal and perivenous, but more strongly in the former. Ethanol further inhibited ketogenesis, but only in the periportal zone. Stimulation was found for the perivenous zone. The predominance of most ethanol effects in the periportal region of the liver is probably related to the fact that its transformation is also clearly predominant in this region, as demonstrated in a previous study. The differential effect on ketogenesis, on the other hand, suggest that the net effects of ethanol are the consequence of a summation of several partial effects with different intensities along the hepatic acini.     

Gluconeogenesis and ketogenesis in perfused liver of rats submitted to short-term insulin-induced hypoglycaemia

Gluconeogenesis and ketogenesis of in situ rat perfused liver submitted to short-term insulin-induced hypoglycaemia (IIH) were investigated. For this purpose, 24-h fasted rats that received intraperitoneal (ip) regular insulin (1.0 U kg(-1)) or saline were compared. The studies were performed 30 min after insulin (IIH group) or saline (COG group) injection. For gluconeogenesis studies, livers from the IIH and COG groups were perfused with increasing concentrations (from basal blood concentrations until saturating concentration) of glycerol, L-lactate (Lac) or pyruvate (Pyr). Livers of the IIH group showed maintained efficiency to produce glucose from glycerol and higher efficiency to produce glucose from Lac and Pyr. In agreement with these results the oral administration of glycerol (100 mg kg(-1)), Lac (100 mg kg(-1)), Pyr (100 mg kg(-1)) or glycerol (100 mg kg(-1)) + Lac (100 mg kg(-1)) + Pyr (100 mg kg(-1)) promoted glycaemia recovery. It can be inferred that the increased portal availability of Lac, Pyr and glycerol could help glycaemia recovery by a mechanism mediated, partly at least, by a maintained (glycerol) or increased (Lac and Pyr) hepatic efficiency to produce glucose. Moreover, in spite of the fact that insulin inhibits ketogenesis, the capacity of the liver to produce ketone bodies from octanoate during IIH was maintained.     

Diltiazem inhibits fatty acid oxidation in the isolated perfused rat liver

The effects of diltiazem on fatty acid metabolism were measured in the isolated perfused rat liver and in isolated mitochondria. In the perfused rat liver diltiazem inhibited oxygen uptake and ketogenesis from endogenous substrates. Ketogenesis from exogenously supplied palmitate was also inhibited. The β-hydroxybutyrate/acetoacetate ratio in the presence of palmitate alone was equal to 3·2. When the fatty acid and diltiazem were present simultaneously this ratio was decreased to 0·93, suggesting that, in spite of the inhibition of oxygen uptake, the respiratory chain was not rate limiting for the oxidation of the reducing equivalents coming from β-oxidation. In experiments with isolated mitochondria, incubated in the presence of all intermediates of the Krebs cycle, pyruvate or glutamate, no significant inhibition of oxygen uptake by diltiazem was detected. Inhibition of oxygen uptake in isolated mitochondria was found only when palmitoyl CoA was the source of the reducing equivalents. It was concluded that a direct effect on β-oxidation may be a major cause for the inhibition of oxygen uptake caused by diltiazem in the perfused liver. © 1997 John Wiley & Sons, Ltd.     

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.     

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 © 2010 John Wiley & Sons, Ltd.     

Ketogenesis evaluation in perfused liver of diabetic rats submitted to short‐term insulin‐induced hypoglycemia

Ketogenesis, inferred by the production of acetoacetate plus ß‐hydroxybutyrate, in isolated perfused livers from 24‐h fasted diabetic rats submitted to short‐term insulin‐induced hypoglycemia (IIH) was investigated. For this purpose, alloxan‐diabetic rats that received intraperitoneal regular insulin (IIH group) or saline (COG group) injection were compared. An additional group of diabetic rats which received oral glucose (gavage) (100 mg kg^?1) 15 min after insulin administration (IIH + glucose group) was included. The studies were performed 30 min after insulin (1.0 U kg^?1) or saline injection. The ketogenesis before octanoate infusion was diminished (p < 0.05) in livers from rats which received insulin (COG vs. IIH group) or insulin plus glucose (COG vs. IIH + glucose group). However, the liver ketogenic capacity during the infusion of octanoate (0.3 mM) was maintained (COG vs. IIH group and COG vs. IIH + glucose group). In addition, the blood concentration of ketone bodies was not influenced by the administration of insulin or insulin plus glucose. Taken together, the results showed that inspite the fact that insulin and glucose inhibits ketogenesis, livers from diabetic rats submitted to short‐term IIH which received insulin or insulin plus glucose showed maintained capacity to produce acetoacetate and ß‐hydroxybutyrate from octanoate. Copyright © 2009 John Wiley & Sons, Ltd.     

Metabolic effects of acetate in perfused rat liver studies on ketogenesis,glucose output,lactate uptake and lipogenesis

1. Livers from fed male rats were perfused in situ in a non-recirculating system with whole rat blood containing acetate at six concentrations, from 0.04 to 1.5 μmol/ml, to cover the physiological range encountered in the hapatic portal venous blood in vivo. 2. Below a concentration of 0.25 μmol/ml there was net production of acetate by the liver, while above it there was ner uptake with a fractional extraction of 40%. 3.No relationship was observed between blood [acetate] and hepatic ketogenesis, the ration [3-hydroxybutyrate]/[acetoacetate] or glucose output, either at low fatty acid concentration s or during oleate infusion. 4. Following the increase in serum fatty acid concentration, induced by oleate infusion, there were suquential incresase in ketogenesis and the ratio of [3-hydroxybutyrate]/[acetoacetate] while glucose output rose and lactate uptake fell significantly after in redox state. 5. There was a highly significant negative correlation between blood [acetate] and hepatic lactate uptake during oleate infusion. At the highest acetate concentration of 1.5 μmol/ml there was a small net hepatic lactate output. After oleate infusion ceased, lactate uptake increased, but the negative correlation between blood [acetate] and hepatic lactate uptake persisted. 6. Livers were also perfused with iether [1-¹⁴C]acetate or [U-¹⁴C]lactate at a concentration of acetate of either 0.3 or 1.3 μmol/ml of blood. With [1-¹⁴C]acetate, most of the radioactivity was recovered as fatty acids at the lower concentration of blood acetate. At the higher blood [acetate] a considerably smaller proportion of the radioactivity was recovered in lipids. With [U-¹⁴C]lactate the reverse pattern obtained i.e., recovery was greater at the high concentration of acetate and fell at the low concentration. Fatty acid biosynthesis, measured with ³H₂O, was stimulated from 2.4 to 6.6 μmol of fatty acid/g of liver per h by high blood [acetate] although the contribution of (acetate+lactate) to synthesis remained constant at 33–38% of the total. 7. These results emphasize the important role of the liver in regulating blood acetate concentrations and indicate that it can be major hepatic substrate. Acetate taken up by the liver appeared to compete directly with lactate, for lipogenesis and metabolism and acetate uptake was inhibited by raised bloodd [lactate].     

Characterization of insulin uptake into subcellular fractions of perfused rat liver using two different iodinated tracers

The binding and uptake of insulin in perfused rat liver has been investigated with specifically labelled 125I-A14-tyrosyl insulin as a tracer and compared with a commercially available iodo-insulin preparation. The commercial preparation did not show saturation uptake kinetics and the clearance from the perfusate remained low and constant throughout a wide concentration range. A14 labelled insulin showed saturation kinetics and high clearance at low carrier concentration, falling rapidly with increasing carrier concentration and reaching a steady state value of 1 ml/min. These results emphasize the importance of using specifically labelled insulin in physiological and biochemical studies of hepatic insulin metabolism. Perfusion with A14 tyrosine-labelled insulin at 4 degrees C showed apparent saturation with binding to the plasma membrane fraction. Perfusion at 37 degrees C also showed apparent saturation with uptake predominantly to the ligandosome fraction. These results implicate the plasma membrane-ligandosome pathway in the hepatic uptake of insulin at both physiological and pharmacological concentrations of the hormone.     

Effects of monensin on vesicular transport pathways in the perfused rat liver

In the rat hepatocyte, the internalization and degradation of asialoglycoproteins and the secretion of plasma and biliary proteins require specific intracellular sorting of vesicles. To aid in the biochemical characterization of these different vesicular pathways, we examined the effects of the ionophore monensin on the uptake and degradation of 125I-asialoorosomucoid (ASOR) and on the secretion of plasma and biliary proteins by the in situ perfused rat liver. In control livers, 77% of injected 125I-ASOR was extracted on first pass; 93% of the extracted radioactivity was released back into the circulation (totally degraded and some intact ASOR was found); and approximately 2% was recovered in the bile, some of which was intact. Monensin treatment decreased first pass uptake of 125I-ASOR to 57% and abruptly blocked the release of radioactivity into the perfusate and the bile. When hepatic proteins were biosynthetically labeled with 3H-leucine, monensin treatment dramatically reduced and delayed the secretion of newly synthesized proteins into both the perfusate and the bile. In contrast with control livers, in which secretion of protein into the perfusate preceded secretion of protein into the bile, TCA-precipitable 3H-protein appeared in bile about 20 min before TCA-precipitable 3H-protein appeared in the perfusate in monensin-treated livers. Thus, monensin treatment in the perfused liver blocked the degradation of asialoglycoproteins and inhibited the secretion of plasma proteins but had less effect on biliary protein secretion. These data document physiologic effects of monensin in an intact organ and suggest that biochemical distinctions between different vesicular pathways exist in the rat hepatocyte.     

Effects of norepinephrine on the metabolism of fatty acids with different chain lengths in the perfused rat liver

The effects of norepinephrine on ketogenesis in isolated hepatocytes have been reported as ranging from stimulation to inhibition. The present work was planned with the aim of clarifying these discrepancies. The experimental system was the once-through perfused liver from fasted and fed rats. Fatty acids with chain lengths varying from 8-18 were infused. The effects of norepinephrine depended on the metabolic state of the rat and on the nature of the fatty acid. Norepinephrine clearly inhibited ketogenesis from long-chain fatty acids (stearate > palmitate > oleate), but had little effect on ketogenesis from medium-chain fatty acids (octanoate and laureate). With palmitate the decrease in oxygen uptake was restricted to the substrate stimulated portion; with stearate, the decrease exceeded the substrate stimulated portion; with oleate, oxygen uptake was transiently inhibited. Withdrawal of Ca²⁺ attenuated the inhibitory effects. ¹⁴CO₂ production from [1-¹⁴C]oleate was inhibited. Net uptake of the fatty acids was not affected by norepinephrine. In livers from fed rats, oxygen uptake and ketogenesis from stearate were only transiently inhibited. The conclusions are: (a) in the fasted state norepinephrine reduces ketogenesis and respiration by means of a Ca²⁺-dependent mechanism; (b) the degree of inhibition varies with the chain length and the degree of saturation of the fatty acids; (c) norepinephrine favours esterification of the activated long-chain fatty acids in detriment to oxidation; (d) in the fed state the stimulatory action of norepinephrine on glycogen catabolism induces conditions which are able to reverse inhibition of ketogenesis and oxygen uptake.     

Metabolic effects of acetaminophen. Studies in the isolated perfused rat liver

The effects of acetaminophen on the metabolism of the isolated perfused rat liver were investigated. The following results were obtained: (1) Acetaminophen increased glucose release and glycolysis from endogenous glycogen (glycogenolysis). (2) Oxygen uptake, gluconeogenesis from either pyruvate or fructose and glycogen synthesis were inhibited. (3) In isolated rat liver mitochondria acetaminophen decreased state III and state IV respiration; it also decreased the ADP/O ratio and the respiratory control ratio. (4) The action of acetaminophen on glycogenolysis was not affected by N-acetylcysteine; this compound, however, increased glycogen synthesis. (5) The effects of acetaminophen are reversible. It was concluded that glycogen depletion by acetaminophen can be produced by two mechanisms. The first, as previously demonstrated by several workers, depends on irreversible binding of a reactive metabolite. The second, however, is reversible and depends primarily on an inhibition of mitochondrial energy metabolism.