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.     

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.     

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.     

Glucagon and insulin control of gluconeogenesis in the perfused isolated rat liver. Effects on cellular metabolite distribution.

The metabolic effects of glucagon and glucagon plus insulin on the isolated rat livers perfused with 10 mM sodium L-lactate as substrate were studied. Glucagon stimulated gluconeogenesis, ketogenesis and ureogenesis at the concentration used of 2.1 nM. The addition of insulin to give a glucagon-to-insulin ratio of 0.2 reversed all the glucagon effects. The glucagon enhancement of gluconeogenesis was accompanied by a rise in cytosolic and mitochondrial state of reduction of the NAD system and a fall in the [ATP]/[ADP] ratio. The analysis of the intermediary metabolite concentrations suggested, as possible sites of glucagon action, the steps between pyruvate and phosphoenolpyruvate as well as the reactions catalyzed by phosphofructokinase and/or fructose bisphosphatase. All the changes in metabolite contents were abolished when insulin was present. Glucagon increased the intramitochondrial concentration of all the metabolites, whose intracellular distribution was calculated. The finding of a significant rise in the calculated intramitochondrial concentration of oxaloacetate points to pyruvate carboxylation as an important site of glucagon interaction with the gluconeogenic pathway. A primary event in the glucagon action redistributing intracellular metabolites seems to be the mitochondrial entry of malate. The possibility is discussed that the changes in metabolite cellular distribution were brought about by the increased cellular state of reduction caused by the hormone.     

The determinants of insulin extraction in the isolated perfused rat liver.

The effect of hepatic blood flow and portal insulin concentration on insulin extraction during one passage through the isolated perfused rat liver was studied. The percentage of insulin extracted was constant over the physiological range of blood flows (4 to 28 ml/min). The total amount of insulin extracted increased as the input concentration was raised from 48 to 4860 microU/ml with the highest level of extraction being approximately 700 microU of insulin per gram of liver per minute. When square wave input pulses of 243 to 4860 microU/ml were presented, about 5% of this insulin was retained and then released by the liver for periods up to 15 minutes after the cessation of the input. The possible roles of glucose and glucagon as regulators of insulin extraction were studied. Glucose (300 mg/dl), as compared with no glucose, led to a significant reduction of insulin extraction (22% vs. 38%, p less than 0.001). Glucagon had no effect on insulin extraction in the presence of constant levels of glucose. It is concluded, therefore, that glucose may increase circulating insulin levels not only by its well known stimulation of insulin secretion by the pancreas, but also by inhibiting insulin extraction by the liver.     

O2 dependence of insulin stimulation of glucose uptake by perfused rat liver: effects of carboxyhaemoglobin and haematocrit

Livers from fed male rats were perfused in a non-recirculating system with undiluted rat blood containing 14 mM glucose. In these experiments there was a substantial uptake of glucose which was stimulated by insulin. Perfusion with blood containing carboxyhaemoglobin at a concentration of 40% of total haemoglobin lowered O2 consumption and abolished hepatic glucose uptake in control and insulin-infused livers, respectively. In experiments with rat erythrocytes resuspended in buffer to haematocrit values of 38 and 22%, O2 consumption and control and insulin-stimulated rates of glucose uptake were similar to corresponding perfusions with undiluted blood and blood containing carboxyhaemoglobin. It is concluded that serum factors are of relatively small importance and that hepatic glucose uptake is dictated by O2 supply.     

Transport,metabolism and distribution space of octanoate in the perfused rat liver

The scope of the present work was to investigate the metabolism and the passage of octanoate from albumin into the phospholipid bilayer of the plasma membrane and from thence into the cell space. The experiments were done in the isolated perfused rat liver with infusions of albumin and octanoate at various concentrations. Once steady-state conditions were attained, trace amounts of [1-¹⁴C]-octanoate, [¹³¹I]-albumin and [³H]-water were injected simultaneously and the effluent perfusate was fractionated. The normalized dilution curves were used for model analysis. The model which gives the best fit to the experimental results and which also produces the most consistent parameters is one that presupposes a rapid distribution of octanoate into the cell membrane and a slow transfer from the cell membrane into the cytosol. The concentration dependence of the distribution between the membrane and the extracellular space is parabolic, suggesting that octanoate changes the properties of the cell membrane when present at higher concentrations. The passage from the cell membrane into the cell space is relatively slow and limits metabolic transformation partly or totally, depending on the octanoate concentration in the plasma membrane. The rapid transfer of octanoate from the albumin space into the plasma membrane corroborates previous measurements of the dissociation of the albumin–octanoate complex. © 1997 John Wiley & Sons, Ltd.     

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.     

In vitro effects of glycosylated insulin and glucagon in perfused liver of the rat.

Using perfused liver of the rat, the hepatic uptake of glycosylated insulin (GI) and glucagon (GG) and its effects on hepatic glucose output were investigated. Insulin and glucagon were glycosylated in ambient high glucose concentration, and GI80 or GG80 (insulin or glucagon incubated with 0.08% glucose), GI350 or GG350 (incubated with 0.35% glucose), and GI1000 or GG1000 (incubated with 1% glucose) were prepared. The liver was perfused with the medium containing 1000 microU/ml insulin and 200 pg/ml glucagon or 200 microU/ml insulin and 1000 pg/ml glucagon. The fractional uptake of insulin or glucagon by perfused liver was not significantly altered by the glycosylation. In the liver perfused with 1000 microU/ml insulin and 200 pg/ml glucagon, glucose output was not changed by the glycosylation of the hormones, while in the liver perfused with 200 microU/ml insulin and 1000 pg/ml glucagon, GI1000 reduced its biological activity, as reflected by insulin-mediated decrease in glucose output. These results suggest that in the liver insulin incubated with markedly high concentration of glucose reduces its biological activity at a physiological concentration in the presence of high concentration of glucagon.     

Characteristics of sinusoidal uptake and biliary excretion of cysteinyl leukotrienes in perfused rat liver

In single-pass perfused rat liver, the sinusoidal uptake of infused 3H-labelled leukotriene (LT) C4 (10 nmol.l-1) was inhibited by sulfobromophthalein. Inhibition was half-maximal at sulfobromophthalein concentrations of approximately 1.2 mumol.l-1 in the influent perfusate and leukotriene uptake was inhibited by maximally 34%. Sulfobromophthalein (20 mumol.l-1) also decreased the uptake of infused [3H]LTE4 (10 nmol.l-1) by 31%. Indocyanine green (10 mumol.l-1) inhibited the sinusoidal [3H]LTC4 uptake by 19%. Replacement of sodium in the perfusion medium by choline decreased the uptake of infused [3H]LTC4 (10 nmol.l-1) by 56%, but was without effect on the uptake of sulfobromophthalein. The canalicular excretion of LTC4, LTD4 and N-acetyl-LTE4 was inhibited by sulfobromophthalein. In contrast, the proportion of polar omega-oxidation metabolites recovered in bile following the infusion of [3H]LTC4 was increased. Taurocholate, which had no effect on the sinusoidal leukotriene uptake, increased bile flow and also the biliary elimination of the radioactivity taken up. With increasing taurocholate additions, the amount of LTD4 recovered in bile increased at the expense of LTC4. Following the infusion of [3H]LTD4 (10 nmol.l-1), a major biliary metabolite was LTC4 indicating a reconversion of LTD4 to LTC4. In the presence of taurocholate (40 mumol.l-1), however, this reconversion was completely inhibited. The findings suggest the involvement of different transport systems in the sinusoidal uptake of cysteinyl leukotrienes. LTC4 uptake is not affected by bile acids and has a sodium-dependent and a sodium-independent component, the latter probably being shared with organic dyes. Sulfobromophthalein also interferes with the canalicular transport of LTC4, LTD4 and N-acetyl-LTE4, but not with the excretion of omega-oxidized cysteinyl leukotrienes. The data may be relevant for the understanding of hepatic leukotriene processing in conditions like hyperbilirubinemia or cholestasis.     

Regulation of tyrosine transminase degradation in the isolated perfused rat liver by cycloheximide and insulin

In the isolated perfused rat liver, synthesis of the enzyme tyrosine transminase (EC 2.6.1.5) is stimulated by either insulin or glucocorticoid hormone. The antibiotic cycloheximide blocks both synthesis and degradation of tyrosine transminase in insulin-treated perfused livers, whereas it blocks only synthesis of the enzyme in glucocorticoid-treated or untreated livers. When insulin is given in additions to cycloheximide, degradation of the enzyme is blocked in glucocorticoid-treated livers as well. The inhibitory effects on degradation are enzyme-specific, since tryptophan oxygenase (EC 1.13.1.12), the synthesis of which is stimulated by glucocorticoid hormone but not by insulin, is degraded at the same rate under all conditions.     

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.     

Residence time distribution of diazepam in the isolated perfused rat liver. Analysis with the axial dispersion model.

The application of the axial dispersion model to diazepam hepatic elimination was evaluated using data obtained for impulse-response experiments with diazepam in the single-pass isolated perfused rat liver preparation. The transient form of the two-compartment dispersion model was applied to the output concentration versus time profile of diazepam after bolus input of a radiolabelled tracer into the hepatic portal vein (n = 4), providing DN and CLint estimates of 0.251 +/- 0.093 and 135 +/- 59 ml min-1, respectively. In contrast, the one-compartment form of the axial dispersion model, which assumes instantaneous transversal distribution of substance to the accessible spaces within the liver, could not adequately describe the residence time distribution (RTD) of diazepam. Furthermore, the magnitude of DN, a stochastic parameter which characterizes the axial spreading of solutes during transit through the liver, is similar to that determined for non-eliminated substances such as erythrocytes, albumin, sucrose and water. These findings suggest that the dispersion of diazepam in the perfused rat liver is determined primarily by the architecture of the hepatic microvasculature.