Comparative acute effects of leptin and insulin on gluconeogenesis and ketogenesis in perfused rat liver

The acute effects of physiological levels of leptin (10 ng ml(-1)) and insulin (20 microU ml(-1)) on hepatic gluconeogenesis and ketogenesis were compared. Leptin or insulin alone decreased (p<0.05) the activation of hepatic glucose, L-lactate and urea production from L-alanine. However, the hepatic glucose production was not modified if leptin was combined with insulin. These results indicated that both, i.e. leptin and insulin, could promote a non-additive reduction in the rate of catabolism of L-alanine. However, in contrast with insulin (p<0.05), leptin did not inhibit the activation of hepatic glucose production from pyruvate or glycerol. On the other hand, activation of hepatic production of acetoacetate and beta-hydroxybutyrate from octanoate was not affected by leptin or insulin. Thus, our data demonstrate that the acute effect of leptin on hepatic metabolism was partially similar to insulin (activation of glucose production from L-alanine and activation of acetoacetate or beta-hydroxybutyrate production from octanoate) and partially different from insulin (activation of glucose production from pyruvate or glycerol).     

Zonation of the metabolic action of vasopressin in the bivascularly perfused rat liver

Predominance of the vasopressin binding capacity in the hepatic perivenous area leads to the hypothesis that the metabolic effects of the hormone should also be more pronounced in this area. Until now this question has been approached solely by experiments with isolated hepatocytes where an apparent absence of metabolic zonation was found. We have reexamined this question using the bivascularly perfused liver. In this system periportal cells can be reached in a selective manner with substrates and effectors via the hepatic artery when retrograde perfusion (hepatic vein --> portal vein) is done. The action of vasopressin (1-10 nM) on glycogenolysis, initial calcium efflux, glycolysis and oxygen uptake were measured. The results revealed that the action of vasopressin in the liver is heterogeneously distributed. Glycogenolysis stimulation and initial calcium efflux were predominant in the perivenous area, irrespective of the vasopressin concentration. Oxygen uptake was stimulated in the perivenous area; in the periportal area it ranged from inhibition at low vasopressin concentrations to stimulation at high ones. Lactate production was generally greater in the perivenous zone, whereas the opposite occurred with pyruvate production. Analysis of these and other results suggests that at least three factors are contributing to the heterogenic response of the liver parenchyma to vasopressin: a) receptor density, which tends to favour the perivenous zone; b) cell-to-cell interactions, which tend to favour situations where the perivenous zone is amply supplied with vasopressin; and c) the different response capacities of perivenous and periportal cells.     

Hepatic heterogeneity in the response to ATP studied in the bivascularly perfused rat liver

The zonation of the purinergic action of ATP 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 ATP was infused according to four different experimental protocols: (A) anterograde perfusion and ATP infusion via the portal vein; (B) anterograde perfusion and ATP via the hepatic artery; (C) retrograde perfusion and ATP via the hepatic vein; (D) retrograde perfusion and ATP via the hepatic artery. The following metabolic parameters were measured: glucose release, lactate production and oxygen consumption. The hemodynamic effects were evaluated by measuring the sinusoidal mean transit times by means of the indicator-dilution technique. ATP was infused during 20 min at four different rates (between 0.06-0.77 µmol min_-1 g liver_-1; 20-200 µM) in each of the four experimental protocols.The results that were obtained allow several conclusions with respect to the localization of the effects of ATP along the hepatic acini: (1) In retrograde perfusion the sinusoidal mean transit times were approximately twice those observed in anterograde perfusion. ATP increased the sinusoidal mean transit times only in retrograde perfusion (protocols C and D). The effect was more pronounced with protocol D. These results allow the conclusion that the responsive vasoconstrictive elements are localized in a pre-sinusoidal region; (2) All hepatic cells, periportal as well as perivenous, were able to metabolize ATP, so that concentration gradients were generated with all experimental protocols. Extraction of ATP was more pronounced in retrograde perfusion, an observation that can be attributed, partly at least, to the longer sinusoidal transit times. In anterograde perfusion, the extraction of ATP was time-dependent, a phenomenon that cannot be satisfactorily explained with the available data; (3) ATP produced a transient initial inhibition of oxygen uptake when protocols A and B were employed. These protocols are the only ones in which the cells situated shortly after the intrasinusoidal confluence of the portal vein and the hepatic artery were effectively supplied with ATP. The decrease in oxygen consumption was more pronounced at low ATP infusions when protocol B was employed. These observations allow the conclusion that the former phenomenon is localized mainly in cells situated shortly after the intrasinusoidal confluence of the portal vein and hepatic artery. Oxygen consumption in all other cells, especially the proximal periportal ones, is increased by ATP; (4) In agreement with previous data found in the literature, glycogenolysis stimulation by ATP was more pronounced in the periportal region. The cells that respond more intensively are not the proximal periportal ones, but those situated in the region of the intrasinusoidal confluence of the portal vein and the hepatic artery.     

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 micromol 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 micromol 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.     

Regional heterogeneities in the production of uric acid from adenosine in the bivascularly perfused rat liver

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 M) 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 M 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.     

Bivascular liver perfusion in the anterograde and retrograde modes: Zonation of the response to inhibitors of oxidative phosphorylation

The action of cyanide (500 μM ), 2,4-dinitrophenol (50 μM ) and atractyloside (100 μM ) on glycogen catabolism and oxygen uptake was investigated in the bivascularly perfused liver of fed rats. Cyanide, 2,4-dinitrophenol and atractyloside were infused at identical rates into the hepatic artery in either the anterograde or retrograde perfusion. The accessible aqueous cell spaces were determined by means of the multiple-indicator dilution technique. Glucose release, oxygen uptake and glycolysis were measured as metabolic parameters. Oxygen uptake changes per unit cell space caused by atractyloside (inhibition) and 2,4-dinitrophenol (stimulation) were equal in the retrograde perfusion (periportal cells) and the anterograde perfusion (space enriched in perivenous cells); the decreases caused by cyanide were higher in the retrograde perfusion. Glucose release from periportal cells was not increased upon inhibition of oxidative phosphorylation, a phenomenon which was independent of the mechanism of action of the inhibitor. There were nearly identical changes in glycolysis in the periportal and perivenous cells. It was concluded that: (1) oxygen concentration in the perfused rat liver, if maintained above 100 μM , had little influence on the zonation of the respiratory activity; (2) in spite of the lower activities of the key enzymes of glycolysis in the periportal hepatocytes, as assayed under standard conditions, these cells were as effective as the perivenous ones in generating ATP in the cytosol when oxidative phosphorylation was impaired; (3) the key enzymes of glycogenolysis and glycolysis in periportal and perivenous cells responded differently to changes in the energy charge.     

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].     

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 metabolism of fructose in the bivascularly perfused rat liver.

The metabolism of fructose was investigated in the bivascularly and hemoglobin-free perfused rat liver. Anterograde and retrograde perfusions were performed. In anterograde perfusion, fructose was infused at identical rates (19 mumols min-1 g-1) via the portal vein (all liver cells) or the hepatic artery (predominantly perivenous cells); in retrograde perfusion fructose was infused via the hepatic vein (all liver cells) or the hepatic artery (only periportal cells). The cellular water spaces accessible via the hepatic artery were measured by means of the multiple-indicator dilution technique. The following results were obtained. (i) Fructose was metabolized to glucose, lactate and pyruvate even when this substrate was infused via the hepatic artery in retrograde perfusion; oxygen consumption was also increased. (ii) When referred to the water spaces accessible to fructose via the hepatic artery in each perfusion mode, the rate of glycolysis was 0.99 +/- 0.14 mumols min-1 ml-1 in the retrograde mode; and, 2.05 +/- 0.19 mumols min-1 ml-1 in the anterograde mode (P = 0.002). (iii) The extra oxygen uptake due to fructose infusion via the hepatic artery was 1.09 +/- 0.16 mumols min-1 ml-1 in the retrograde mode; and, 0.51 +/- 0.08 mumols min-1 ml-1 in the anterograde mode (P = 0.005). (iv) Glucose production from fructose via the hepatic artery was 2.18 +/- 0.18 mumols min-1 ml-1 in the retrograde mode; and, 1.83 +/- 0.16 mumols min-1 ml-1 in the anterograde mode (P = 0.18). (v) Glucose production and extra oxygen uptake due to fructose infusion did not correlate by a single factor in all perfusion modes. It was concluded that: (a) rates of glycolysis are lower in the periportal area, confirming previous views; (b) extra oxygen uptake due to fructose infusion is higher in the periportal area; (c) a predominance of glucose production in the periportal area could not be demonstrated; and (d) extra oxygen uptake due to fructose infusion is not a precise indicator for glucose synthesis.     

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.     

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.     

Influence of tamoxifen on gluconeogenesis and glycolysis in the perfused rat liver

The actions of tamoxifen, a selective estrogen receptor modulator used in chemotherapy and chemo-prevention of breast cancer, on glycolysis and gluconeogenesis were investigated in the isolated perfused rat liver. Tamoxifen inhibited gluconeogenesis from both lactate and fructose at very low concentrations (e.g., 5 μM). The opposite, i.e., stimulation, was found for glycolysis from both endogenous glycogen and fructose. Oxygen uptake was unaffected, inhibited or stimulated, depending on the conditions. Stimulation occurred in both microsomes and mitochondria. Tamoxifen did not affect the most important key-enzymes of gluconeogenesis, namely, phosphoenolpyruvate carboxykinase, pyruvate carboxylase, fructose 1,6-bisphosphatase and glucose 6-phosphatase. Confirming previous observations, however, tamoxifen inhibited very strongly NADH- and succinate-oxidase of freeze–thawing disrupted mitochondria. Tamoxifen promoted the release of both lactate dehydrogenase (mainly cytosolic) and fumarase (mainly mitochondrial) into the perfusate. Tamoxifen (200 μM) clearly diminished the ATP content and increased the ADP content of livers in the presence of lactate with a diminution of the ATP/ADP ratio from 1.67 to 0.79. The main causes for gluconeogenesis inhibition are probably: (a) inhibition of energy metabolism; (b) deviation of intermediates (malate and glucose 6-phosphate) for the production of NADPH required in hydroxylation and demethylation reactions; (c) deviation of glucosyl units toward glucuronidation reactions; (d) secondary inhibitory action of nitric oxide, whose production is stimulated by tamoxifen; (e) impairment of the cellular structure, especially the membrane structure. Stimulation of glycolysis is probably a compensatory phenomenon for the diminished mitochondrial ATP production. The multiple actions of tamoxifen at relatively low concentrations can represent a continuous burden to the overall hepatic functions during long treatment periods.