The kinetic parameters of the uptake of very-low-density lipoprotein remnant cholesteryl esters by perfused rat livers

The aim of this study was to determine the kinetic parameters of the hepatic uptake of VLDL remnant cholesteryl esters. Rat livers were perfused in situ with a broad range of remnant [3H]cholesteryl ester concentrations of known specific radioactivity. Following exactly 3 min of perfusion, hepatic lipids were extracted and labelled cholesteryl esters were separated by thin-layer chromatography and counted. The rate of cholesteryl ester uptake was a saturable process and the apparent kinetic parameters were determined from the Lineweaver-Burk plot of the data. Km and Vmax were calculated to be 72 microM and 35 nmol cholesteryl ester/min per g liver, respectively. For the purpose of comparison, we have expressed our kinetic parameters in terms of number of particles (Vmax = 0.022 nmol particles/min per g liver and Km = 45 nM) and compared our values with those obtained with chylomicron remnants by another group of investigators (Sherrill, B.C., Innerarity, T.L. and Mahley, R.W. (1980) J. Biol. Chem. 255, 1804-1807). We found that the maximal capacity for the removal of VLDL particles was similar to what was observed with rat chylomicron remnants. In contrast, the Km for the uptake process of VLDL remnant particles was approximately four times higher than that of rat chylomicron remnant particles. Our results are consistent with the hypothesis that hepatic removal of both chylomicron and VLDL remnants is mediated by the same receptor, but suggest that the affinity of VLDL remnants for the hepatic removal process is substantially lower, possibly due to structural differences between the two remnant particles.     

Stimulation by 3-mercaptopicolinate of net glusose uptake by perfused livers from diabetic rats

Glucose uptake/production was studied as a function of varied glucose loadsin isolated perfused livers from glucagon-treated alloxan-diabetic rats. Uptake of D-[U-¹⁴C]glucose was seen at all levels studied - 9.5–71 mM. In studies with unlabelled D-glucose carried out in the absence of 3-mercaptopicolinate, livers of diabetic rats showed a net production of glucose with perfusate glucose levels less than 22 mM. Above this level, these livers exhibited a time- and concentration-dependent net uptake of glucose for the period of 20–30 min. When 4 mM 3-mercaptopicolinate, which inhibited gluconeogenesis from endogenous substrates, was included in perfusates, a continuous net uptake of unlabelled glucose was observed at all levels above 4 mM. This lowering of the null-point, cross-over glucose concentration was shown to relate mechanistically to the observed reduction in steady hepatic glucose 6-phosphate level produced by mercaptopicolinate. The need for supplemental mechanisms of glucose utilization by high K_ww hepatic enzyme(s) operative in the virtual absence of insulin-dependent glucokinase also is indicated by these observations and by kinetic analysis.     

Activation of glycogenolysis in perfused rat livers by glucagon and metabolic inhibitors

The activation of hepatic glycogenolysis by glucagon and metabolic inhibitors was studied in isolated perfused livers from fed rats. Glucose production rates and phosphorylase activity were increased by all these agents. If iodoacetate (1 mM) and cyanide (1 mM) were infused simultaneously, glycogenolysis was activated to the same extent as by glucagon (1 nM). The effects of the hormone were additive to those of cyanide, but not to those of iodoacetate. When glycogen breakdown was maximally activated by cyanide plus glucagon, additional iodoacetate was inhibitory. The glucagon-induced release of cyclic AMP into the perfusate was partially suppressed by iodoacetate. The inhibitors caused various degrees of depletion of the tissue ATP content and parallel augmentation of the AMP levels. ADP rose to a lesser extent. Indirect evidence suggested that of a progressive lowering of the cellular ATP levels was accompanied by an inhibition of enzyme dephosphorylation as well as of phosphorylation processes. However, dephosphorylation appeared to be more sensitive to changes of the energy balance, resulting in an activation of phosphorylase in response to the metabolic inhibitors.     

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 was 10 μM and the K_i of the l-glycerol 3-phosphate inhibition was 50 μM. The maximum activity (V) was 3.70 μmoles/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, 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.     

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.     

Impact of flow rate on lactate uptake and gluconeogenesis in glucagon-stimulated perfused livers

The impact of reduced hepatic flow on lactate uptake and gluconeogenesis was examined in isolated glucagon-stimulated perfused livers from 24-h-fasted rats. After surgical isolation, livers were perfused (single pass) for 30 min with Krebs-Henseleit (KH) bicarbonate buffer, fresh bovine erythrocytes (hematocrit approximately 20%), and no added substrate. After this "washout" period, steady-state perfusions were initiated with a second reservoir containing the KH buffer, bovine erythrocytes, [U-(14)C]lactate (10,000 dpm/ml), lactate (2.5 mM), and glucagon (250 microg/ml). Perfusion flow rate was adjusted to one of five rates (i.e., 1.8, 2.7, 3.9, 7.4, and 11.0 ml.min(-1).100 g body wt(-1)). After the perfusion, the liver was dissected out and weighed so as to establish the actual flow rate per gram of liver. The resulting flow rates ranged from 0.52 to 4.03 ml.min(-1).g liver(-1). As a function of flow rate, lactate uptake rose in a hyperbolic fashion to an apparent plateau of 2.34 micromol.min(-1).g liver(-1). Fractional extraction (FX) of lactate from the perfusate demonstrated an exponential decline with increased flow rates (r=0.97). At flow rates above 1.0 ml.min(-1).g liver(-1), adjustments in FX compensated for changes in lactate delivery, resulting in steady rates of lactate uptake and gluconeogenesis. Below 1.0.min(-1).g liver(-1) the increased FX was unable to compensate for the decline in lactate delivery and lactate uptake declined rapidly. Gluconeogenesis demonstrated similar kinetics to lactate uptake, reflecting its dominant role among pathways for lactate removal under the current conditions.     

Biliary protein output by isolated perfused rat livers. Effects of bile salts.

The output of proteins into bile was studied by using isolated perfused rat livers. Replacement of rat blood with defined perfusion media deprived the liver of rat serum proteins (albumin, immunoglobulin A) and resulted in a rapid decline in the amounts of these proteins in bile. When bovine serum albumin was incorporated into the perfusion medium it appeared in bile within 20 min and the amount in the bile was determined by the concentration of the protein in the perfusion medium. The use of a defined perfusion medium also deprived the livers of bile salts and the amounts of these, and of plasma-membrane enzymes [5'-nucleotidase (EC 3.1.3.5) and phosphodiesterase I], in bile declined rapidly. Introduction of micelle-forming bile salts (taurocholate or glycodeoxycholate) to the perfusion medium 80 min after liver isolation markedly increased the output of plasma-membrane enzymes but had no effect on the other proteins. The magnitude of this response was dependent on the bile salt used and its concentration in bile; there was little effect on plasma-membrane enzyme output until the critical micellar concentration of the bile salt had been exceeded in the bile. A bile salt analogue, taurodehydrocholate, which does not form micelles, did not produce the enhanced output of plasma-membrane enzymes. This work supports the view that the output of plasma-membrane enzymes in bile is a consequence of bile salt output and also provides evidence for mechanisms by which serum proteins enter the bile.     

Stochastic vs. deterministic uptake of dodecanedioic acid by isolated rat livers

Deterministic and stochastic differential equations models of the uptake of dodecanedioic acid (C12) are fitted to experimental data obtained on nine isolated, perfused rat livers. 11500 μg of C12 were injected as a bolus into the perfusing liver solution. The concentrations of C12 in perfusate samples taken over 2 h from the beginning of the experiments were analyzed by High Performance Liquid Chromatography (HPLC). A two-compartment deterministic model is studied. To include spontaneous erratic variations in the metabolic processes the parameter for the uptake rate is randomized to obtain a stochastic differential equations model. Parameters are estimated in a two-step procedure: first, parameters in the drift part are estimated by least squares; then, the diffusion parameter is estimated using Monte-Carlo simulations to approximate the unknown likelihood function. Parameter estimation is carried out over a wide range of reasonable measurement error variances to check robustness of estimates. It is concluded that the kinetics of dodecanedioic acid, in the experimental conditions discussed, is well approximated by a model including spontaneous erratic variations in the liver uptake rate.     

Myocardial uptake of thiopental enantiomers by the isolated perfused rat heart

Myocardial uptake of thiopental enantiomers by an isolated perfused rat heart preparation was examined after perfusion with protein-free perfusate. Outflow perfusate samples were collected at frequent intervals for 20 min during single-pass perfusion with 10 μg/ml racemic thiopental (washin phase) and for another 45 min during perfusion with drug-free perfusate (washout phase). (+)- and (−)-thiopental concentrations were assayed by chiral high-performance liquid chromatography. Heart rate, perfusion pressure, and electrocardiogram were also monitored. During the washin phase, there was no significant difference between the mean values of the equilibration rate constants of (+)- and (−)-thiopental enantiomers (0.44 ± 0.07 min⁻¹ and 0.43 ± 0.09 min⁻¹, respectively, P > 0.05). Mean volumes of distribution of (+)- and (−)-thiopental enantiomers were similar (6.34 ± 1.20 and 6.45 ± 1.29 ml/g for the washin phase and 7.22 ± 0.71 and 7.47 ± 0.81 ml/g for the washout phase, respectively, P > 0.05). This indicates that tissue accumulation of thiopental enantiomers in the isolated perfused rat heart was not stereoselective. Uptake of thiopental by the heart was perfusion flow rate-limited and independent of capillary permeability. These findings suggest that myocardial tissue concentration of racemic thiopental should be an accurate predictor of myocardial drug effect. © 1996 Wiley-Liss, Inc.     

Kinetics of lipid peroxidation in isolated surviving rat livers

The kinetics of accumulation of lipid peroxidation products (hydroperoxides as primary products and malonic dialdehyde and "fluorescent pigments" as secondary ones) was investigated in an isolated non-perfused and preliminarily perfused liver during aerobic incubation. In the course of surviving there takes place an intensive accumulation of primary, secondary and final products of lipid peroxidation whose kinetics is of an extreme character. The rate of this process in a non-perfused liver is considerably higher than in a preliminarily perfused liver.     

Amino acid release by isolated perfused cirrhotic livers

Livers of normal and cirrhotic rats were perfused in vitro with and without amino acid substrates (2.3 mM ornithine, 10 mM glutamine or 20 mM alanine) in order to assess urea formation and amino acid release. The rates of urea production were lower in the livers of cirrhotic rats when compared to those of controls only in perfusions with added substrates. The release of several amino acids by livers of cirrhotic rats was higher than that of controls although the pattern of amino acids in the perfusate was different from that reported in plasma during hepatic insufficiency.     

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.     

Secretion and uptake of nascent hepatic very low density lipoprotein by perfused livers from fed and fasted rats

Livers from fed or 24-hr fasted male rats were perfused in a recycling system. VLDL labeled with [1-14C]oleate (95% in triglyceride), produced in separate perfusions of livers from fed rats, was added to the medium as a pulse. Uptake of VLDL 14C-labeled triglyceride by livers from fasted rats was less than that from fed rats regardless of addition of oleate. During the interval in which radioactive triglyceride was taken up, the mass of triglyceride in the medium increased, indicative of the synthesis and net secretion of triglycerides. The rates of secretion of VLDL and uptake of VLDL were both more rapid in livers from fed rats in comparison to those from fasted animals. It was calculated that about 50% of the triglyceride synthesized and secreted by the liver was taken back by livers from fed rats. The VLDL from livers of fasted rats did not contain any apoE detectable by SDS gel electrophoresis or by radioimmunoassay when no fatty acid or 166 mumol of oleic acid was infused. In contrast, apoE comprised 6% of the VLDL apoprotein derived from perfusion of livers from fed animals in the absence of added fatty acid, and 20% when the fed livers were infused with 166 mumol of oleic acid. However, the net output (accumulation) of apoE by fasted liver was only two-thirds that from fed livers. When lipoprotein-free rat plasma containing apoE (4 mg/dl) was used in place of bovine serum albumin, the VLDL secreted by livers from either fed or fasted rats contained apoE and was taken up to a similar extent by such livers. These data suggested that the apoE of the d greater than 1.21 g/ml fraction was transferred to newly secreted VLDL which then stimulated uptake of the VLDL by livers from fasted rats. With further stimulation of secretion of VLDL triglyceride by infusion of 332 mumol of oleic acid/hr, the percent of apoE in the VLDL secreted by livers from fasted rats increased to 20%, which was similar to that of the VLDL produced by livers from fed rats when either 166 or 332 mumol/hr was infused. These data suggest a relationship between rates of hepatic secretion of VLDL (TG) and apoE, and the association of apoE with the secreted VLDL. During fasting, reduced secretion of both VLDL and apoE resulted in a VLDL particle that was considerably diminished in content of apoE and, therefore, that would be taken up by the liver at a reduced rate, in comparison to that observed in the fed animal.