1.1
Introduction
The liver is the largest organ of the body. Its weight (1.5–1.8 kg) represents about 2% of the total body weight. The anatomical location is of course linked to its function. The liver function is comparable to that of the stomach, intestine, pancreas and kidney together. In fact all nutrients resulting from the di- gestion of the food are taken up by the intestine and then by the liver. Furthermore the liver is responsi- ble for the synthesis of the serum proteins and by this means for the oncotic pressure and for the re- tention of water within the vessels. The liver stores nutrients and the energy derived from the oxidation of the nutrients.
However, the liver is not only a power plant but also a cleaning device. In fact its direct relationship with the intestine is not without danger. Despite the reabsorption of water, the large intestine contains an enormous number of bacteria and a large quanti- ty of their products. The bacteria and their products can reach the venous blood and the liver sinusoid, where they are taken up and digested.
Although the liver is made up of several cell pop- ulations (Table 1.1), the most abundant cell type by mass and by number is the hepatocyte. The human liver is made of 80×10
9hepatocytes. To understand the functions of the hepatocytes it is useful to look at the laboratory findings of a young student who was admitted to the university clinic with jaundice and Hb
SAg positivity. His total bilirubin level was 20 mg/dl. His serum transaminase level was in- creased and thromboplastin time was strongly de- creased. During the next few days, his total protein concentration decreased below the lower normal level, the thromboplastin time did not increase, the transaminase level decreased but the serum bi- lirubin continued to be elevated. Liver volume de- creased continuously (as measured by echography), while the spleen size increased. The patient devel- oped a confusional state; the serum ammonia con- centration increased from normal to 190 mg/dl. The patient was considered for urgent transplantation.
Administration of albumin and fresh frozen plasma allowed normal blood pressure and kidney func- tion to be maintained. Although his serum ammo- nia concentration was lowered with lactulose, only successful liver transplantation led to the complete recovery of his cerebral edema and to the normali- zation of the pathologic serum parameters. The ex- planted liver weight was 649 g, which is about 50%
of the assumed normal weight for that patient.
The case is paradigmatic for the consequence of the lack of sufficient functional liver cell mass (Fig. 1.1).
1.2
Hepatocyte Development
The hepatoblast is considered to be the primitive hepatocyte precursor. However, it has not been es- tablished conclusively whether there is one type of
Hepatocytes
Giuliano Ramadori, Bernhard Saile
1
Table 1.1 Cell types resident in the liver.
Parenchymal cells Hepatocytes
Resident immune cells Kupffer cells NK-lymphocytes [51]
T-lymphocytes (e.g. NK1.1 Ag+ T cells) Specialized cells Sinusoidal endothelial
cells
Cholangiocytes Mesenchymal cells with
fibrogenic potential
Hepatic stellate cells Liver myofibroblasts Portal fibroblasts
hepatoblast or whether there is a hierarchy of line- age progression consisting of primitive hepatoblasts and stronger committed bipotential precursors.
The hepatoblast derives from the endodermal out- growth of the hepatic diverticulum. It is supposed to give rise to the hepatocyte and to parenchymal cells. In man, the progenitor cells are immunore- active for cytokeratins 8, 18, 19 and 14. Most of the progenitor cells develop to adult hepatocytes while losing cytokeratin 14 and 19; the latter is no longer detectable in human hepatoblasts at week 20 of ges- tation [27, 64]. Characteristic for the hepatoblast is the synthesis of alpha-fetoprotein (AFP), which be- gins in human liver as early as day 29. Although it is known that albumin gene expression starts later and increases in parallel with the decrease of AFP gene expression, which stops almost completely at birth, several reports claim that albumin gene expression begins as early as AFP expression [53, 54]. However, so far no study has been performed showing de novo synthesis of the protein.
During fetal life the liver cell plates are three to five cells thick. At birth the plates are two cells thick and one cell-thick plate of the adult human liver is reached at 5 years of age.
During fetal development hepatocytes exhibit considerable DNA synthesis and cell replication.
Two hours after birth DNA synthesis rates are el- evated, with 18% of the hepatocytes incorporating [
3H]thymidine into DNA. Three weeks later only 9%
of the hepatocytes show evidence of DNA synthesis.
This activity declines continuously after birth until at 6 weeks of age only a few hepatocytes (0.1%–0.4%) show evidence of DNA synthesis [6].
Within the first 3 weeks after birth, liver mass increases together with hepatocellular DNA synthe- sis, but no increase of mitosis in hepatocytes can be observed. This may mean that increased metabolic requirements induce a hypertrophy of the hepato- cytes characterized by DNA synthesis and enlarge- ment of the hepatocytes and consequently of liver growth. A similar phenomenon can be observed in the adult animal after a period of fasting [38, 49].
In the fetal liver, DNA synthesis is quite strong, but only mononuclear diploid cells are observed, with a third of the nuclei being in the S phase in the suckling phase; in young adult animals DNA syn- thesis strongly decreases and the number of diploid nuclei decreases to 50%, most being polyploid (44%
tetraploid) [17].
The hepatocyte polyploidy parallels increasing cell size and cytoplasmic complexity, which corre- spond to the adaptation of the cell to the increasing metabolic demand of the adult status. In early life, diurnal variability of the hepatic DNA synthesis can be observed. This phenomenon is linked to feeding;
in fact, reversal of food intake schedules alters the profiles of DNA synthesis. In lower animals, food intake induces polyploidy in most gut cells.
1.3 Structure
The hepatocyte is one of the largest cells of the body.
It has a size of 20–30 µm with a volume of 11,000 µm
3(estimations vary between 10,000 and 60,000 µm
3).
The size, however, can vary considerably depending on age, location and therefore on the blood flow and metabolic load.
The hepatocyte is an unbelievably complex sys- tem, which has to fulfil several complex functions at the same time. These different functions are ac- complished by means of very effectively function- ing structures and organelles. A hepatocyte can be compared with the picture of the “Potsdamer Platz” in Berlin at the peak time of reconstruction or with the “big dig” in Boston, where it is difficult to believe that all the people and the machines work well, to give rise to a well-organized end product.
Hepatocytes are long-lived cells with little turnover in the absence of cell loss. Some people believe that liver tissue is renewed entirely approximately once a year.
The hepatocyte is polyhedric and possesses 5–12 facettes. Of these, one to three are in contact with the sinusoidal blood, whereas four to nine are in contact with the biliary pole of the neighboring cell.
Fig. 1.1. CD31 staining of the liver of a patient with acute liver failure. Note the huge necrotic areas and only small regions of intact liver tissue as visible by intact sinusoids. Line: borders of the necrotic area. Arrows: CD31-positive macrophages in the necrotic area. Arrowheads: CD31-positive cells of the liver sinu- soid (sinusoidal endothelial cells, Kupffer cells)
1.4
Plasma membrane
The plasma membrane of the hepatocyte has a thick- ness of 7 µm. The hepatocyte is a polarized cell pos- sessing three different specialized membrane do- mains: (a) the basolateral or sinusoidal domain, (b) the canalicular domain and (c) the lateral domain.
1.4.1
Basolateral or Sinusoidal Domain
The basolateral or sinusoidal domain faces the si- nusoids and the perisinusoidal space of Disse. This domain is also called the vascular pole of the hepa- tocyte and constitutes 70% of total cell surface. It presents itself with 25–50 microvilli/µm, each meas- uring 0.5 µm in length and 0.1 µm in diameter. How- ever, they are not uniformly distributed, as there are clusters of thinner and longer microvilli on con- cavities existing on the basolateral domain that face toward concavities on the surface of the opposite hepatocyte, which also contains these long, slender microvilli. The microvilli pervade the space of Disse and protrude through the fenestrae of the sinusoidal endothelial cells into the sinusoids. For this reason they are thought to play a pivotal role in maintain- ing the integrity of the space of Disse [35]. However, endo- and exocytosis are the major functions of the basolateral domain. For this reason the basolateral domain shows indentations or pits. Whereas some of them represent exocytosis by secretory vesiculi, others represent so-called coated pits, which are in- volved in receptor-mediated endocytosis. The Na
+/ K
+-adenosine triphosphate (ATPase) ion pump, the Na
+/H
+-exchanger, as well as the Na
+:HCO
3-co- transporter are located at the basolateral domain, maintaining a substantial ion gradient and trans- membrane potential necessary for driving these transports across the cell membrane [19, 60].
1.4.2
Canalicular or Apical Domain
The canalicular or apical domain is also called the biliary pole of the hepatocyte. This domain consti- tutes 15% of the total hepatocyte surface and forms the bile canaliculus along with the canalicular do- main of the opposite hepatocyte. The bile canalicu- lus is a half tubule cut into the hepatocyte surface.
Laterally it is restricted by a smooth surface with junctional complexes. The diameter of the bile canal- iculi changes with site in the liver lobule. In acinar
zone 3 it is 0.5–1 µm and in acinar zone 1, 1–2.5 µm [35]. The canaliculus contains microvilli, which are more abundant at the edges of the half tubulus. In the cytoplasm around the canaliculus there is also a network of contractile microfilaments changing the caliber of the canaliculus, thereby regulating the bile flow. In the canalicular domain the apical bile acid transporter, organic ion transporters and P-glycoproteins are located, being responsible for the primary triphosphate (ATP)-dependent trans- port of organic components [8, 57].
1.4.3
Lateral Domain
The lateral domain of the hepatocyte ranges from the edge of the canalicular domain to the edge of the sinusoidal domain, representing about 15% of the total cell surface. The border between the lateral and the canalicular domains is represented by junc- tional complexes that include: (a) tight junctions, (b) gap junctions and (c) desmosomes.
1.4.4
Tight Junctions
The tight junctions represent the barrier between the canaliculus and the rest of the intercellular space. They are composed of belt-like zones made up of three to five parallel strands, whereby the cohesiveness of the tight junctions depends on the number of strands. The gap junctions are patches of close approximation of adjacent membranes, which are separated by a gap of 2–4 nm. The gap is bridged by transmembrane protein particles, which protrude from the membrane and contain a central pore. Two of those protrusions from opposite cells, both containing a central pore, serve as channels of intercellular communication [52]. In addition, the lateral surface also contains so-called “press-stud”
or “snap-fastener” types of intercellular junctions that consist of membrane protrusions that interact with membrane indentations on the opposite cell.
1.5 Organelles
As noted above, the hepatocyte is one of the most metabolically active cell types of the body. This sug- gests that it contains a large amount of organelles.
The most abundant are the endocytoplasmic re-
ticulum (ER), mitochondria, lysosomes and peroxi- somes (Fig. 1.2).
1.5.1
Endocytoplasmic Reticulum
The ER represents 15% of total cell volume; how- ever, its surface area is about 35-fold that of the cell membrane. The ER represents a complex system of membrane-bound channels. Two different types of ER can be distinguished according to their appear- ance on electron microscopy – the rough ER (RER), so called because of its association with ribosomes, which give it a rough appearance, and the smooth ER (SER), which consists of smooth membrane- bound channels and is less abundant than RER. The relative amount of the two types in the hepatocyte is not constant but varies with the location of the hepatocyte in the acinus and its physiologic state;
e.g. the surface area of the ER in zone 3 is twice that
in zone 1. The RER appears as parallel profiles of flattened cisternae distributed randomly in the cy- toplasm. It communicates on the one hand with the nuclear envelope, and on the other hand with the SER. The SER in turn communicates with the RER and the Golgi complex but not with the nuclear en- velope and consists of anastomosing and interlinked channels. It is noteworthy that neither the RER nor the SER communicates with the plasma mem- brane. The synthesis of proteins takes place in the polyribosomes that are attached to the RER. From here they finally reach the cisternae of the RER and are transported to the SER and finally to the Golgi complex. In the Golgi complex they are packed into the secretory Golgi vesicles. Many functions of the hepatocyte are accredited to the ER: synthesis of se- cretory proteins, synthesis of structural membrane proteins, metabolism of fatty acids, phospholipids and triglycerides, production and metabolism of cholesterol, xenobiotic metabolism, ascorbic acid synthesis and heme degradation. All these func-
Fig. 1.2. Hepatocyte metabolism. SER smooth endocytoplas- mic reticulum, RER rough endocytoplasmic reticulum, Nu nucle- us (the nuclei in the chart are small because of lack of space), GC Golgi complex, Po peroxisomes, VLDL very low-density lipopro- teins, Li lipid droplets, Lf lipofuscin. Note that all physiological functions of the hepatocyte are regulated by a fine network of transcriptional regulators and their receptors with hepatocyte
nuclear factors acting as master regulators. (Modified from Drenckhahn D, Fahimi D, Fleischhauer K. Leber und Gallenbla- se. In: Drenckhahn D, Zenker W, eds. Benninghoff. Anatomie.
Makroskopische Anatomie, Embryologie und Histologie des Menschen. Munich, Vienna, Baltimore: Urban und Schwarzen- berg, 1994:216)
tions are localized to specialized domains of the ER.
Structural proteins can also be synthesized by free ribosomes, especially during liver development and regeneration. Whereas the RER is mainly involved in protein synthesis, in the SER there are enzymes needed for drug metabolism, cholesterol biosynthe- sis and conversion of cholesterol to bile acids. For example, the well-known cytochrome P450 enzyme system is located in the ER. When this system is in- duced, this leads to hypertrophy of the SER, whose histological correlate is a “ground glass” appearance of the cytoplasm. Another very important enzyme, glucose-6-phosphatase, is also associated with the ER. During glycogenesis and glycogenolysis, the SER proliferates. As noted above there is a close connection between the RER and the mitochondria and these complexes are important for synthesis of membranes and heme. Heme itself is an important component of the cytochromes [24, 63].
1.5.2
Golgi Complex
Two to four per cent of the total cell volume is made up of the Golgi complex, which is located close to the bile canaliculus and the nucleus. It is made up of about 50 interconnected complexes, each being composed of three to five parallel cisternae with associated vesicles and lysosomes. The surface of the cisternae can be separated into the cis surface (convex surface) and the trans surface (concave sur- face). The cis surface faces toward the ER and the vesicles from the ER transport proteins from the ER to the cell surface. The trans surface is associated with vesicles containing osmophilic spheres corre- sponding to very low-density lipoproteins (VLDL), demonstrating that the Golgi complex of the hepa- tocytes is important for VLDL synthesis. In addition the Golgi complex is responsible for the terminal glycosylation of secretory protein and recycling of membrane glycoprotein receptors [9, 12, 59].
1.5.3
Mitochondria
The mitochondria make up 13%–20% of the total hepatocyte cytoplasmic volume. About 1,000 mito- chondria can be found in a hepatocyte. They are the power plant of the hepatocyte, generating the ener- gy required for the metabolic functions of the hepa- tocyte, e.g. fatty acid oxidation. The mitochondria are 1.5 µm in diameter and 4 µm in length. They can change their shape, fuse and move in the cytoplasm, in association with microtubules. Like the ER, there
are more mitochondria per cell in acinar zone 3 compared with acinar zone 1. They consist of an outer and an inner membrane. The outer membrane has no enzymatic activity but contains a transport protein called porin. Porin is able to form channels that are permeable to proteins of <2 kDa. The inner membrane is highly convoluted and folded to form so-called cristae that are closely associated with ma- trix granules. The inner membrane contains the en- zymes of the respiratory chain, responsible for oxi- dative phosphorylation and the generation of ATP.
The matrix of the mitochondria contains a small circular DNA, ribosomes and enzymes of the cit- ric acid cycle, urea cycle and beta oxidation of fatty acids. In addition calcium stores are found, which form electron-dense granules. Whereas the mito- chondrial DNA encodes for mitochondrial proteins synthesized by the mitochondrial ribosomes, most of the mitochondrial proteins are imported from the nucleus-derived transcription. Interestingly, the mitochondria are able to self-replicate and the half life is about 10 days [21, 48, 62, 68].
1.5.4
Primary Lysosomes
The primary lysosomes are enveloped by a single membrane. In their interior they contain enzymes such as acid hydolases, acid phosphatase, aryl sul- fatase, esterase and β-glucuronidase. By these means they serve as storage granules and seques- ter enzymes produced by the ER and packed by the Golgi. So-called secondary lysosomes are formed when primary lysosomes fuse with other mem- brane-bound vesicles containing endogenous cel- lular material or exogenous material destined for degradation. These are called autophagic vesicles.
The secondary lysosomes are mainly located near
the Golgi complex and the canalicular domain. In
electron microscopy they appear as so-called peri-
biliary dense bodies. The secondary lysosomes vary
in size and number and contain a variety of mate-
rials. During starvation, regeneration and develop-
ment they are particularly numerous. For example,
lysosomes accumulate lipofuscin, ferritin in iron
overload states, copper in cases of Wilson's disease
and glycogen during glycogenolysis. Coated endo-
cytic vesicles, the so-called endosomes, are derived
from receptor-mediated endocytosis (after internal-
ization of receptor–ligand complexes on the basola-
teral domain). Such ligands are insulin, low-density
lipoproteins, transferrin, immunoglobulins (IgA)
and asialoglycoproteins. The ligands themselves
may be processed before exocytosis, degraded (in
autophagic vesicles made by fusion with lysosomes)
or transported across the cell along microtubules.
The latter process is called transcytosis.
1.5.5 Peroxisomes
The peroxisomes are small, ovoid membrane-bound granula of 0.1–0.2 µm diameter, each hepatocyte containing 300–600. They contain oxidases and catalases that are responsible for 20% of the total oxygen consumption of the hepatocyte. The oxygen is used to oxidize numerous substrates and to pro- duce energy. The hydrogen peroxide formed during this process is hydrolyzed by the peroxisomal cata- lase. Ethanol is also metabolized in the liver by the peroxisomal catalase. The importance of the peroxi- somes becomes obvious by their proliferation dur- ing liver development and liver regeneration (e.g.
recovery from partial hepatectomy and various liver diseases) as well as after administration of salicylate and clofibrate [21, 36, 70].
1.6
Nucleus/Polyploidy
The onset of polyploidy in the liver has been known for quite a long time and has been studied widely in hepatocytes. While most hepatocytes (see above) contain a single nucleus, up to 40%–50% contain two or more nuclei. Multinucleated hepatocytes are thought to develop in response to completion of DNA synthesis and mitosis but failure of cell divi- sion by cytokinesis, which would normally generate daughter cells containing single nuclei. It has also been hypothesized that polyploid cells originate from the fusion of multinucleated cells, especially because studies have shown that the prevalence of multinucleated cells declines in response to simulta- neous induction of hepatic polyploidy. However, this has not yet been validated and other mechanisms resulting in loss of multinucleated cells have not yet been excluded, e.g. cells containing two nuclei could divide into two mononuclear cells or undergo apop- tosis [7, 37]. There is another concept regarding the fate of mammalian cells when mitotic progression is perturbed, e.g. in cases of disruption of the cen- tromere assembly. In this situation cells are able to continue to synthesize DNA and generate additional complements of chromosomes in the same nucleus with loss of cytokinesis ability [5].
During postnatal liver growth, hepatocytes un- dergo polyploidization. Analysis of DNA content in nuclei of hepatocytes demonstrated that DNA syn-
thesis was prominent in the fetal liver with almost one third of all nuclei being in the S phase. This de- creased soon after birth, with fewer than 5% being in the S phase. In young adults, DNA synthesis was even less and only 1% of nuclei were in the S phase.
In contrast, the liver of fetal and suckling animals contained only diploid cells, whereas the prevalence of diploid nuclei in young adults declined to 53%, with the remainder showing progression toward polyploidy. The ploidy class here is predominantly tetraploid (44%) [55]. This suggests that the post- natal liver growth is mainly due to hypertrophy, during which hepatocytes accumulate increased amounts of DNA, probably as an adaptive response to increasing metabolic requirements [65].
1.7 Physiology
The liver exerts many functions that comprise stor- age, metabolism, production and secretion. Besides the functions mentioned above, the processing of absorbed nutrients and xenobiotics, as well as bile acid synthesis and bile formation, the hepatocytes are also responsible for maintenance of glucose, amino acid, ammonia and bicarbonate homeostasis in the body, the synthesis of most plasma proteins, the storage and processing of signal molecules and, last but not least, they participate in the acute phase reaction. At this point it is mandatory to keep in mind that the well-organized physiological function of the hepatocyte largely depends not only on the proper function of hepatocyte nuclear factors and their receptors [40], but also on the proper function of the excretory apparatus.
1.7.1
Lipid/Lipoprotein, Cholesterol and Bile Metabolism
The liver plays a central role in lipoprotein metabo-
lism. The hepatocytes, like the parenchymal cells
of many other tissues, possess the low-density li-
poprotein (LDL) receptor, which is capable of tak-
ing up cholesterol. In addition, hepatocytes possess
another receptor that binds and internalizes apoE-
containing lipoproteins. Cholesterol is the basis for
bile acid synthesis or is directly shifted through the
cell and secreted into the bile. Whereas the forma-
tion of primary bile acids like cholic and chenode-
oxcholic acid takes place exclusively in the liver, the
formation of secondary bile acids (deoxycholic acid,
lithocholic acid) occurs by the metabolism by intes-
tinal bacteria in which the primary bile acids are conjugated with amino acids, sulfate or glucuronic acid. This leads to a decrease in toxicity and an in- crease in water solubility. The rate-controlling step in bile acid synthesis is the microsomal P
450enzyme cholesterol-7 α-hydroxylase [66]. This, as well as the hydroxymethylglutaryl-CoA reductase (HMG-CoA reductase; rate-limiting enzyme of hepatocyte cho- lesterol synthesis) underlie a feedback inhibition by bile acids at the levels of transcription and activity.
The bile acids undergo an enterohepatic circu- lation so that only small amounts of bile salts ex- creted by the liver are derived from a de novo syn- thesis. Therefore an efficient uptake mechanism is required, realized by transporters described earlier in this chapter. Little is known about the intracel- lular transport of bile acids from the sinusoidal to the canalicular region in the hepatocyte. Whereas diffusion may be involved, three cytosolic bile-acid- binding proteins, which also function as proteins binding 3- α-hydroxysteroid dehydrogenase, glu- tathione-S-transferase or fatty acids, have been de- scribed. Their role in bile acid transport is, however, controversial. Since electron microscopy detects vesicles that contain bile acids, there might also be a vesicular bile acid transport. Bile acids could enter these microsomal or Golgi-derived vesicles driven by the positive membrane potential. Although these findings support the concept of vesicular bile acid transport, it could also be that these vesicles trans- port the canalicular bile acid carrier to the canalicu- lar membrane domain and not free bile acids. Thus the vesicular and non-vesicular bile acid transport is still unclear. However, transcytotic transport proc- esses have been shown for protein excretion into the bile [4]. Hereby the newly synthesized membrane proteins, including those for the canalicular mem- brane, are initially targeted from the Golgi complex and the ER to the sinusoidal membrane. Here, those destined to the canalicular membrane are re-sort- ed, endocytosed and transported to the canalicu- lar membrane via transcytosis. Other proteins and polysaccharides can also be taken up by receptor- mediated or fluid-phase endocytosis and also reach the canalicular membrane via transcytosis. A ve- sicular transport and exocytosis are also thought to be responsible for the excretion of cholesterol and phospholipids into the bile [33]. The canalicular secretion of bile acids and other organic anions or cations generates an osmotic gradient that drives the osmotic water flow into the bile canaliculi. It is, however, noteworthy that for bile acid excretion, the rate-limiting point is not the basolateral uptake, but the capacity of canalicular excretion [20].
1.7.2
Glucose Metabolism
The liver plays a pivotal role in glucose metabo- lism of the organism, regulating the blood glucose level by removing glucose if it is present in excess by glycogen synthesis or glycolysis and lipogenesis or supplying glucose if it is needed by glycogenoly- sis or gluconeogenesis. The glucose uptake and re- lease across the hepatocellular plasma membrane is guaranteed by a bidirectional transporter, the Glut- 2 transporter. On the other hand it should be noted that the plasma membrane transport of glucose is not the major regulatory site of glucose metabolism.
Several factors are capable of controlling the revers- ible switch between glycogenolysis/gluconeogenesis and glycogen synthesis/glycolysis, such as substrate concentrations, hormone levels, hepatic nerves, the hepatocellular hydration and zonal hepatocyte het- erogeneity [22, 23]. Glycogen synthesis and glycoly- sis are predominantly regulated by the portal glu- cose concentration, with insulin and parasympathic nerves being auxiliary factors. Glycogenolysis and gluconeogenesis, on the other hand, are initiated by glucagon and sympathic nerves but inhibited by high portal glucose concentration. Three substrate cycles, the glucose/glucose-6-P cycle, the fructose- 6-P/fructose-1,6-bisphosphate cycle and the pyru- vate/phosphoenolpyruvate cycle are involved in regulating the hepatic gluconeogenesis/glycolysis.
The nature of these enzymes allows a rapid switch of flux through the pathways determining whether the liver becomes a producer or consumer of glucose [41].
1.7.3
Amino Acid and Ammonia Metabolism
The liver also participates in the amino acid home- ostasis of the body. Regarding this function, cel- lular hydration leading to hepatocellular swell- ing or shrinking seems to play an important role.
Such short-term modulation of cell volume within
a narrow range is known to serve as a potent signal,
which modifies cellular metabolism and gene ex-
pression. An increased portal amino acid load leads
to swelling of the hepatocytes and consecutively to
an increase of the amino acid flux across the plasma
membrane. This stimulates the amino acid break-
down and utilization for protein synthesis as well
as glycogen synthesis and, simultaneously, inhibits
amino acid generation from proteolysis. However, it
has also to be noted that hepatic amino acid extrac-
tion is not equipotent for all amino acids. Highest
amino acid extraction is observed for the glucone- ogenic amino acids alanine, serine and threonine.
Also most essential amino acids are extracted by the liver. On the other hand, branched amino acids such as leucine, valine and isoleucine, which are only used for protein synthesis, are not catabolized.
The portal blood contains high concentrations of ammonia, which is derived from the intestinal mucosa from glutamine and from intestinal micro- organisms. However, ammonia is also produced by the hepatocytes during the processing of amino ac- ids. The detoxification of ammonia occurs by both liver-specific urea synthesis and glutamine syn- thesis. Failure of hepatocellular function leads to increased serum levels of the neurotoxin ammonia and thereby to encephalopathy and neuropsychiat- ric disorder.
1.7.4
Protein Synthesis
Apart from immunoglobulins, the liver provides most circulating plasma proteins, which are syn- thesized by the hepatocytes, except von Willebrand factor, which is produced by endothelial cells [29], IGFBP3 [34] and probably C1q, which are synthe- sized by macrophages [3]. Among these serum pro- teins are albumin, factors of the coagulation system (Factor I [fibrinogen], Factor II [prothrombin], Fac- tor V, Factor VI, Factor IX, Factor X), as well as com- pounds of the complement cascade [44–46]. The major protein secreted by the hepatocytes is albu- min (50% of the secreted proteins!). The individual hepatocyte, however, is not specialized for produc- tion of a specific plasma protein. In fact, the single hepatocytes are able to synthesize the whole spec- trum of proteins in comparable amounts. However, for some plasma proteins a heterogeneity of produc- tion dependent on zonal location of the hepatocyte can be observed, e.g. for albumin in vivo. Albumin production by the hepatocytes is especially high:
12 g per day are produced in man and this amount could be increased up to fourfold upon albumin loss. Except for albumin and C-reactive protein, all proteins produced by the hepatocytes are glyco- proteins. On the other hand, specific receptors for galactose- or mannose-terminated glycoproteins al- low the hepatocytes to take part in the clearance of the glycoproteins [15].
The hepatocytes secrete proteins constitutively, meaning that they continuously produce and se- crete proteins and contain no stores for the proteins synthesized. Therefore, the rate of protein secretion depends on the protein synthesis. Examples of fast- secreted proteins are albumin (albumin is secreted
so fast that it is difficult to find it intracellularly), fibronectin or α
1-protease inhibitor; examples of slow-secreted proteins are fibrinogen or transferrin.
However, protein synthesis of the hepatocytes un- derlies a certain control by amino acid concentra- tion, cell swelling, growth and thyroid hormones, glucagons and vasopressin.
1.7.5
Acute Phase Response
The liver is the site of synthesis of acute phase pro- teins. They comprise a heterogeneous group of plasma proteins, the concentrations of which rap- idly change upon tissue injury at extrahepatic sites.
The synthesis of these proteins in the liver is medi- ated by cytokines (acute phase mediators) such as interleukin-6 (IL-6), interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF- α), which are secreted by inflammatory cells. Among these, IL-6 seems to be the most important mediator. Its secretion by mono- cytes, macrophages and endothelial cells underlies the control of IL-1, TNF- α or endotoxin and viruses.
IL-6 possesses a specific receptor on the hepatocyte surface leading to an increased transcription of genes encoding for acute phase proteins. However, it is also noteworthy that the presence of glucocorti- coids is necessary for the cytokine-induced synthe- sis of acute phase proteins. The increased synthesis of acute phase proteins leads to increased plasma levels that range from 1.5-fold (ceruloplasmin, com- plement C3) to up to several hundred-fold (C-reac- tive protein [CRP], serum amyloid A) and also their effects are large. Proteins such as α
1-antitrypsin and α
1-antichymotrypsin are protease inhibitors, thus restricting the protease activity of enzymes of inflammatory cells. Others are immunomodulators like α
1-acid glycoprotein or take part in the clearance of xenobiotic material and microorganisms (CRP, serum amyloid A) and in host defense (complement system) [32, 43, 45]. The level of some secreted and of some intracellular proteins of the hepatocytes can also be regulated by acute phase mediators. Exam- ples are the upregulated expression of IGFBP-1 and IGFBP-4 of hepatocytes by IL-6, the upregulation of hemoxygenase-1 (HO-1) expression by IL-1 or the inhibition of phosphoenolpyruvate carboxykinase (PCK), the gluconeogenic key enzyme, by IL-6.
1.7.6
Iron Metabolism
The hepatocyte is able to accumulate iron by several
routes. It expresses both classic transferrin receptors
(TfR1 and TfR2) and is thought to possess ferritin receptors. TfR1 regulation is highly iron dependent, but its role in liver iron uptake is unclear and this re- ceptor is expressed in only small amounts on hepa- tocytes. The TfR2, however, is highly liver specific.
It occurs in two forms. As it is missing the iron- responsive elements (IRS), the expression of the α form is not regulated by iron. The β form is widely expressed at low level and may be a secreted protein.
Upon binding, the Tf–TfR complex is internalized into endosomes. Here the Fe is then released from Tf by a reduction of endosomal pH. Once released, the Fe is transported through the endosomal membrane and into the cell via Nramp2 or the divalent metal transporter 1 (DMT1). From this point, Fe can be used for a variety of metabolic processes or stored within the protein ferritin.
However, excessive iron uptake, e.g. in all non- transfusion iron overload diseases, is due to the up- take of non-Tf-bound iron by the hepatocyte. Fol- lowing this, the deposition of iron in the liver oc- curs in any case in which the iron-binding capacity of Tf is exceeded. However, little is known about this transport system; e.g. it is unclear whether it recog- nizes Fe
2+and/or Fe
3+. On the other hand the non-Tf iron transport system seems to be always active and is not downregulated by iron as it is for TfR1 [1, 2].
1.7.7
Hepatocyte as an Endocrine Cell
The liver produces almost all circulating insulin- like growth factor-1 (IGF-1). Production by the hepatocyte of IGF-1 as well as IGF-1 binding protein, which is important for maintaining IGF-1 in the cir- culation, is controlled by cooperation of multiple cytokine pathways (e.g. the MAPK pathway) and cy- tokines including IL-6, insulin, TGF- α and TGF-β [34, 67]. However, although it has long been suggest- ed that circulating IGF-1 is also responsible for body growth, newer studies have clearly indicated that lo- cally produced IGF-1 is involved in growth but only to a much lesser extent than circulating IGF-1 [58].
On the other hand, lack of the IGF acid-labile subu- nit, which is also produced by the hepatocytes and regulated by growth hormone, may be responsible for growth impairment [10]. Furthermore, a recent publication suggests that the hepatocyte-derived IGF-1 has an important role in maintaining a fine balance between growth hormone and insulin to promote normal carbohydrate and lipid metabolism [69]. In addition, hepatocyte IGF-1 production, not alone, but in concert with hepatocyte growth factor (HGF), may be an important factor in progression of hepatocellular carcinoma (HCC) [42].
Fig. 1.3. Hepatobiliary transport systems. Note that the canalic- ular transporters are driven by ATP. OATP organic anion transport- ing polypeptide family, NTCP Na+-taurocholate co-transporting polypeptide, OCT organic cation transporter, OAT organic anion
transporter, BSEP bile salt excretion protein, MDR multidrug re- sistance glycoprotein, MRP multidrug resistance protein family, ABC ATP-binding cassette transporters, GSH glutathione. (Cour- tesy of D. Keppler, DKFZ, Heidelberg, Germany)
1.7.8
Transport Systems
The hepatocellular transport systems can be divided into basolateral and canalicular transport systems (for an overview, see Fig. 1.3).
The main basolateral transport systems com- prise the Na
+-dependent bile salt uptake, the Na
+- independent uptake of amphipathic substrates and the basolateral efflux pumps.
Na
+-Dependent Bile Salt Uptake
Studies on rat liver and hepatocytes as well as on ba- solateral plasma membrane vesicles indicated that more than 80% of taurocholate, but less than 50% of cholate uptake into hepatocytes is sodium-depend- ent. Whereas unconjugated bile salts are uncharged molecules that can transverse membranes by pas- sive non-ionic diffusion, conjugation with glycine or taurine requires the presence of a specific carrier protein for hepatocellular uptake. The main uptake system for conjugated bile salts in mammalian liv- er is called the Na
+-taurocholate co-transporting polypeptide (NTCP), which is exclusively expressed at the basolateral domain of the hepatocyte. The only non-bile salt substrates that are transported by NTCP are selected sulfated steroid conjugates like estrone-3-sulfate and dehydroepiandrosterone sulfate (DHEA). NTCP is structurally related to the intestinal bile salt transporter (IBAT), which also mediates Na
+-dependent uptake of bile salts, and is expressed not only in the ileum but also in the kid- ney and in cholangiocytes [31].
Na
+-Independent Hepatic Uptake of Amphipathic Substrates:
the Organic Anion Transporting Polypeptide Family
Whereas uptake of conjugated bile salts into the liver is largely a Na
+-dependent process mediated by the NTCP, numerous other endogenous or xenobiotic compounds, including non-bile salt organic anions and drugs, are cleared from the sinusoidal blood by carrier-mediated uptake into the hepatocyte. Fol- lowing hepatocellular uptake, many of these com- pounds are biotransformed in two phases. Phase I is mediated by cytochrome P450 enzymes and prepares the drug for conjunction by creating polar groups.
Phase II conjugates drugs with glucuronate, sulfate, glycine or methyl group, thereby representing a de- toxification step. These conjugates can then be ex- creted into the bile or the urine. The Na
+-independ- ent hepatocellular uptake of bile salts and non-bile
salt amphipathic compounds cannot be attributed to a single transport protein. In fact it is guaranteed by a family of transport proteins called the “organic anion transporting polypeptides” (OATP). So far three members of the OATP family have been iden- tified in rat hepatocytes, called OATP1, OATP2 and OATP4. OATP1 is localized at the basolateral mem- brane of hepatocytes and the apical membranes of kidney proximal tubular cells and the choroid plex- us epithelial cells. Several lines of evidence suggest that OATP1 is a “multispecific bile acid transport- er”. OATP1 has been shown to mediate hepatocellu- lar uptake of bile salts, bromosulfophathalin (BSP), conjugated steroids, thyroid hormones, leukotriene C
4, bilirubin monoglucuronide, ouabain, ochratox- in A, the anionic magnetic resonance imaging agent gadoxetate, the angiotensin-converting enzyme in- hibitors enalapril and temocaprilat, the HMG-CoA reductase inhibitor pravastatin and even oligopep- tides including the thrombin inhibitor CRC-220 and opioid receptor antagonists. The driving force for OATP-mediated substrate transport is not yet fully understood, although it has been shown that OATP1 can mediate bidirectional transport of BSP and an- ion exchange of taurocholate/HCO
3–. An important driving force for the OATP1-dependent organic an- ion uptake may be the counter transport of reduced glutathione.
OATP2 is located at the basolateral membrane of the hepatocytes and has also been detected in the retina, in endothelial cells of the blood–brain bar- rier and at the basolateral plasma membrane of the choroids plexus epithelial cells. OATP2 is a close homolog of OATP1 and transports bile salts, car- diac glycosides (e.g. digoxin) and cyclic peptides.
The most important difference between OATP1 and OATP2 is their location in the liver acinus. Whereas OATP1 is distributed homogenously within the liver acinus, OATP2 is predominately expressed in the perivenous hepatocytes excluding the innermost two cell layers around the central vein. Interesting- ly, drugs like phenobarbital or digoxin and thyroxin lead to a significant increase of OATP2 expression, even in the innermost layer of the central vein.
OATP4 can also mediate Na
+-independent uptake of bile salts. It transports numerous organic anions in- cluding taurocholate, BSP, conjugated steroids and thyroid hormones.
In human liver at least four OATPs have been
identified, called OATP-A, OATP-B, OATP-C and
OATP8. OATP-C and OATP8 are exclusively ex-
pressed on the basolateral domain of the hepatocyte
and are 80% homologous. The closest homolog in
the rat liver is OATP4, which is 64% and 66% homol-
ogous. Therefore the substrate specificity of the hu-
man OATP-C and OATP8 and that of the rat OATP4
are very similar. Transport substrates of OATP-C are taurocholate, bilirubin monoglucuronide, DHEAS, pravastatin and BSP. OATP8 has a similar substrate specificity, but additionally transports digoxin and cholecystokinin. OATP-B is strongly expressed in the human liver and additionally expressed in spleen, placenta, lung, kidney, heart, ovary, small intestine and brain. OATP-B has a limited substrate specificity for the organic anions BSP, estrone-3- sulfate and DHEAS when compared to OATP-C and OATP8. OATP-A is expressed in relatively low levels in human hepatocytes. Although it was originally isolated from human liver, it is predominantly ex- pressed in human cerebral endothelial cells. OATP- A transports bile salts, BSP, estrone-3-sulfate DH- EAS, opioid receptor antagonists, antihistamines and amphipathic organic cations. Thus, in contrast to the preference of OATP-B, OATP-C and OATP8 for organic anions, OATP-A additionally trans- ports amphipathic organic cations, indicating that they might mediate substrate uptake independently from their charge. Taken together, the OATP family of transporters plays a central role in organic anion and drug clearance of the hepatocyte [28, 31].
Na
+-Independent Hepatic Uptake
of Hydrophilic Organic Cations and Anions:
the Organic Ion Transporter Family
In addition to the NCTP and the OATPs, a third family, called the organic anion transporter family, mediates substrate uptake. This family comprises the organic anion transporter (OAT), the organic cation transporter (OCT) and the organic cation transporter novel type (OCTN)/carnitine trans- porter families. Whereas Oat1 is expressed only in rat kidney, Oat2 is expressed exclusively and Oat3 predominantly in rat liver. In human liver only Oat2 has been found so far. Oat2 mediates the sodium- independent transport of α-ketoglutarate and sali- cylates, whereas Oat3 transports para-amino-hip- purate (PAH), estrone-3-sulfate and the cationic compound cimetidine.
The organic cation transporter called OCT1 is expressed in rat kidney, small intestine enterocytes and the basolateral domain of the hepatocytes. In humans, hOCT1 is exclusively expressed in the liver and mediates the hepatic clearance of type I cations such as dopamine, choline, tetraethylammonium or N-methylnicotinamide [14, 16, 39].
Basolateral Efflux Pumps
The basolateral membrane of the hepatocyte also possesses members of the multidrug resistance pro- tein family (MRP) belonging to the superfamily of
ATP-binding cassette (ABC) transporters. MRP1 mediates ATP-dependent efflux of glutathione-S- conjugates, leukotriene C
4, steroid conjugates or bile salt conjugates. MRP1 is normally expressed at low levels in hepatocytes. MRP3 mediates the baso- lateral efflux of the organic anions estradiol-17- α-
D- glucuronide and S (2,4-dinitrophenyl) glutathione, the anti-cancer drugs methotrexate and etoposide and even monovalent bile salts. MRP5 is suggested to be an anion transporter; however, its expression in adult liver is very low. MRP6 is localized at the lateral membrane of the hepatocytes and transports the cyclic pentapeptide and endothelin antagonist BQ-123 [25, 26, 56].
The canalicular transport systems manage one of the major functions of the liver, the excretion of bile salt, non-bile salt organic anions and copper.
The importance of this function becomes obvi- ous from the view of the endoscopist who finds the green color of the bile to be predominant in many parts of the gut.
Bile Salt Excretion
The canalicular excretion is the rate-limiting step in the secretion of bile salts from blood into the bile.
Whereas bile salt concentrations in hepatocytes are in the micromolar range, canalicular bile salt con- centrations are 1,000-fold higher, which requires active transport across the canalicular hepatocyte membrane. The characterization of ATP-dependent taurocholate transport in canalicular membrane vesicles indicated the presence of a specific carrier system for monovalent bile salts. The main transport system that mediates excretion of monovalent bile salts is the so-called “bile salt export pump” (BSEP).
The sequence is more homologous with the multid- rug resistance (MDR) family than with the MRPs.
BSEP is expressed on the surface of canalicular mi- crovilli as indicated by electron microscopic studies.
Rat BSEP mediates the ATP-dependent transport of taurocholate, glycocholate, taurochenodeoxychola- te and tauroursodeoxcholate. The human BSEP gene locus has been identified as the positional candidate for progressive familial intrahepatic cholestasis type 2 (PFIC2), a progressive liver disease characterized by low biliary bile salt concentrations. In PFIC2, BSEP is absent from the canalicular membrane and biliary salt concentrations are less than 1% of nor- mal [47, 61] (see Chapter 34).
Excretion of Non-bile Salt Organic Anions
The excretion of non-bile salt organic anions into
the bile is mediated by the canalicular multidrug
resistance protein 2 (MRP2). Both rat and human
MRP2 are expressed predominantly in the liver with exclusive localization in the canalicular membrane.
The spectrum of organic anions is qualitatively sim- ilar to that of MRP1. It includes glutathione conju- gates, glucuronides, leukotriene C
4and divalent bile salts, but not monovalent bile salts. A role for MRP2 in the canalicular excretion of reduced glutathione (GSH), a major driving force for the maintenance of bile salt-independent bile flow, has also been dem- onstrated. In addition it is suggested that various structurally and functionally unrelated xenobiotics, e.g. probenecid, glibenclamide and rifampicin, in- hibit organic anion excretion [30, 56].
Phospholipid Excretion
The major lipid that is excreted together with choles- terol is phosphatidylcholine. The constant delivery of phosphatidylcholine from the inner to the outer hemileaflet of the canalicular membrane is mediat- ed by several ATP-dependent and ATP-independent phosphatidylcholine flippases. The ATP-dependent flippase is a class III MDR glycoprotein called MDR2 in mice or MDR3 in humans. Mouse MDR2 and hu- man MDR3 are highly expressed in the canalicular membrane of the hepatocytes [11, 13].
Copper Excretion
The liver is the central organ of copper homeosta- sis and has great capacity to store and excrete this metal. The degree of copper excretion is directly proportional to the size of the hepatic copper pool, suggesting that hepatocytes can sense the copper status in the cytoplasm and then regulate copper ex- cretion into the bile. The biliary excretion of heavy metals such as copper is an important detoxifying mechanism of the liver. Copper excretion is medi- ated by a copper-transporting P-type ATPase called ATP7B, predominantly expressed in the liver. It is localized to the trans-Golgi network where it medi- ates the incorporation of copper into cuproenzymes such as coeruloplasmin. An isoform of ATP7B is lo- cated in the mitochondria, which might be a reason for the abnormalities of the mitochondria morphol- ogy in Wilson’s disease. The ATP7B was also found in the so-called late endosomes. Copper incorpo- rated into late endosomes is probably transported to lysosomes and subsequently excreted into bile by a process known as biliary lysosomal excretion.
Copper is probably taken up into the hepatocytes via the copper transporters hCTR1 and hCTR2.
As the copper concentration of the hepatocyte in- creases, ATP7B redistributes from the trans-Golgi network to a cytoplasmic vesicular compartment and to pericanalicular vacuoles. After copper deple-
tion, ATP7B returns to the trans-Golgi network. By these means copper can induce redistribution of its own transporter from the trans-Golgi network to the apical membrane, where it may mediate biliary copper excretion. Copper-induced redistribution of ATP7B may provide a mechanism to preserve cop- per when it is scarce and to prevent copper toxicity when levels become too high [18, 50].
References
1. Anderson GJ. Ironing out disease: inherited disorders of iron homeostasis. IUBMB Life 2001;51(1):11–17.
2. Angelucci E, Brittenham GM, McLaren CE et al. Hepatic iron concentration and total body iron stores in thalassemia ma- jor. N Engl J Med 2000;343(5):327–331.
3. Armbrust T, Nordmann B, Kreissig M et al. C1Q synthesis by tissue mononuclear phagocytes from normal and from damaged rat liver: up-regulation by dexamethasone, down- regulation by interferon gamma, and lipopolysaccharide.
Hepatology 1997;26(1):98–106.
4. Bartles JR, Hubbard AL. Biogenesis of the rat hepatocyte plasma membrane. Methods Enzymol 1990;191:825–841.
5. Bernat RL, Borisy GG, Rothfield NF et al. Injection of anti- centromere antibodies in interphase disrupts events re- quired for chromosome movement at mitosis. J Cell Biol 1990;111(4):1519–1533.
6. Brodsky VY, Delone GV. Functional control of hepatocyte proliferation. Comparison with the temporal control of car- diomyocyte proliferation. Biomed Sci 1990;1(5):467–470.
7. Brodsky WY, Uryvaeva IV. Cell polyploidy: its relation to tis- sue growth and function. Int Rev Cytol 1977;50:275–332.
8. Crocenzi FA, Mottino AD, Roma MG. Regulation of synthe- sis and trafficking of canalicular transporters and its altera- tion in acquired hepatocellular cholestasis. Experimental therapeutic strategies for its prevention. Curr Med Chem 2004;11(4):501–524.
9. Dixon JL, Ginsberg HN. Hepatic synthesis of lipoproteins and apolipoproteins. Semin Liver Dis 1992;12(4):364–372.
10. Domene HM, Bengolea SV, Martinez AS et al. Deficiency of the circulating insulin-like growth factor system associated with inactivation of the acid-labile subunit gene. N Engl J Med 2004;350(6):570–577.
11. Elferink RP, Tytgat GN, Groen AK. Hepatic canalicular mem- brane 1: the role of mdr2 P-glycoprotein in hepatobiliary lipid transport. FASEB J 1997;11(1):19–28.
12. Erlinger S. Do intracellular organelles have any role in trans- port of bile acids by hepatocytes? J Hepatol 1996;24(Suppl 1):88–93.
13. Erlinger S. Mechanisms of hepatic transport and bile secre- tion. Acta Gastroenterol Belg 1996;59(2):159–162.
14. Gorboulev V, Ulzheimer JC, Akhoundova A et al. Cloning and characterization of two human polyspecific organic cation transporters. DNA Cell Biol 1997;16(7):871–881.
15. Gross V, Steube K, Tran-Thi T et al. The role of N-glycosylation for the plasma clearance of rat liver secretory glycoproteins.
Eur J Biochem 1987;162(1):83–88.
16. Grundemann D, Gorboulev V, Gambaryan S et al. Drug ex- cretion mediated by a new prototype of polyspecific trans- porter. Nature 1994;372(6506):549–552.
17. Gupta S. Hepatic polyploidy and liver growth control. Semin Cancer Biol 2000;10(3):161–171.
18. Harada M, Kawaguchi T, Kumemura H et al. Where is the site that ATP7B transports copper within hepatocytes? Gastro- enterology 2003;125(6):1911–1912.
19. Haussinger D. Liver fibrosis and NA(+)/H(+) exchange. Gas- troenterology 2001;120(2):572–575.
20. Haussinger D, Hallbrucker C, Saha N et al. Cell volume and bile acid excretion. Biochem J 1992;288(Pt 2):681–689.
21. Jones AL, Spring-Mills E, Felts JM. Ultrastructural concepts of drug metabolism. III. The hepatocyte: membranes, lipids, and alcohol. Am J Drug Alcohol Abuse 1974;1(3):421–444.
22. Jungermann K, Thurman RG. Hepatocyte heterogeneity in the metabolism of carbohydrates. Enzyme 1992;46(1–3):33–
58.
23. Kaiser S. Cell volume regulates liver phosphoenolpyruvate carboxykinase and fructose-1,6-bisphosphatase genes. Am J Physiol 1998;274(3 Pt 1):G509–G517.
24. Kanamura S, Kanai K, Watanabe J. Fine structure and func- tion of hepatocytes during development. J Electron Microsc Tech 1990;14(2):92–105.
25. Kauffmann HM, Keppler D, Kartenbeck J et al. Induction of cMrp/cMoat gene expression by cisplatin, 2-acetylaminoflu- orene, or cycloheximide in rat hepatocytes. Hepatology 1997;26(4):980–985.
26. Keppler D, Leier I, Jedlitschky G et al. The function of the multidrug resistance proteins (MRP and cMRP) in drug con- jugate transport and hepatobiliary excretion. Adv Enzyme Regul 1996;36:17–29.
27. Kiiasov AP, van Euken P, van Pelt J et al. [The expression of de- smin and cytokeratins No. 8 and 18 in the non-hematopoi- etic cells of the rat liver]. Ontogenez 1997;28(3):217–222.
28. Kim RB. Organic anion-transporting polypeptide (OATP) transporter family and drug disposition. Eur J Clin Invest 2003;33(Suppl 2):1–5.
29. Knittel T, Neubauer K, Armbrust T et al. Expression of von Willebrand factor in normal and diseased rat livers and in cultivated liver cells. Hepatology 1995;21(2):470–476.
30. Konig J, Nies AT, Cui Y et al. Conjugate export pumps of the multidrug resistance protein (MRP) family: localization, sub- strate specificity, and MRP2-mediated drug resistance. Bio- chim Biophys Acta 1999;1461(2):377–394.
31. Kullak-Ublick GA, Stieger B, Meier PJ. Enterohepatic bile salt transporters in normal physiology and liver disease. Gastro- enterology 2004;126(1):322–342.
32. Kushner I, Rzewnicki DL. The acute phase response: general aspects. Baillieres Clin Rheumatol 1994;8(3):513–530.
33. Lanzini A, Northfield TC. Biliary lipid secretion in man. Eur J Clin Invest 1991;21(3):259–272.
34. Lelbach A, Scharf JG, Ramadori G. Regulation of insulin-like growth factor-I and of insulin-like growth factor binding protein-1, -3 and –4 in cocultures of rat hepatocytes and Kupffer cells by interleukin-6. J Hepatol 2001;35(5):558–567.
35. Macchiarelli G, Motta PM. The three-dimensional micro- structure of the liver. A review by scanning electron micros- copy. Scan Electron Microsc 1986;(Pt 3):1019–1038.
36. Makita T. Molecular organization of hepatocyte peroxi- somes. Int Rev Cytol 1995;160:303–352.
37. Medvedev ZA. Age-related polyploidization of hepatocytes:
the cause and possible role. A mini-review. Exp Gerontol 1986;21(4–5):277–282.
38. Michalopoulos G. Control of hepatocyte proliferation in regeneration, augmentative hepatomegaly, and neoplasia.
Prog Clin Biol Res 1991;369:227–236.
39. Nagel G, Volk C, Friedrich T et al. A reevaluation of substrate specificity of the rat cation transporter rOCT1. J Biol Chem 1997;272(51):31953–31956.
40. Odom DT, Zizlsperger N, Gordon DB et al. Control of pan- creas and liver gene expression by HNF transcription factors.
Science 2004;303(5662):1378–1381.
41. Pilkis SJ, Granner DK. Molecular physiology of the regulation of hepatic gluconeogenesis and glycolysis. Annu Rev Physiol 1992;54:885–909.
42. Price JA, Kovach SJ, Johnson T et al. Insulin-like growth factor I is a comitogen for hepatocyte growth factor in a rat model of hepatocellular carcinoma. Hepatology 2002;36(5):1089–
1097.
43. Ramadori G, Christ B. Cytokines and the hepatic acute-phase response. Semin Liver Dis 1999;19(2):141–155.
44. Ramadori G, Rasokat H, Burger R et al. Quantitative deter- mination of complement components produced by purified hepatocytes. Clin Exp Immunol 1984;55(1):189–196.
45. Ramadori G, Sipe JD, Dinarello CA et al. Pretranslational modulation of acute phase hepatic protein synthesis by murine recombinant interleukin 1 (IL-1) and purified human IL-1. J Exp Med 1985;162(3):930–942.
46. Ramadori G, Tedesco F, Bitter-Suermann D et al. Biosynthe- sis of the third (C3), eighth (C8), and ninth (C9) complement components by guinea pig hepatocyte primary cultures. Im- munobiology 1985;170(3):203–210.
47. Redinger RN. The coming of age of our understanding of the enterohepatic circulation of bile salts. Am J Surg 2003;185(2):168–172.
48. Reuben A. Absolute mitochondrion. Hepatology 2003;37(5):1228–1231.
49. Rozga J. Hepatocyte proliferation in health and in liver fail- ure. Med Sci Monit 2002;8(2):RA32–RA38.
50. Schilsky ML, Irani AN, Gorla GR et al. Biliary copper excretion capacity in intact animals: correlation between ATP7B func- tion, hepatic mass, and biliary copper excretion. J Biochem Mol Toxicol 2000;14(4):210–214.
51. Seki S, Habu Y, Kawamura T et al. The liver as a crucial or- gan in the first line of host defense: the roles of Kupffer cells, natural killer (NK) cells and NK1.1 Ag+ T cells in T helper 1 immune responses. Immunol Rev 2000;174:35–46.
52. Seseke FG, Gardemann A, Jungermann K. Signal propaga- tion via gap junctions, a key step in the regulation of liver metabolism by the sympathetic hepatic nerves. FEBS Lett 1992;301(3):265–270.
53. Shiojiri N. Development and differentiation of bile ducts in the mammalian liver. Microsc Res Tech 1997;39(4):328–335.
54. Shiojiri N, Lemire JM, Fausto N. Cell lineages and oval cell progenitors in rat liver development. Cancer Res 1991;51(10):2611–2620.
55. Sigal SH, Gupta S, Gebhard DF, Jr. et al. Evidence for a ter- minal differentiation process in the rat liver. Differentiation 1995;59(1):35–42.
56. Silverman JA. Multidrug-resistance transporters. Pharm Bio- technol 1999;12:353–386.
57. Sippel CJ, McCollum MJ, Perlmutter DH. Bile acid transport by the rat liver canalicular bile acid transport/ecto-ATPase protein is dependent on ATP but not on its own ecto-ATPase activity. J Biol Chem 1994;269(4):2820–2826.
58. Sjogren K, Liu JL, Blad K et al. Liver-derived insulin-like growth factor I (IGF-I) is the principal source of IGF-I in blood but is not required for postnatal body growth in mice. Proc Natl Acad Sci USA 1999;96(12):7088–7092.
59. Strange RC. Hepatic bile flow. Physiol Rev 1984;64(4):1055–
1102.
60. Strazzabosco M, Boyer JL. Regulation of intracellular pH in the hepatocyte. Mechanisms and physiological implica- tions. J Hepatol 1996;24(5):631–644.
61. Thompson R, Strautnieks S. BSEP: function and role in pro- gressive familial intrahepatic cholestasis. Semin Liver Dis 2001;21(4):545–550.
62. Treem WR, Sokol RJ. Disorders of the mitochondria. Semin Liver Dis 1998;18(3):237–253.
63. van de WG, Lange A, Newgard C et al. New lessons in the regulation of glucose metabolism taught by the glucose 6- phosphatase system. Eur J Biochem 2000;267(6):1533–1549.
64. Van Eyken P, Sciot R, Callea F et al. The development of the intrahepatic bile ducts in man: a keratin-immunohisto- chemical study. Hepatology 1988;8(6):1586–1595.
65. Viola-Magni MP, Rossi R, Biondi R et al. Base composition changes in hepatocyte nuclei DNA of rats at different ages.
Biochim Biophys Acta 1978;520(1):38–51.
66. Vlahcevic ZR. Regulation of cholesterol 7 alpha-hydroxylase by different effectors. Ital J Gastroenterol 1996;28(6):337–
339.
67. Voci A, Arvigo M, Massajoli M et al. IGF-I production by adult rat hepatocytes is stimulated by transforming growth fac- tor-alpha and transforming growth factor-beta1. Eur J Endo- crinol 1999;140(6):577–582.
68. Watford M. The urea cycle: a two-compartment system. Es- says Biochem 1991;26:49–58.
69. Yakar S, Setser J, Zhao H et al. Inhibition of growth hormone action improves insulin sensitivity in liver IGF-1-deficient mice. J Clin Invest 2004;113(1):96–105.
70. Yokota S. Degradation of normal and proliferated peroxi- somes in rat hepatocytes: regulation of peroxisomes quan- tity in cells. Microsc Res Tech 2003;61(2):151–160.
