Liver parenchyma heterogeneity in the response to extracellular NAD+

The perfused rat liver responds intensely to NAD+ infusion (20-100 microM). Increases in portal perfusion pressure and glycogenolysis and transient inhibition of oxygen consumption are some of the effects that were observed. The aim of the present work was to investigate the distribution of the response to extracellular NAD+ along the hepatic acinus. The bivascularly perfused rat liver was used. Various combinations of perfusion directions (antegrade and retrograde) and infusion routes (portal vein, hepatic vein and hepatic artery) were used in order to supply NAD+ to different regions of the liver parenchyma, also taking advantage of the fact that its extracellular transformation generates steep concentration gradients. Oxygen uptake was stimulated by NAD+ in retrograde perfusion (irrespective of the infusion route) and transiently inhibited in antegrade perfusion. This indicates that the signal causing oxygen uptake inhibition is generated in the periportal area. The signal responsible for oxygen uptake stimulation is homogenously distributed. Stimulation of glucose release was more intense when NAD+ was infused into the portal vein or into the hepatic artery, indicating that stimulation of glycogenolysis predominates in the periportal area. The increases in perfusion pressure were more pronounced when the periportal area was supplied with NAD+ suggesting that the vasoconstrictive elements responding to NAD+ predominate in this region. The response to extracellular NAD+ is thus unequally distributed in the liver. As a paracrine agent, NAD+ is likely to be released locally. It can be concluded that its effects will be different depending on the area where it is released.     

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.     

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.     

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.     

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.     

Metabolic effects of acetaminophen. Studies in the isolated perfused rat liver

The effects of acetaminophen on the metabolism of the isolated perfused rat liver were investigated. The following results were obtained: (1) Acetaminophen increased glucose release and glycolysis from endogenous glycogen (glycogenolysis). (2) Oxygen uptake, gluconeogenesis from either pyruvate or fructose and glycogen synthesis were inhibited. (3) In isolated rat liver mitochondria acetaminophen decreased state III and state IV respiration; it also decreased the ADP/O ratio and the respiratory control ratio. (4) The action of acetaminophen on glycogenolysis was not affected by N-acetylcysteine; this compound, however, increased glycogen synthesis. (5) The effects of acetaminophen are reversible. It was concluded that glycogen depletion by acetaminophen can be produced by two mechanisms. The first, as previously demonstrated by several workers, depends on irreversible binding of a reactive metabolite. The second, however, is reversible and depends primarily on an inhibition of mitochondrial energy metabolism.     

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.     

Deciphering the physiological response of Escherichia coli under high ATP demand

One long‐standing question in microbiology is how microbes buffer perturbations in energy metabolism. In this study, we systematically analyzed the impact of different levels of ATP demand in Escherichia coli under various conditions (aerobic and anaerobic, with and without cell growth). One key finding is that, under all conditions tested, the glucose uptake increases with rising ATP demand, but only to a critical level beyond which it drops markedly, even below wild‐type levels. Focusing on anaerobic growth and using metabolomics and proteomics data in combination with a kinetic model, we show that this biphasic behavior is induced by the dual dependency of the phosphofructokinase on ATP (substrate) and ADP (allosteric activator). This mechanism buffers increased ATP demands by a higher glycolytic flux but, as shown herein, it collapses under very low ATP concentrations. Model analysis also revealed two major rate‐controlling steps in the glycolysis under high ATP demand, which could be confirmed experimentally. Our results provide new insights on fundamental mechanisms of bacterial energy metabolism and guide the rational engineering of highly productive cell factories.     

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.     

Modelization and experimental studies on the control of the glycolytic-glycogenolytic pathway in rat liver

In this paper we construct a model of the glycolytic-glycogenolytic converging pathway in rat liver, by integrating experimental data obtained in anin vitro system and information available from the literature. The model takes the mathematical expression of an S-system representation within the power law formalism (Savageau, 1976. Biochemical System Analysis: A study of function and design in Molecular Biology. Addison-Wesley, Reading, Mass.). By using this theoretical framework a model analysis was carried out that allowed us a) the assessment of the quality of the model in terms of its consistency and robustness, b) the steady state analysis and control characterization of the system, and c) the study of the dynamics of the system after changes in the level of two magnitudes of biological significance: the glucose concentration and the phosphofructokinase enzyme activity. Model predictions are compared with experimental measurements referred to Logarithmic Gains through fluxes and substrates concentrations showing that there is a good correlation between the model predictions and the experimentally determined values.     

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.     

Modulation by oxygen of zonal gene expression in liver studied in primary rat hepatocyte cultures

The different endowment with key enzymes and thus different metabolic capacities of periportal and perivenous cell types led to the model of "metabolic zonation." The periportal and perivenous hepatocytes receive different signals owing to the decrease of substrate concentrations including O2 and hormone levels during passage of blood through the liver sinusoids. These different signal patterns should be important for the short-term regulation of metabolism and also for the long-term induction and maintenance of the different enzyme pathways by control of gene expression. The periportal to perivenous drop in oxygen tension was considered to be a key regulator in the zonated expression of carbohydrate-metabolizing enzymes. In primary hepatocyte cultures, glucagon activated the phosphoenolpyruvate carboxykinase (PCK) gene to higher levels under arterial than under venous oxygen. The insulin-dependent activation of the glucokinase (GK) gene was reciprocally modulated by oxygen. Exogenously added hydrogen peroxide mimicked the effects of arterial oxygen on both the glucagon-dependent PCK gene and the insulin-dependent GK activation. Therefore, the oxygen sensor could be a hydrogen peroxide-producing oxidase which could contain a heme group for "measuring" the O2 tension. This notion was corroborated by the finding that CO mimicked the positive effect of O2 on PCK gene activation. Transfection of PCK promoter-CAT gene constructs into primary hepatocytes showed that the oxygen modulation of the PCK gene activation occurred in the region -281/+69. The modulation by O2 was not mediated by isolated cAMP-responsive elements. Nuclear protein extracts prepared from hepatocytes cultured under venous Po2 as compared to arterial Po2 showed an enhanced binding activity to the promoter fragment -149/-43. Oxidative conditions such as H2O2 reduced the DNA-binding activity, thus supporting the role of H2O2 as a mediator in the O2 response of the PCK and GK genes.     

Uptake of asialoglycophorin-liposomes by the perfused rat liver

Asialoglycophorin-containing liposomes and their contents (¹²⁵I-labeled bovine serum albumin) were taken up by a perfused rat liver with subsequent digestion of their protein components. The uptake of these liposomes required Ca²⁺ as well as desialylation. The process was inhibited partially by asialofetuin and completely by further addition of asialoglycophorin to the perfusate.     

Investigation of the hepatic respiration and liver zonation on rat hepatocytes using an integrated oxygen biosensor in a microscale device

The development of an in vitro functional liver zonation model is a major issue to reproduce physiological liver features. Oxygen concentration is one of the potential explanations of a primary regulating factor of zonation. In this frame, we investigated the oxygen gradient inside a microfluidic device containing rat hepatocyte cultures. The device integrated a platinum (Pt) (II) octaethylporphyrin sensor, allowing a 2D mapping of the oxygen concentration. After 3 hr adhesion of the hepatocytes, the sensor indicated an intense oxygen depletion, leading to an oxygen shortage in the center of the device. After a 30 min perfusion of the culture medium, we monitored the formation of the oxygen gradient along the culture due to cellular respiration. The profile of the oxygen gradient was modulated and controlled by increasing either the perfusion flow rate or the device thickness. In addition, the oxygen gradient was time dependent as far as it decreased with the time of culture. Perivenous and periportal liver patterns were characterized by the immunostaining of the hepatic markers. We put in evidence a spatio temporal hepatic organization. We observed the overexpression since 24 hr of perfusion of the APC and PCK1 proteins upstream in the oxygen-rich area of the device. The overexpression of GS, GCK, CYP1A, and HIFα proteins were observed downstream in the oxygen-poor area. Then, CYP3A2 and β-catenin spatial reorganization was achieved after 48 hr of culture. The results presented a partial zonation-like pattern that was superimposed with an oxygen gradient profile.     

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.     

Redox modulation of the response of NADH oxidase activity of rat liver plasma membranes to cyclic AMP plus ATP

NADH oxidase activity of rat liver plasma membranes was inhibited by lowconcentrations (1-100 nM) of ATP. The inhibition was amplified by additionof nanomolar concentrations (0.1-10) of cyclic AMP. The inhibition wascomplex and related to a marked increase in the Km for NADH at high NADHconcentrations together with a concomitant decrease in the Vmax. In theabsence of added or residual ATP, cyclic AMP was without effect. Theresponse of cyclic AMP + ATP was inhibited by low concentrations of theselective inhibitor of cyclic AMP-dependent protein kinase, H-89 but not bystaurosporin. The Vmax but not the Km was modified by treating the plasmamembranes with a mild oxidizing agent, N-chlorosuccinamide, or with thereducing agent, dithiothreitol. In the presence of dithiothreitol, the Vmaxwas reduced by cyclic AMP + ATP. In contrast, in the presence ofN-chlorosuccinamide, the Vmax was increased by cyclic AMP + ATP relative tocyclic AMP + ATP alone. Thus, the effect of cyclic AMP + ATP on the Vmaxcould be either an increase or a decrease depending on whether the membraneswere oxidized or reduced. The results demonstrate regulation of NADH oxidaseactivity of rat liver plasma membranes through cyclic AMP-mediatedphosphorylation by membrane-located protein kinase activities where thefinal response is dependent on the oxidation-reduction status of the plasmamembranes.     

Actions of p‐synephrine on hepatic enzyme activities linked to carbohydrate metabolism and ATP levels in vivo and in the perfused rat liver

p‐Synephrine is one of the main active components of the fruit of Citrus aurantium (bitter orange). Extracts of the bitter orange and other preparations containing p‐synephrine have been used worldwide to promote weight loss and for sports performance. The purpose of the study was to measure the action of p‐synephrine on hepatic enzyme activities linked to carbohydrate and energy metabolism and the levels of adenine mononucleotides. Enzymes and adenine mononucleotides were measured in the isolated perfused rat liver and in vivo after oral administration of the drug (50 and 300 mg/kg) by using standard techniques. p‐Synephrine increased the activity of glycogen phosphorylase in vivo and in the perfused liver. It decreased, however, the activities of pyruvate kinase and pyruvate dehydrogenase also in vivo and in the perfused liver. p‐Synephrine increased the hepatic pools of adenosine diphosphate and adenosine triphosphate. Stimulation of glycogen phosphorylase is consistent with the reported increased glycogenolysis in the perfused liver and increased glycemia in rats. The decrease in the pyruvate dehydrogenase activity indicates that p‐synephrine is potentially capable of inhibiting the transformation of carbohydrates into lipids. The capability of increasing the adenosine triphosphate‐adenosine diphosphate pool indicates a beneficial effect of p‐synephrine on the cellular energetics.     

Uptake of asialo-glycophorin by the perfused rat liver and isolated hepatocytes

Perfused rat livers took up asialo-glycophorin, a glycoprotein derived from human erythrocyte membraneds, with a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ for the clearance of 7 min. As a comparison, asialo-orosomucoid was taken up by this system with a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 3.5 min. Both proteins were digested and their ¹²⁵I labels were released to the perfusate as free ¹²⁵I^?. EGTA completely inhibited uptake of these glycoproteins, but not uptake of denatured bovine serum albumin. Addition of Ca²⁺ reversed the inhibition nearly completely. Isolated hepatocytes had an uptake rate of approximately 3 ng/min per 10⁶ cells for the asialo forms of glycophorin, orosomucoid and fetuin. Cellular uptake of each of these asialoglycoproteins could be inhibited by one of the other proteins. Asialo-fetuin caused a 95% inhibition of the uptake rate of asialo-orosomucoid by the perfused liver. This fetal calf glycoprotein had a similar inhibitory effect on asialo-glycophorin, but only after an initial 40% of the asialo-glycophorin had been taken up by the liver at an almost normal rate during the first 30 min of perfusion. The possiblity of an alternative hepatic removal system for asialo-glycophorin is suggested.     

Hepatic endothelial lipase activity in neonatal rat liver

Hepatic endothelial lipase (HEL) activity is as high in the neonatal (1-day old) rat liver as in adults. Most of the HEL activity is located at the capillaries since 75% of the total activity is released by heparin or collagenase perfusion. The residual activity (non-releasable) is located in hepatocytes and not in hemopoietic cells, which are the major cell type in neonatal liver. Per mg of protein, the HEL activity is 50% higher in neonatal than in adult hepatocytes. We suggest that neonatal hepatocytes have an increased capacity to synthesize and secrete HEL activity, so maintaining a high activity in the whole organ. it might contribute to the hepatic uptake of cholesterol from circulating lipoproteins, in a period in which endogenous cholesterol synthesis is known to be inhibited in the liver.     

Insulin uptake by rat liver endothelium studied in fractionated liver cell suspensions

Summary Cellular distribution of insulin receptors was studied in fractionated rat liver cell suspensions using ¹²⁵1-insulin and a visual probe consisting of latex beads covalently linked to insulin (minibeads). Fractionation was done on metrizamide gradients which yielded two cellular fractions. The large cell fraction consisted mostly of hepatocytes and the small cell fraction consisted of 37% endothelial cells as well as Kupffer cells. The magnitude of insulin uptake by the endothelium-rich small cell fraction was at least double that of the uptake by the hepatocyte-rich fraction. The minibead technique demonstrated that in the small cell fraction only endothelial cells, and not Kupffer cells, were responsible for the insulin uptake. Our findings suggest that liver endothelium may be responsible for the uptake of circulating insulin and its transport to hepatocyte. This emphasizes the presence of a tissue-blood barrier in the liver.Abbreviations PRS phosphate-buffered saline - SEM scanning electron microscopy - TEM transmission electron microscopy