Kinetics of glycerol uptake by the perfused rat liver. Membrane transport, phosphorylation and effect on NAD redox level.

The kinetics of glycerol uptake by the perfused rat liver were determined according to a model which includes membrane transport, intracellular phosphorylation and competitive inhibition of glycerol phosphorylation by L-glycerol 3-phosphate. The membrane transport obeys first-order kinetics at concentrations below 10 mM in the affluent medium. The K-m of the glycerol phosphorylation was 10 muM and the K-i of the L-glycerol 3-phosphate inhibition was 50 muM. The maximum activity (V) was 3.70 mumoles/min per g liver wet wt. These results are similar to in vitro kinetics of the glycerol kinase, except that K-i was found to be somewhat lower in the intact organ. At low glycerol concentrations, a steep concentration gradient exists across the liver cell membrane. The increase in the lactate to pyruvate concentration ratio during glycerol metabolism is related to the actual concentration of L-glycerol 3-phosphate, not to the rate of glycerol uptake.     

The effect of blood on the uptake of liposomal lipid by perfused rat liver

Liposomes have been prepared from dipalmitoylphosphatidylcholine (DPPC) and its mixtures with phosphatidylinositol (PI) and stearylamine. The absorption of the liposomes by perfused rat liver has been studied as a function of blood level (0-7% haematocrit). It has been found that the rate constant for uptake of liposomes (perfusion constant, kp) is markedly reduced by addition of blood to the perfusate particularly in the haematocrit range 0-3%. The perfusion constant is dependent on the liposome composition and decreases with incorporation of PI and increases with incorporation of stearylamine into DPPC liposomes, but is independent of the initial size of the liposomes in the range of weight-average diameter from 40-400 nm. The possible effects of blood components on the liposomes and their subsequent absorption by the liver are discussed.     

Determination of the kinetic constants of fructose transport and phosphorylation in the perfused rat liver

The kinetics of fructose uptake was determined in perfused rat liver during steady-state fructose elimination. On the basis of the corresponding values of fructose concentration in the affluent and in the effluent medium, and the fructose and ATP concentration in biopsies, the kinetics of membrane transport and intracellular phosphorylation in the intact organ was calculated according to a model system. Carrier-mediated fructose transport has a high $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ (67 mM) and $\text{V}$ (30 μmoles · min^?1 ·g^?1). The calculated kinetic constants of the intracellular phosphorylation were compared with values obtained with an acid-treated rat liver high speed supernatant (values given in parentheses). $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ with fructose 1.0 mM (0.7 mM), $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ with ATP 0.54 mM (0.37 mM), $\text{V}$ 10.3 μmoles · min^?1 · g^?1 (10.1 μmoles · min^?1 · g^?1, calculated on the basis of the highest measured rate of fructose uptake correcting the ATP concentration to saturating values). The kinetics of fructose uptake reveals that at Physiological fructose concentrations the membrane transport limits the rate of fructose uptake, thus protecting the liver from severe depletion of adenine nucleotides.     

Effect of insulin and glucagon on the uptake of carnitine by perfused rat liver

The possible direct effects of insulin and glucagon on carnitine uptake by perfused rat liver were studied with L-[3H]carnitine of an initial concentration of 50 microM in the perfusate. Insulin (10 nM) did not significantly affect the uptake by livers from fed animals. However, insulin could reverse the stimulated transport by livers from 24-h fasted animals, reducing the uptake rate from 852 +/- 54.1 to 480 +/- 39.9 (mean +/- S.E.), P less than 0.01 (rates are expressed as nmol per h per 100 g body wt). Glucagon (50 nM) stimulated the uptake rate when livers were either from fed (551 +/- 40.1 vs. 915 +/- 55.3, P less than 0.01) or from fasted animals (852 +/- 54.1 vs. 1142 +/- 88.1, P less than 0.02). Based on these and earlier observations, we propose that the carnitine concentration in rat liver is controlled by insulin and glucagon via cellular transport processes.     

The effects of dietary conditions and glycerol concentration on glycerol uptake by rat liver and kidney-cortex slices

1. Glycerol utilization by rat liver and kidney-cortex slices was studied in an attempt to define factors that might be important in the regulation of glycerol utilization by these tissues in vivo; the formation of glucose from glycerol by kidney-cortex slices was also studied. 2. The rate of glycerol uptake by liver slices was not changed (in comparison with the normal fed control) by starvation (48hr.), feeding with a low-carbohydrate diet (4-8 days) or feeding with a diet containing 25% glycerol (up to 18 days). Similarly, starvation or a low-carbohydrate diet had no effect on uptake by kidney-cortex slices; however, feeding with the glycerol diet increased glycerol uptake by kidney-cortex slices. 3. The rates of glycerol uptake by slices from both tissues were increased on raising the glycerol concentration from 0.2mm to 2.5 or 5.0mm. 4. Starvation increased the conversion of glycerol into glucose by kidney-cortex slices, but there was no effect of the low-carbohydrate diet; the rate of glucose formation was increased by feeding with the 25%-glycerol diet and was proportional to the increase in glycerol uptake. The rate of glucose production by these slices was increased by raising the glycerol concentration in the incubation medium from 0.2mm to 1.0mm, but, except for the slices from animals receiving the 25%-glycerol diet, there was no effect above 1.0mm-glycerol. 5. The significance of plasma glycerol concentration in regulating glycerol uptake by these tissues is discussed.     

Membrane transport in relation to net uptake of glucose in the perfused rat hindlimb. Stimulatory effect of insulin, hypoxia and contractile activity.

The assay for the fucose-binding protein described by Lehrman & Hill [(1983) Methods Enzymol. 98, 309-319] was adapted for the measurement of the asialoglycoprotein receptor in rat liver. The amount of ligand bound to the plasma-membrane-associated or affinity-purified receptor was acutely sensitive to the concentrations of Triton X-100 and NaCl in the assay: 0.02% (v/v) Triton X-100 increased ligand binding to the two preparations by 100% and 40% respectively. Higher concentrations of detergent progressively decreased binding, and in 0.32% Triton X-100 it was about 30% of the value obtained in detergent-free buffer. The addition of increasing concentrations of NaCl to the assay progressively inhibited ligand binding to the membrane-associated receptor, whereas there was a 60% increase in binding to the pure receptor in the presence of 0.1-0.2 M-NaCl. These effects could not be identified in the original assay procedure described by Hudgin, Pricer, Ashwell, Stockert & Morell [(1974) J. Biol. Chem. 249, 5536-5543]. Using optimal assay conditions the binding of 125I-beta-D-galactosyl-bovine serum albumin to both the membrane-associated and purified receptor was inhibited by 50% by 1 nM-beta-D-galactosyl-bovine serum albumin and -asialoorosomucoid and by approx. 100 microM-beta-L-fucosyl-bovine serum albumin, whereas beta-D-galactose, lactose and beta-L-fucose had no effect on ligand binding up to concentrations of 1 mM, 500 microM and 5 mM respectively. KD values of 0.94 and 1.25 nM and Bmax. values of 40 and 1660 pmol of D-galactosyl-bovine serum albumin bound/mg of receptor were obtained for the membrane-bound and purified receptor respectively. Hill-plot analysis of the same data gave slopes of 0.96 and 1.01. Scatchard analysis of saturation-binding studies with other subcellular fractions indicated that the receptor was distributed in the proportions 72:23:2.5:2.5 between total microsomal fractions, plasma membrane, Golgi and canalicular membrane respectively. The receptor was about 1% of the total protein in each compartment and was estimated to be about 0.3% of the total liver protein.     

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.     

Hepatocyte heterogeneity in glutamate uptake by isolated perfused rat liver

Glutamate is simultaneously taken up and released by perfused rat liver, as shown by 14CO2 production from [1-14C]glutamate in the presence of a net glutamate release by the liver, turning to a net glutamate uptake at portal glutamate concentrations above 0.3 mM. 14CO2 production from portal [1-14C]glutamate is decreased by about 60% in the presence of ammonium ions. This effect is not observed during inhibition of glutamine synthetase by methionine sulfoximine. 14CO2 production from [1-14C]glutamate is not influenced by glutamine. Also, when glutamate accumulates intracellularly during the metabolism of glutamine (added at high concentrations, 5 mM), 14CO2 production from [1-14C]glutamate is not affected. If labeled glutamate is generated intracellularly from added [U-14C]proline, stimulation of glutamine synthesis by ammonium ions did not affect 14CO2 production from [U-14C]proline. After induction of a perivenous liver cell necrosis by CCL4, i.e. conditions associated with an almost complete loss of perivenous glutamine synthesis but no effect on periportal urea synthesis, 14CO2 production from [1-14C]glutamate is decreased by about 70%. The results are explained by hepatocyte heterogeneity in glutamate metabolism and indicate a predominant uptake of glutamate (that reaches the liver by the vena portae) by the small perivenous population of glutamine-synthesizing hepatocytes, whereas glutamate production from glutamine or proline is predominantly periportal. In view of the size of the glutamine synthetase-containing hepatocyte pool [Gebhardt, R. and Mecke, D. (1983) EMBO J. 2, 567-570], glutamate transport capacity of these hepatocytes would be about 20-fold higher as compared to other hepatocytes.     

Hepatic lipase-mediated hydrolysis versus liver uptake of HDL phospholipids and triacylglycerols by the perfused rat liver

Hepatic triacylglycerol-lipase-mediated hydrolysis and liver uptake of high-density lipoprotein (HDL) lipid components were studied in a recirculating rat liver perfusion, a situation where the enzyme is physiologically expressed and active at the vascular bed. Human native HDL were labelled with tri-[3H]oleoylglycerol, [N-methyl-3H]dipalmitoylphosphatidylcholine (DPPC), 1-palmitoyl,2-[14C]linoleoylphosphatidylcholine (PLPC), 1-palmitoyl,2-[14C]linoleoylphosphatidyl-ethanolamine (PLPE) and 1-palmitoyl,2-[14C]palmitoylphosphatidylethanolamine (DPPE). (1) Relative degradation rates of phosphatidylethanolamine molecular species were 2- to 10-fold higher than those of phosphatidylcholine. Considering [14C] PLPC and [14C] PLPE as representative of HDL phosphatidylcholine and phosphatidylethanolamine, respectively, the amounts of lysophosphatidylcholine and lysophosphatidylethanolamine generated after a 60 min perfusion were comparable. The enzyme showed a clear preference for the molecular species bearing an unsaturated fatty acid at the 2 position of glycerol; this was the most pronounced in the case of phosphatidylethanolamine molecular species. (2) Relative liver uptake of HDL-phosphatidylethanolamine was 4- to 5-fold higher than that of HDL-phosphatidylcholine, irrespective of the constitutive fatty acids. Nevertheless, mass estimation indicated that 3 times more molecules of phosphatidylcholine than of phosphatidylethanolamine were transferred. No correlation could be found between the relative degradation rates of phospholipids and their relative liver uptake, indicating a dissociation between the two processes. (3) Perfusate decay and relative liver uptake of labelled HDL-triacylglycerol were higher than that of any phospholipid class. No circulating radiolabelled free fatty acids accumulated in the perfusate, but they were found acylated into liver cell phospholipids and triacylglycerols. (4) A prior 10-12-min washout of the liver vascular bed with heparin removed over 80% of the hepatic lipase activity, as assessed by specific immunoinhibition. Hepatic lipase-depleted liver displayed impaired phospholipid hydrolysis and triacyglycerol uptake, whereas the transfer of HDL phospholipids to liver tissue was unaffected.     

Kinetics of sulfation in the rat in vivo and in the perfused rat liver

Sulfation of phenols and similar low-molecular-weight substrates in the rat in vivo is a rather complex process. Besides enzyme kinetic parameters, cosubstrate availability (indirectly measured by serum sulfate concentration) and competition with glucuronidation also play a role. For some substrates extensive extrahepatic sulfation occurs, accounting for more than 50% of the total-body sulfation capacity. However, the hepatic contribution may be under-estimated when drugs are administered into the hepatic portal vein, because saturation of hepatic metabolism may occur under those conditions. Inside the liver, sulfation is located primarily in zone 1, the periportal area. This can be shown in the single-pass perfused rat liver by perfusion in either the normal or retrograde flow direction. In the rat sulfate conjugates are eliminated preferentially in urine, whereas glucuronides are excreted to a high extent in bile. Therefore, it is important to collect both bile and urine in the characterization of pharmacokinetics of conjugation in vivo. Selective inhibition of sulfation by pentachlorophenol and 2,6-dichloro-4-nitrophenol facilitates studies of the role of sulfation in elimination of its substrates, and the competition between sulfation and glucuronidation for the same substrate.     

Kinetics of lipopolysaccharide clearance by Kupffer and parenchyma cells in perfused rat liver

We studied the kinetics of [3H]lipopolysaccharide ([3H]LPS) (endotoxin) binding to Kupffer cells and hepatocytes at the level of the microtubular system after treatment with gadolinium chloride (GdCl(3)) and colchicine. Liver perfusion in Sprague-Dawley rats involves both portal vein and thoracic inferior vena cava cannulations as inlet and outlet, respectively. The subhepatic inferior vena cava is ligated to prevent perfusate leakage. Buffer containing 2% serum and [3H]LPS is administered at 1 ml/min and collected for 50 min. Rate constants for hepatocellular clearance of [3H]LPS in controls, colchicine-treated rats, GdCl(3)-treated rats, and colchicine plus GdCl(3)-treated rats are assessed using a simplified mathematical model. Forward-binding, reversal-binding, residency time, and influx rate constants are estimated. Results show that in GdCl(3)-treated rats, the hepatocytes effectively clear endotoxin from the circulation, and its ultimate binding affinity at the hepatocyte site is somewhat reduced compared to the Kupffer cells. In colchicine-treated rats, the disruption of the microtubule network altered [3H]LPS binding with Kupffer cells, suggesting that the microfilament-microtubular network also affects Kupffer cell function. Simultaneous treatments with colchicine and GdCl(3) increased the influx rate constant, suggesting that the compiled morphological alterations up-regulated endotoxin clearance by the liver, as indicated by a drastic increase in cellular vacuolation. In conclusion, the kinetics of the trafficking process of [3H]LPS clearance are regulated by apical-sinusoidal endocytotic and canalicular routes.