Zonation of hepatic fatty acid metabolism - The diversity of its regulation and the benefit of modeling.

A pronounced heterogeneity between hepatocytes in subcellular structure and enzyme activities was discovered more than 50years ago and initiated the idea of metabolic zonation. In the last decades zonation patterns of liver metabolism were extensively investigated for carbohydrate, nitrogen and lipid metabolism. The present review focuses on zonation patterns of the latter. We review recent findings regarding the zonation of fatty acid uptake and oxidation, ketogenesis, triglyceride synthesis and secretion, de novo lipogenesis, as well as bile acid and cholesterol metabolism. In doing so, we expose knowledge gaps and discuss contradictory experimental results, for example on the zonation pattern of fatty acid oxidation and de novo lipogenesis. Thus, possible rewarding directions of further research are identified. Furthermore, recent findings about the regulation of metabolic zonation are summarized, especially regarding the role of hormones, nerve innervation, morphogens, gender differences and the influence of the circadian clock. In the last part of the review, a short collection of models considering hepatic lipid metabolism is provided. We conclude that modeling, despite its proven benefit for understanding of hepatic carbohydrate and ammonia metabolisms, has so far been largely disregarded in the study of lipid metabolism; therefore some possible fields of modeling interest are presented.

[The ecology of ticks, tick-borne diseases and biological tick control in Baden-Württemberg]. / Ökologie von Zecken als Überträger von Krankheitserregern in Baden-Württemberg und biologische Zeckenbekämpfung.

Ticks and tick-borne diseases are of great significance for the health of humans and animals. However, the factors influencing their distribution and dynamics are inadequately known. In a project financed by the Baden-Württemberg Ministry of the Environment, Climate and Energy Industry, as part of the program BWPLUS, interdisciplinary specialists work together to determine the influence of weather, (micro)climate, habitat, land use, human activities, and the population dynamics of host animals on the distribution and abundance of ticks and the diseases that they transmit in Baden-Württemberg. The project comprises four modules: the large-scale distribution of ticks in Baden-Württemberg (module 1), detailed studies of host-tick-pathogen interaction in relation to the microclimate (module 2), and the spatial occurrence of important tick-borne pathogens (module 3). The fourth module involves the comprehensive analysis and synthesis of all data in order to determine the relative importance of the factors studied and to develop a risk model. Recently, intensive investigations into tick control have been undertaken using various entomopathogenic fungi and nematodes as well as a parasitoid wasp. Our aim was to determine whether these natural enemies could be used to effectively reduce the number of free-living ticks.

[Smoke-free by ramadan: experience with a low-threshold prevention offer on smoking cessation for persons with migration background]. / Rauchfrei zu Ramadan. Erfahrungen mit einem niederschwelligen Präventionsangebot zur Raucherentwöhnung bei Personen mit Migrationshintergrund.

BACKGROUND: Persons with migration background exhibit higher smoking rates in comparison to the general population.These smokers often cannot be reached by prevention measures at the family doctor's office. METHODS: In summer 2011 the health campaign "Smoke-free by Ramadan" was launched in 11 German cities. Measures included the training of doctors on smoking cessation methods, general bilingual information flyers, and in some cases lectures on smoking, specifically for imams. A number of local events, especially for individuals with Turkish migration background were initiated. For these health events a specially equipped health bus of the BKK-vor-Ort was used, in which visitors were offered following elements: systematic data collection about age, sex and smoking behavior, a test to determine of the severity of nicotine dependence (Fagerström test, FTNA), as well as spirometric lung function test. Smokers were generally motivated to stop smoking. Data were anonymously collected and analysed in a documentation and communication sheet in Turkish language, and test results were handed over to participants on a printed information sheet. RESULTS: Data of 1012 people collected on 8 health days were analysed (70% men, mean age 46.5 years). The percentage of smokers was 41.5% (men) or 30% (women). Of 294 male smokers, according to FTNA 43.6% had low, 24.8% had moderate, and 31.6% strong nicotine dependence; in the 91 female smokers the corresponding rates were 54.9%, 30.8% and 14.3%. The distribution pattern of the dependency levels was statistically significantly different between genders (p = 0.006). Reduced lung function (FEV, < 80%) occurred in smokers more often than in nonsmokers (30% versus 21%). CONCLUSIONS: These results reinforce the importance of low-threshold prevention measures. By screening, here shown by the example of individuals with Turkish migration background, a significant number of smokers was identified who had in addition to strong nicotine addiction also significantly impaired lung function. As the odds for successful cessation without support are below 5%, evidence-based smoking cessation was advised to all smokers.

[Smoking cessation in pneumological routine care]. / Tabakentwöhnung in der pneumologischen Routineversorgung.

Continuous cigarette smoking clearly influences the course and prognosis of diseases like COPD/emphysema and asthma bronchiale in an adverse manner. However smoking cessation as a therapy measure is not a common part of general health-care in Germany as reimbursement of the central component of psychosocial support (behavioural therapy - BT) is allowed only to a minor degree and of pharmacotherapy support (nicotine replacement, varenicline, bupropione) is completely excluded by the legislator. This prospective "real-life" study with 198 participants shows, that with the abolition of the reimbursement barrier for cognitive behavioural therapy in the setting of a pneumological practice/clinic a high long-term abstinence of 45.4 % (point prevalence after 12 months) can be achieved. Apart from the reimbursement of BT, predominant success factors were the implementation of the measure in the practice/clinic, where patients are under long-term treatment and the application of a two-stage motivational model for the participation. Reimbursement of smoking cessation pharmacotherapy was not possible in this study. Thus, pharmacotherapy was applied to fewer than necessary patients and was predominantly too short and in a too low dosage.

Proton beam quality enhancement by spectral phase control of a PW-class laser system.

We report on experimental investigations of proton acceleration from solid foils irradiated with PW-class laser-pulses, where highest proton cut-off energies were achieved for temporal pulse parameters that varied significantly from those of an ideally Fourier transform limited (FTL) pulse. Controlled spectral phase modulation of the driver laser by means of an acousto-optic programmable dispersive filter enabled us to manipulate the temporal shape of the last picoseconds around the main pulse and to study the effect on proton acceleration from thin foil targets. The results show that applying positive third order dispersion values to short pulses is favourable for proton acceleration and can lead to maximum energies of 70 MeV in target normal direction at 18 J laser energy for thin plastic foils, significantly enhancing the maximum energy compared to ideally compressed FTL pulses. The paper further proves the robustness and applicability of this enhancement effect for the use of different target materials and thicknesses as well as laser energy and temporal intensity contrast settings. We demonstrate that application relevant proton beam quality was reliably achieved over many months of operation with appropriate control of spectral phase and temporal contrast conditions using a state-of-the-art high-repetition rate PW laser system.

Nicotinamide phosphoribosyltransferase (NAMPT/PBEF/visfatin) is constitutively released from human hepatocytes.

Circulating NAMPT (PBEF/visfatin) has pleiotropic functions and is secreted from adipocytes. Since it is doubtful whether serum levels can be explained by secretion from adipocytes alone, we asked whether hepatocytes are also able to liberate NAMPT. Using HepG2 cells and primary rat and human hepatocytes, release of NAMPT into the cell culture supernatant was found to occur constitutively in a time-dependent manner. In primary human hepatocytes, secretion within 24h was far higher than the cellular content, but was neither influenced by inhibitors of secretion nor by glucose, insulin or TNFalpha. As determined by size exclusion chromatography, HepG2 lysates and supernatants primarily contained the dimeric form of NAMPT which exhibited similar in vitro specific enzymatic activity. In primary human hepatocytes, secreted NAMPT was less active. Our results demonstrate that human hepatocytes are a potential source of circulating NAMPT.

Heterogeneous expression of cholesterol 7 alpha-hydroxylase and sterol 27-hydroxylase genes in the rat liver lobulus.

We investigated the lobular localization and molecular level of expression of cholesterol 7 alpha-hydroxylase and sterol 27-hydroxylase, two key enzymes in bile acid synthesis, in isolated periportal and pericentral hepatocytes and by in situ hybridization of rat liver. Enzyme activity, mRNA, and gene transcription of cholesterol 7 alpha-hydroxylase were predominant in pericentral hepatocytes of control rats, being 7.9-, 9.9-, and 4.4-fold higher than in periportal hepatocytes, respectively. Similar localization was found for sterol 27-hydroxylase: 2.9-, 2.5-, and 1.7-fold higher enzyme activity, mRNA, and gene transcription, respectively, was found in pericentral hepatocytes. Interruption of the enterohepatic circulation with colestid resulted in upregulation of these parameters for both enzymes, as a consequence of stimulated gene expression mainly in the periportal zone. In contrast, mRNA levels and gene transcription of 3-hydroxy-3-methylglutaryl CoA reductase showed opposite lobular distribution. Selective periportal expression for the latter was enhanced, but remained local, after colestid treatment. In situ hybridization showed unambiguously that cholesterol 7 alpha-hydroxylase mRNA is localized exclusively in the pericentral zone and that sterol 27-hydroxylase mRNA is expressed preferentially in the pericentral region, though less pronounced. Administration of colestid led to expression of both genes within a larger area of the liver lobulus. In conclusion, we suggest that cholesterol 7 alpha-hydroxylase and sterol 27-hydroxylase are coordinately regulated by the bile acid gradient over the lobulus, resulting in predominant expression in the pericentral zone. Opposite lobular localization of cholesterol and bile acid synthesis provides an alternative view to interregulation of these metabolic pathways.

Demonstration of a beam loaded nanocoulomb-class laser wakefield accelerator.

Laser-plasma wakefield accelerators have seen tremendous progress, now capable of producing quasi-monoenergetic electron beams in the GeV energy range with few-femtoseconds bunch duration. Scaling these accelerators to the nanocoulomb range would yield hundreds of kiloamperes peak current and stimulate the next generation of radiation sources covering high-field THz, high-brightness X-ray and γ-ray sources, compact free-electron lasers and laboratory-size beam-driven plasma accelerators. However, accelerators generating such currents operate in the beam loading regime where the accelerating field is strongly modified by the self-fields of the injected bunch, potentially deteriorating key beam parameters. Here we demonstrate that, if appropriately controlled, the beam loading effect can be employed to improve the accelerator's performance. Self-truncated ionization injection enables loading of unprecedented charges of ∼0.5 nC within a mono-energetic peak. As the energy balance is reached, we show that the accelerator operates at the theoretically predicted optimal loading condition and the final energy spread is minimized.Higher beam quality and stability are desired in laser-plasma accelerators for their applications in compact light sources. Here the authors demonstrate in laser plasma wakefield electron acceleration that the beam loading effect can be employed to improve beam quality by controlling the beam charge.

Altered acinar distribution of glutamine synthetase and different growth response of cultured enzyme-positive and -negative hepatocytes after partial hepatectomy.

Partial hepatectomy (PH) results in the persistent drop of the specific activity of glutamine synthetase (GS) (EC 6.3.1.2). This drop correlates with the reduced proportion of GS+ hepatocytes and with the reduced GS+ area surrounding the central veins such that GS+ hepatocytes are arranged in a single cell layer only. Cultivation of hepatocytes isolated at various times after PH revealed considerable differences in the growth characteristics of GS+ and GS- hepatocytes discriminated by immunocytochemistry. In the absence or presence of epidermal growth factor and insulin, the labeling index of GS- hepatocytes peaked in cultures established 48 h after PH at 10% and 50%, respectively, while that of GS+ cells was much lower (2% and 6%). In cultures established at later times after PH the labeling index of GS- cells decreased gradually, while that of GS+ hepatocytes increased continuously, reaching about 20% and more than 50% for controls and epidermal growth factor/insulin-treated cultures, respectively, in cultures established 72 after PH. Norepinephrine stimulated the labeling index of both cell populations during the first 24 h only, but again GS- hepatocytes responded somewhat earlier than did GS+ hepatocytes. These results demonstrate that the differences in the growth characteristics of GS+ and GS- hepatocytes are due to different priming of these cells in vivo and may result in the different expansion of the respective cell populations during regeneration after PH.

Cytochrome P-450-induced cytotoxicity of mitoxantrone by formation of electrophilic intermediates.

Recent studies of our group have shown that the oxidation of the substituted anthraquinone skeleton is involved in the biotransformation of mitoxantrone. In this report the importance of this process with regard to the mode of action of the drug is investigated. This communication describes a new high performance liquid chromatography separation for mitoxantrone and its metabolites allowing the direct coupling of high performance liquid chromatography to mass spectrometry. Application of this technique to bile of mitoxantrone-treated pigs reveals the formation of several metabolites in addition to the drug-derived compounds found in urine. Seven biliary metabolites are identified as thioether derivatives of mitoxantrone and its side chain oxidation products. Independent synthesis and structural elucidation of 3 thioether conjugates of the drug provides unequivocal evidence that the hydroquinone moiety of mitoxantrone is the site of reaction with glutathione. Furthermore, the formation of the thioether conjugates in HepG2 hepatoma cells and in rat hepatocytes during cell incubations is demonstrated. Inhibition of cytochrome P-450 with metyrapone prevents the formation of the thioether conjugates and leads to a complete loss of the cytotoxicity of mitoxantrone in HepG2 cells and rat hepatocytes up to concentrations of 200 to 400 microM thereby indicating that mitoxantrone has a negligible effect by itself. Rat hepatocytes were found to be more susceptible for the oxidation-induced cytotoxicity than HepG2 cells. These results demonstrate that the acute cytotoxicity of mitoxantrone depends on prior oxidation of its 1,4-dihydroxy-5,8-diaminoanthraquinone moiety.

Inhibition of cholesterol biosynthesis by allicin and ajoene in rat hepatocytes and HepG2 cells.

Exposure of primary rat hepatocytes and human HepG2 cells to allicin and ajoene resulted in the concentration-dependent inhibition of cholesterol biosynthesis at different steps of this metabolic pathway. At low concentrations of ajoene sterol biosynthesis from [14C]acetate in rat hepatocytes was decreased by 18% with an IC50-value of 15 microM, while allicin was almost uneffective. In HepG2 cells, both compounds significantly inhibited sterol biosynthesis by 14% and 19% with IC50-values of 7 and 9 microM for allicin and ajoene, respectively. This inhibition was exerted at the level of HMG-CoA-reductase as revealed by the absence of inhibition, if [14C]acetate was replaced by [14C]mevalonate as a precursor, and by direct determination of enzyme activity. At somewhat higher concentrations inhibition of cholesterol biosynthesis by both, allicin and ajoene, was also observed at late steps resulting in the accumulation of the precursor lanosterol. Alliin instead was completely inactive. In the case of allicin, small amounts of dihydrolanosterol and 7-dehydrocholesterol were formed at intermediate concentrations of 5-10 microM. From these results it is concluded that a major point of inhibition at the late steps occurs at the level of lanosterol 14 alpha-demethylase.

Isolation of rat pgp3 cDNA: evidence for gender and zonal regulation of expression in the liver.

Distinct differences exist in the function and regulation of the individual p-glycoprotein (pgp) members in many species. In order to study regulation of individual pgp mRNA isoforms it is, therefore, necessary to have probes that can distinguish between the various pgp isoforms. However, to date few studies examining hepatic gene expression in rat liver have used pgp gene specific probes. Towards this end we screened a cDNA library constructed from a normal rat liver with a human pgp1 cDNA and isolated a partial cDNA for class III pgp, rat pgp3. By comparison of the sequence of this new rat pgp3 cDNA with genomic and cDNA sequences for rat pgp1 and rat pgp2 we selected oligonucleotide probe sequences that would allow us to differentiate between the highly homologous rat pgp2 and pgp3 genes on Northern blots and by polymerase chain reaction (PCR). We found that pgp3, for both male and female rats, was the predominant form of pgp expressed in normal rat liver with males consistently expressing several-fold lower levels of pgp3 than females. Because many genes are zonally expressed in the hepatic acinus we examined the possibility that pgp3 might show heterogeneous distribution as well. We found, by in situ hybridization of paraformaldehyde-fixed rat liver sections that pgp3 was distributed non-uniformly across the hepatic acinus with a gradient that showed the highest expression toward the terminal hepatic venule. We confirmed this finding by selectively isolating hepatocytes from either the terminal hepatic venular or periportal zones using a digitonin/collagenase perfusion procedure. Application of specific pgp3 PCR primers to RNA isolated from hepatocytes from these areas confirmed that pgp3 mRNA was the predominant form in the hepatocytes surrounding the terminal hepatic venule. Finally, we examined pgp3 expression in a variety of tissues by Northern blot analysis and found that pgp3 was most highly expressed in the liver and gastrointestinal tract (with a gradient of expression from small to large intestine), while low levels were found in the kidney, heart and brain. Pgp3 mRNA was undetectable in the adrenal gland and in skeletal muscle. In summary, using rat pgp gene specific oligonucleotide probes we found that pgp3 gene expression is regulated by anatomic location with the highest mRNA expression in organs that are involved in drug detoxification. Our results also demonstrate heterogeneity of hepatic rat pgp3 gene expression, which is influenced by both gender and by acinar location.

Enhanced matrix degradation after withdrawal of TGF-beta1 triggers hepatocytes from apoptosis to proliferation and regeneration.

TGF-beta1 is a profibrogenic cytokine participating in deposition of extracellular matrix in fibrotic disorders. In liver, its anti-proliferative/apoptotic effect on hepatocytes promotes fibrosis. The tetracycline-controlled double-transgenic TA(LAP-2)/p(tet)TGF-beta1 mouse provides a model for reversible liver fibrosis. In livers of TGF-beta1-expressing mice, hepatocytes showed synchronous apoptosis detected by DNA laddering and active caspase-3 staining that disappeared when expression of transgenic TGF-beta1 was switched off. In these 'off' mice, perisinusoidal liver fibrosis resolved within 21 days accompanied by elevated proliferation of hepatocytes. Here, we have specified the intermediary stages (2-3 days off and 6 days off) in terms of (i) proliferation (by immunohistochemical staining of proliferating cell nuclear antigen and expression of cyclin D1 mRNA) and (ii) extracellular matrix remodelling processes (by measuring mRNA expression of matrix metalloproteinases-2 and -13 (mmp-2 and mmp-13) and tissue inhibitor of matrix metalloproteinases 1 (timp-1) and quantitative morphometric analysis. In summary, we show a rapidly declining timp-1 mRNA level together with lastingly high mmp-2 and mmp-13 mRNA levels after 2-3 days, suggesting that high matrix-degrading potential represents a prerequisite for the markedly enhanced proliferation of hepatocytes in the early stages after switching off transgenic TGF-beta1.

Metabolic zonation of the liver: regulation and implications for liver function.

Liver parenchyma shows a remarkable heterogeneity of the hepatocytes along the porto-central axis with respect to ultrastructure and enzyme activities resulting in different cellular functions within different zones of the liver lobuli. According to the concept of metabolic zonation, the spatial organization of the various metabolic pathways and functions forms the basis for the efficient adaptation of liver metabolism to the different nutritional requirements of the whole organism in different metabolic states. The present review summarizes current knowledge about this heterogeneity, its development and determination, as well as about its significance for the understanding of all aspects of liver function and pathology, especially of intermediary metabolism, biotransformation of drugs and zonal toxicity of hepatotoxins.

Pressure-induced dissociation of casein micelles: size distribution and effect of temperature.

Pressure-induced dissociation of a turbid solution of casein micelles was studied in situ in static and dynamic light scattering experiments. We show that at high pressure casein micelles decompose into small fragments comparable in size to casein monomers. At intermediate pressure we observe particles measuring 15 to 20 nm in diameter. The stability against pressure dissociation increased with temperature, suggesting enhanced hydrophobic contacts. The pressure transition curves are biphasic, compatible with a temperature (but not pressure)-dependent conformational equilibrium of two micelle species. Our thermodynamic model predicts an increase in structural entropy with temperature.

High pressure near infrared study of the mutated light-harvesting complex LH2.

The pressure sensitivities of the near infrared spectra of the light-harvesting (LH2) complex and a mutant complex with a simplified BChl-B850 binding pocket were compared. In the mutant an abrupt change in the spectral properties occurred at 250 MPa, which was not observed with the native sample. Increased disorder due to collapse of the chromophore pocket is suggested.

Acinar heterogeneity of the epidermal growth factor receptor in the liver of male rats.

Epidermal growth factor is cleared from the circulation by the liver, forming a very steep portal-to-central sequestration gradient. It was unknown whether this was due to the position within the liver acinus or whether it was due to functional differences in the hepatocytes. Experiments were undertaken to elucidate the lobular distribution and heterogeneity of the epidermal growth factor receptor in rat liver. Immunocytochemistry showed a predominantly higher staining density over periportal localized hepatocytes. Receptor binding studies with isolated, cultured hepatocytes, enriched in periportal or perivenous located cells, were performed. Our data revealed high- and low-affinity binding sites with a kd of 26 pM and 0.87 nM, respectively, for periportal hepatocytes. The high-affinity receptors were restricted to the periportal hepatocytes only, whereas the number of low-affinity receptors showed a 3 to 4-fold concentration gradient between both cell populations.

Primary cultures of rat hepatocytes as a model system of canalicular development, biliary secretion, and intrahepatic cholestasis. IV. Disintegration of bile canaliculi and disturbance of tight junction formation caused by vinblastine.

Treatment of cultured hepatocytes with vinblastine or colchicine caused striking perturbations of the structural organization of the biliary pole and of the junctional complexes. During the early hours of cultivation the reassociation of the bile canaliculi was impaired by the drug, whereas at later times in culture preformed canaliculi were disintegrated to small vesicular remnants lacking microvilli. Vinblastine did not impair tight junction formation per se. However, under the influence of the drug, tight junctional strands associated in an atypic manner perpendicular to the upper surface of the hepatocytes, whereas those strands lining the canaliculi were decomposed to smaller entities and dislocated within the lateral membrane. Concomitantly to the structural disintegration of the biliary pole an accumulation of vesicles in the pericanalicular cytoplasm was noted. As indicated by numerous filipin-induced lesions, they were characterized by a high content of membrane cholesterol. The apical pole and the contiguous membrane on the other hand contained only very few filipin-cholesterol lesions. These findings suggest that antimicrotubular drugs impair the fusion of pericanalicular vesicles with the luminal membranes of the canaliculi, thus interrupting the delivery of membraneous material to the apical pole. In addition, microtubules seem to play an important role in the coordinated development and the structural fixation of the biliary pole of cultured hepatocytes.

Primary cultures of rat hepatocytes as a model system of canalicular development, biliary secretion, and intrahepatic cholestasis. II. Taurolithocholate-induced alterations of canalicular morphology and of the distribution of filipin-cholesterol complexes.

Hepatocytes in primary monolayer culture treated with taurolithocholate showed distinct ultrastructural changes localized primarily to the bile canalicular membrane. These alterations comprised disturbance and proliferation of tight junctions, dilatation of the canaliculi, loss of microvilli, thickening of the pericanalicular ectoplasm, and bizarre lamellar transformations of canaliculi. In freeze-fracture replicas the lamellae projecting into the canalicular lumen were found to be devoid of intramembranous particles. Localization by the use of filipin of cholesterol in plasma membranes of taurolithocholate affected hepatocytes revealed an extensive accumulation of cholesterol in membranes of dilated canaliculi, and also in outpouchings of the contiguous membrane in the ultimate vicinity. Furthermore, a pronounced segregation of cholesterol-rich membrane material into the lumen of dilated canaliculi and into enlargements of the intercellular space could be observed in thin sections. In contrast, a total absence of cholesterol was noted in the lamellar projections of the bizarre transformed canaliculi. The reliability of these findings and their consequences for the mechanism of taurolithocholate-induced cholestasis are discussed and it is suggested that the incorporation of cholesterol into the canalicular membrane reflects only one aspect of the cholestatic effect of taurolithocholate. An additional aspect seems to comprise the dislocation of membrane bound proteins and perhaps other membrane components. This is probably caused by independent mechanisms.

Primary cultures of rat hepatocytes as a model system of canalicular development, biliary secretion, and intrahepatic cholestasis. I. Distribution of filipin-cholesterol complexes during de novo formation of bile canaliculi.

Hepatocytes in primary monolayer culture reconstitute structural intact bile canaliculi sealed by tight junctions. Using filipin as a cytochemical marker for cholesterol-like membrane components in conjunction with the techniques of freeze fracture and thin sectioning, we have studied the distribution of cholesterol during the development of the biliary pole of cultured hepatocytes. It was found that the development of bile canaliculi is characterized in its very early stage by huge accumulations of filipin-cholesterol complexes located at distinct domains of the contiguous membrane. They were surrounded by junction formation zones almost devoid of these complexes, in which the alignment of intramembranous particles takes place. Maturation of the bile canaliculi was accompanied by dispersion of cholesterol within the canalicular membrane and its removal by segregation of cholesterol-rich membrane whorls and vesicles into the lumen. Finally, the luminal membranes, and particularly the areas studied with microvilli, contained only very few filipin-cholesterol complexes. In some cases, these seemed to be still arranged in small clusters. These alterations suggest a crucial role of cholesterol-rich membrane domains during initiation of a biliary polarity. On the other hand, cholesterol-poor (thus probably more fluid) areas might be required for the assembly of tight junctions, and appear to constitute the secretory active apical membrane present in the mature bile canaliculus.