Inhibitory action of isovaleryl-L-carnitine on proteolysis in perfused rat liver

Isovaleryl-l-carnitine inhibits the proteolysis induced by amino acid deprivation in the perfused rat liver to an extent equivalent, or, below 0.4 mM, even greater than that previously found for 1-leucine (Ref. 1). Also the typical concentration-response curve previously found for leucine (Ref. 1) is mimicked by isovaleryl-l-carnitine. The maximum inhibition (approximately 50% of the control) occurred for both l-leucine and isovaleryl-l-carnitine above 0.8 mM. Only at these high concentrations also 1-carnitine and isobutyryl-l-carnitine exhibit a significant, albeit lower, degree of inhibition. The possible mechanism of this proteolysis inhibition is discussed.     

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.     

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.     

The action of alpha-adrenergic agonists on plasma-membrane calcium fluxes in perfused rat liver

The effect of alpha-adrenergic agonists on Ca2+ fluxes was examined in the perfused rat liver by using a combination of Ca2+-electrode and 45Ca2+-uptake techniques. We showed that net Ca2+ fluxes can be described by the activities of separate Ca2+-uptake and Ca2+-efflux components, and that alpha-adrenergic agonists modulate the activity of both components in a time-dependent manner. Under resting conditions, Ca2+-uptake and -efflux activities are balanced, resulting in Ca2+ cycling across the plasma membrane. The alpha-adrenergic agonists vasopressin and angiotensin, but not glucagon, stimulate the rate of both Ca2+ efflux and Ca2+ uptake. During the first 2-3 min of alpha-agonist administration the effect on the efflux component is the greater, the net effect being efflux of Ca2+ from the cell. After 3-4 min of phenylephrine treatment, net Ca2+ movements are essentially complete, however, the rate of Ca2+ cycling is significantly increased. After removal of the alpha-agonist a large stimulation of the rate of Ca2+ uptake leads to the net accumulation of Ca2+ by the cell. The potential role of these Ca2+ flux changes in the expression of alpha-adrenergic-agonist-mediated effects is discussed.     

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.     

Selective actions of growth hormone on rat liver estrogen binding proteins

Levels of hepatic estrogen receptor were 9.0 ± 2.4 fmoles/mg cytosol protein in intact females compared to 3.4 ± 2.2 in hypophysectomized females. Likewise, levels of receptor were 9.8 ± 1.5 fmoles/mg cytosol protein in intact males and 2.7 ± 1.8 in hypophysectomized males. Hypophysectomy abolished the sex differences in a second class of binding sites termed higher capacity lower affinity binding sites by increasing female levels and decreasing male levels. Treatment of hypophysectomized male or female rats with growth hormone (2 units/kg body wt, two times daily) restored normal levels of hepatic estrogen receptor. Administration of growth hormone to hypophysectomized rats did not reverse the effects of hypophysectomy on higher capacity lower affinity binding sites. These studies demonstrate that growth hormone exerts selective actions on different forms of hepatic estrogen binding proteins.     

The action of zymosan on octanoate transport and metabolism in the isolated perfused rat liver

The effects of zymosan on transport, distribution, and metabolism of octanoate in the perfused rat liver were investigated using the multiple‐indicator dilution technique. Livers were perfused with 300 µM octanoate in the absence or in the presence of 100 µg/mL zymosan. Tracer amounts of [1‐¹⁴C]octanoate, [³H] water, and [¹³¹I]albumin were injected into the portal vein, and the effluent perfusate was fractionated. The normalized dilution curves were analyzed by means of a space‐distributed variable transit time model. Zymosan decreased the space into which octanoate undergoes flow‐limited distribution, possibly the first cellular exchanging pool represented by plasma membranes and their adjacencies. However, the rate of transfer of octanoate from the plasma membrane into the rest of the cell was not modified as indicated by the similar values of the influx rates and also the net uptake of octanoate per unit of accessible cellular volume. However, when referred to the wet weight of the liver, the net uptake of octanoate was 37.5% reduced, a value corresponding to the diminution of the cellular accessible space. It can be concluded that an exclusion of a fraction of the liver parenchyma from the microcirculation is the main mechanism by which zymosan reduces the metabolism of exogenous octanoate. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:155–165, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20269     

Degradation of serum amyloid A by isolated perfused rat liver

Degradation of serum amyloid A (SAA) was studied in the isolated perfused rat liver. Radioiodinated SAA was reconstituted with high density lipoproteins (HDL) and administered to rats. Plasma was taken 1 h later, and the HDL were isolated for use as tracer. HDL-bound 125I-SAA was cleared from the plasma of intact animals at a rate similar to SAA in native human HDL. Catabolism of SAA and HDL apoproteins was studied in parallel in the perfused liver. In a 3-h perfusion, 21% of SAA was degraded in contrast to 13% of apoC-III, 7% of apoA-I, and 6% of apoA-II. SAA1 (47% in 3 h) was degraded more rapidly than SAA5 (37%) although their in vivo clearance rates were similar. Degradation of SAA was inhibited when lipoproteins were added to the perfusate. At a protein concentration of 0.15 mg/ml, low density lipoproteins inhibited 47%, HDL 62%, and SAA-rich HDL 75%. Lipid-free normal HDL (0.3 mg/ml perfusate) did not appreciably affect SAA degradation; however, delipidated SAA-rich HDL (0.3 mg of protein/ml; 0.02 mg of SAA/ml) inhibited SAA degradation by 40%. Isolated perfused mouse liver proved more effective than rat liver in degrading SAA (5.3% versus 2.8%/g of liver/h). Degradation appeared to be mediated by cell-associated enzymes since perfusate, which had been recirculated through the liver for 3 h, accounted for less than 15% of the total degradation. Partial (38%) hepatectomy did not significantly reduce apoA-I clearance but reduced that of SAA by 16%, providing additional evidence for hepatic SAA catabolism. We conclude from these studies that SAA is catabolized independently of other HDL proteins, that association with lipoproteins retards SAA clearance, and that SAA catabolism is, in part, a specific process.     

Mechanism of action of sympathetic hepatic nerves on carbohydrate metabolism in perfused rat liver

In the perfused rat liver stimulation of the hepatic nerves around the portal vein and the hepatic artery was previously shown to increase glucose output, to shift lactate uptake to output, to decrease and re-distribute intrahepatic perfusion flow and to cause an overflow of noradrenaline into the hepatic vein. The metabolic effects could be caused directly via nerve hepatocyte contacts or indirectly by the hemodynamic changes and/or by noradrenaline overflow from the afferent vasculature into the sinusoids. Evidence against the indirect modes of nerve action is presented. Reduction of perfusion flow by lowering the perfusion pressure from 2 to 1 ml X min-1 X g-1--as after nerve stimulation--or to 0.35 ml X min-1 X g-1--far beyond the nerve stimulation-dependent effect--did not change glucose output and lowered lactate uptake only slightly. Only re-increase of flow to 2 ml X min-1 X g-1 enhanced glucose and lactate release transiently due to washout of glucose and lactate accumulated in parenchymal areas not perfused during low perfusion flow. In chemically sympathectomized livers nerve stimulation decreased perfusion flow almost normally but without changing the intrahepatic microcirculation; yet it enhanced glucose and lactate output only insignificantly and caused noradrenaline overflow of less than 10% of normal. Conversely, in the presence of nitroprussiate (III) nerve stimulation reduced overall flow only slightly without intrahepatic redistribution but still increased glucose and lactate output strongly and caused normal noradrenaline overflow.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gluconeogenesis in the perfused rat liver

1. A modification of the methods of Miller and of Schimassek for the perfusion of the isolated rat liver, suitable for the study of gluconeogenesis, is described. 2. The main modifications concern the operative technique (reducing the period of anoxia during the operation to 3min.) and the use of aged (non-glycolysing) red cells in the semi-synthetic perfusion medium. 3. The performance of the perfused liver was tested by measuring the rate of gluconeogenesis, of urea synthesis and the stability of adenine nucleotides. Higher rates of gluconeogenesis (1mumole/min./g.) from excess of lactate and of urea synthesis from excess of ammonia (4mumoles/min./g. in the presence of ornithine) were observed than are likely to occur in vivo where rates are limited by the rate of supply of precursor. The concentrations of the three adenine nucleotides in the liver tissue were maintained within 15% over a perfusion period of 135min. 4. Ca(2+), Na(+), K(+), Mg(2+) and phosphate were found to be required at physiological concentrations for optimum gluconeogenesis but bicarbonate and carbon dioxide could be largely replaced by phosphate buffer without affecting the rate of gluconeogenesis. 5. Maximal gluconeogenesis did not decrease maximal urea synthesis in the presence of ornithine and ammonia and vice versa. This indicates that the energy requirements were not limiting the rates of gluconeogenesis or of urea synthesis. 6. Addition of lactate, and especially ammonium salts, increased the uptake of oxygen more than expected on the basis of the ATP requirements of the gluconeogenesis and urea synthesis.     

Effects of ranolazine on fatty acid transformation in the isolated perfused rat liver

It has been proposed that in the heart, ranolazine shifts the energy source from fatty acids to glucose oxidation by inhibiting fatty acid oxidation. Up to now no mechanism for this inhibition has been proposed. The purpose of this study was to investigate if ranolazine also affects hepatic fatty acid oxidation, with especial emphasis on cell membrane permeation based on the observations that the compound interacts with biological membranes. The isolated perfused rat liver was used, and [1-¹⁴C]oleate transport was measured by means of the multiple-indicator dilution technique. Ranolazine inhibited net uptake of [1-¹⁴C]-oleate by impairing transport of this fatty acid. The compound also diminished the extra oxygen consumption and ketogenesis driven by oleate and the mitochondrial NADH/NAD⁺ ratio, but stimulated ¹⁴CO₂ production. These effects were already significant at 20 μM ranolazine. Ranolazine also inhibited both oxygen consumption and ketogenesis driven by endogenous fatty acids in substrate-free perfused livers. In isolated mitochondria ranolazine inhibited acyl-CoA oxidation and β-hydroxybutyrate or α-ketoglutarate oxidation coupled to ADP phosphorylation. It was concluded that ranolazine inhibits fatty acid uptake and oxidation in the liver by at least two mechanisms: inhibition of cell membrane permeation and by an inhibition of the mitochondrial electron transfer via pyridine nucleotides.     

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.     

Effects of haematocrit value and glucagon on the metabolism of perfused rat liver

Liver from adult male rats were perfused in situ for 30 min with either undiluted, defibrinated rat blood (haematocrit value 38%) or the same blood diluted with buffer to give a haematocrit of 20%. Perfusion with diluted blood lowered the PO2 of the effluent perfusate but this was insufficient to prevent the fall in O2 consumption due to the reduction in haematocrit. Glucagon (5 X 10(-9) M) increased hepatic O2 consumption with whole blood but not with diluted blood. perfusate K+ was increased by perfusion with diluted blood and glucagon. Bile flow was depressed and biliary K+ increased by glucagon but only in experiments with whole blood. Perfusate glucose was raised by lowering of hepatic O2 consumption but the hormonal stimulation of glucose output was the same at both haematocrits. Net ketogenesis was increased with perfusion with diluted blood and by glucagon. In the absence of glucagon there was a net secretion of triacylglycerols which was depressed by lowering of the haematocrit. Glucagon inhibited triacylglycerol secretion and the effect was greater with whole blood so that there was net uptake. While effects of glucagon were obtained during perfusion at a lower haematocrit, it would appear that whole blood was the medium that allowed their fullest expression.     

Effects of cycloheximide on protein degradation and gluconeogenesis in the perfused rat liver.

Cycloheximide at concentrations above 18 muM produced a 93% inhibition of total protein synthesis measured by valine incorporation in the perfused rat liver. Rates of protein degradation were estimated by perfusing livers prelabeled in vivo with L-[1-14C]valine with medium containing 15 mM L-valine. Thus labeled valine released from liver protein during perfusion was greatly diluted and reincorporation of label was minimized. Cycloheximide at 18 muM inhibited protein degradation by over 60%, after a delay of 15-20 min. Associated with these effects were dose-dependent increases in the rates of glucose and urea production. Glucose production increased 3 fold, from 0.54 +/- 0.07 in control to 1.85 +/- 0.24 mumol/min/100 g rat in cycloheximide-treated livers. Urea production increased from 0.24 +/- 0.02 to 0.62 +/- 0.06 mumol/min/100 g rat. No changes in liver glycogen or cyclic AMP content were seen. The data suggest that inhibition of protein synthesis provides an increased availability of intra-cellular amino acids and that many of these are rapidly degraded, yielding urea and glucose. This is supported by the fact that intracellular alanine levels were significantly increased following cycloheximide treatment. It is possible that the inhibition of protein degradation by cycloheximide is due to altered intra-cellular pools of amino acids or their metabolites.