Physiologic significance and regulation of hepatocellular heterogeneity

GASTROENTEROLOGY

REVIEW

1988;95:1130-43

ARTICLE

Physiologic Significance and Regulation of Hepatocellular Heterogeneity PETER G. TRABER, JOSE CHIANALE,

and JORGE J. GUMUCIO

Department of Internal Medicine, Division of Gastroenterology, Medical Center, University of Michigan, Ann Arbor, Michigan

S

ince the concept of hepatocellular heterogeneity was proposed a large body of evidence has accumulated that supports the notion that hepatocytes differ according to their location in the hepatic acinus. Several reviews (l-4) have summarized the numerous studies that have provided evidence that there is morphologic, biochemical, and functional (transport and metabolic) heterogeneity of hepatocytes. What is lacking, however, is a broad understanding of the physiologic meaning of this heterogeneity and the mechanisms by which it is established and maintained. Therefore, the objective of this review is twofold. First, several areas of liver function in which zonal differences in hepatocytes have been established will be analyzed to determine how the observed heterogeneity of hepatocytes may be important for the normal physiologic function of the liver. The areas to be discussed include the metabolism of glucose and ammonia, uptake of substances from sinusoidal blood, and bile secretion. Second, the mechanisms that may be responsible for the regulation of hepatocyte heterogeneity will be discussed. This analysis will draw mainly on data derived from experiments investigating the molecular mechanisms of the heterogeneous expression of within the hepatic the cytochrome Ph5” enzymes acinus. We have attempted to provide not an exhaustive review of the abundant descriptive data on hepatocellular heterogeneity, but rather a biological framework for understanding the functional significance of liver cell heterogeneity, while acknowledging that aspects of this proposal are speculative.

Metabolic Compartmentation Functional Unit of the Liver Functional

Unit

of Hepatic

Within the

Parenchyma

Hepatocytes within the liver are organized in tridimensional microvascular units or hepatic acini (Figure 1). The simple hepatic acinus represents the structural and functional unit of the liver paren-

Veterans

Administration

chyma (5,6); it is at the level of this microvascular unit that liver function is performed. Within the hepatic acinus, blood flows from the terminal portal venule and hepatic arteriole at the acinar core to the terminal hepatic venules at the periphery. The perfusion of hepatocytes in individual hepatic acini, therefore, proceeds in a sequential manner from those hepatocytes surrounding the terminal portal venules to those surrounding the terminal hepatic venules. Based on this microcirculatory pattern, the hepatic acinus has been arbitrarily divided into three hepatocytes located around zones: zone 1 represents the terminal portal venule and receives blood containing the largest solute load; zone 2 represents hepatocytes intermediate between those of zones 1 and 3; zone 3 represents hepatocytes surrounding the terminal hepatic venule. Solutes enter the hepatic acinus via the terminal portal venule and terminal hepatic arteriole, are sequentially distributed among hepatocytes, and exit this unit, in some instances as modified or manufactured material, via the terminal hepatic venules, bile, or lymph. This sequential perfusion may be crucial to the development of hepatocyte heterogeneity as well as to the distribution of certain liver functions within the hepatic acinus. Carbohydrate Heterogeneity Regulation

Metabolism: Hepatocyte Enables Short-Term

One of the metabolic pathways in which the influence of hepatocyte heterogeneity has been best studied is that of carbohydrate metabolism. Jungermann and Katz (73) have done extensive work on the subject. According to these authors, the crucial issue of the short-term regulation of carbohydrate Abbreviations used in this paper: BSP, bromosulphthalein; mRNA, messenger ribonucleic acid. 0 1988 by the American Gastroenterological Association 0016-5085188/$3.50

C)ctober19118

HEPATOCELLULAK

. ,

Figurr,

,

/

I. Hepatic acinus. Thrt acinar axis is formed by the terminal branches of the portal venule (‘I’PV). hepatic arteriole [HA], and bile ductule (BD). Blood enters the acinar sinusoids in zone 1 and flows sequentialI> through zone :! and into zone 3, where it exits via the terminal hepatic: venulc (HV).

metabolism in liver is the modulation of the metabolic shift from glycogen synthesis and glycolysis, occurring during the absorptive state, to glycogenolysis and gluconeogenesis, occurring during the postabsorptive state (8-10). Hepatocyte heterogeneity is one of four factors suggested to be involved in the regulation of this metabolic shift. The other three factors proposed are substrate concentration, hormone levels (particularly insulin and glucagon), and the activity of hepatic nerves (11-14). The hepatocytes in zone 1 contain a relatively greater amount of the enzymes necessary for glycogen degradation and gluconeogenesis, whereas the hepatocytes in zone 3 contain relatively more of the enzymes involved in glycogen synthesis and glycolysis. As shown in Figure 2. hepatocytes of zone 1 seem to be forming and secreting glucose, whereas those of zone 3 are predominantly glucose-removing hepatocytes. An analysis of the flux rate of the glucose/glucose-6 phosphate cycle using available enzyme kinetic data has been used to validate this model (7,10). This analysis was consistent with the concept that, in the shift from the absorptive to postabsorptive state, glucose formation by hepatocytes in zone 1 would increase, whereas the glucose utilization by hepatocytes in zone 3 would decrease. The net result would be a shift from glucose uptake to glucose production by the liver. In recent years it has been established that the majority of glycogen synthesis in the liver arises from three carbon compounds, such as lactate, rather than directly from ingested glucose (15). This phenomenon has been termed “the glucose paradox.” Although the source of this lactate is unknown, most of the lactate production is splanchnic-including

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1131

the liver, which may simultaneously produce lactate and glycogen from different hepatic zones (8,15). The zonation model of hepatic glucose metabolism provides a potential physiologic explanation for these findings. Glucose would be taken up by hepatocytes located in zone 3 and lactate released into the hepatic vein which, upon reentry into the portal circulation, would be converted to glycogen by zone 1 hepatocytes (8,15). Therefore, the substrate for glycogen synthesis in hepatocytes of zone 3 may be predominantly glucose (Figure 2). The zonal heterogeneity for carbohydrate metabolism is dynamic and changes with the nutritional status of the animal. For instance, during starvation, the glucogenic zone is enlarged, whereas the glycolytic zone appears to decrease concomitantly with an increase in gluconeogenic enzymes and a decrease in the content of glycolytic enzymes (3,~). Therefore, the compartmentation of the processes involved in carbohydrate metabolism is a good example of how acinar heterogeneity contributes to the regulation of the levels of glucose in the terminal hepatic venules. and consequently in the systemic: circulation.

Ammonia,

Urea,

and

Glutumine

Synthesis

The two major metabolic pathways for ammonia removal by the liver are the synthesis of urea and of glutamine (16). Although these two synthetic processes have been well defined, the acinar organization responsible for the synthesis of urea and glutamine has been clarified only recently by Haussinger and Gerok (17,18). A schematic: view of this metabolic organization is depicted in Figure 3. Arterial ammonia levels, -30 PM (191, are determined by the rate of ammonia production by various ZONE

1

ZONE 2

Glycogen

Degradation

Glycogen

Synthesis

Figure

2

from

to Glucose lactate

ZONE 3

Glycogen Glycogen

Synthesis

from Glucose

Degradation

to lactate

Zonal carbohydrate metabolism The glucose-forming processes of gluconeogenesis ant1 glycogen degradation to glucose seem to occur predominantly in hepatocytes located in zone 1 of the hepatic. acinus. On the other hand. the glucose-utilizing processes of glycolysis and glycogen synthesis from gluc:osc~ appear to occur predominantly in hepatocptes lo(:ated in zone :j of the hepatic acinus. In addition. lar.tate produced b\ hepatocytes located in zone 3 may bc> taken up a&l converted to glycogen by hrpatoc:ytes IIE zone I. This arrangement allows shifts in Larbohvdrate metabolism to occur without reversing the metabolic machinery ot individual hepatocytes.

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ET AL.

GAS’I’ROENTEROLOC;Y

HEPATIC ACINIJS ZONE I

ZONE 3

THY

TPV

UREA N&f GLU TAMINE

Figure

GLUTAMINE

3. Zonal metabolism of ammonia, urea, and glutamine. Urea and glutamine synthesis, two major pathways responsible for ammonia removal, are performed sequentially by hepatocytes of zones 1 and 3, respectively. Whereas urea synthesis seems to be distributed in all of the hepatocytes of zone 1 and in some of those in zone 2, glutamine synthetase is present only in a few hepatocytes surrounding the terminal hepatic venules (THV). Urea synthesis is a pathway with a high capacity and low affinity for ammonia, which is distributed in hepatocytes of zones I and 2. In contrast, glutamine synthesis is a process with a high affinity for ammonia and takes place in hepatocytes surrounding terminal hepatic venules. These hepatocytes receive a relatively lower concentration of ammonia than those more proximally located. On recirculation, glutamine is taken up by the action of by hepatocytes of zone 1 where, glutaminase, ammonia and glutamate are formed. This arrangement enables the acinus to regulate the concentrations of ammonia, urea, and glutamine. as well as the pH of sinusoidal blood at the acinar exit. This figure is a modification of an illustration by Haussinger and Gerok (17). TPV, terminal portal venules.

tissues as well as by the rate of ammonia removal. Although the small and large intestines and the kidneys are major contributors to ammonia producof the ammonia load tion, the liver removes -95% via urea synthesis (17). Glutamine synthesis, however, can become quantitatively important under cmditions of impaired urea synthesis (17). Haussinger (18) has proposed that glutamine synthesized and secreted by hepatocytes of zone 3 is taken up on recirculation by hepatocytes of zone 1. Thus, glutamate and ammonia are released by the action of glutaminases in zone 1 hepatocytes, and the ammonia can be used for urea synthesis; this recycling would avoid the accumulation of glutamine in the systemic circulation (18). Urea cycle enzymes have been shown to be predominantly localized in hepatocytes of zones 1 and 2 when examined by immunohistochemcial techniques (17). In contrast, Gebhardt and Mecke (20) have shown that glutamine synthetase is exclusively localized in a l-%cell-thick layer of hepatocytes surrounding the terminal hepatic venules. This is the only enzyme reported to date to be solely present in one of the acinar zones (20).The evidence for compartmentation of urea and glutamine synthesis

Vol. 95. No. 4

in the hepatic acinus is based not only on the localization of enzymes, but also on studies performed using the isolated perfused rat liver (18). When rat liver was perfused with ammonia under conditions in which ammonia availability for the synthesis of urea and glutamine was rate-limiting, it was observed that ammonia was preferentially used for the synthesis of urea when the perfusion was in the portal to hepatic vein direction (anterograde). Under these conditions, ammonia would have been taken up predominantly by the hepatocytes of zone 1. In contrast, during retrograde perfusion of the liver ammonia was utilized preferentially for glutamine synthesis. When the experimental conditions were changed so that the ammonia concentration was no longer rate-limiting, or so that one of the pathways was inhibited and there was no competition between the rates of urea and glutamine synthesis, the differences in the rates at which hepatocytes urea and glutamine of zones 1 and 3 synthesized were no longer apparent (17).These studies strongly suggest that hepatocytes of zone 1 are, under physiologic conditions, mainly involved in urea synthesis, whereas those of zone 3 contribute predominantly to glutamine synthesis. In addition, there is evidence that the liver releases ammonia from the of hepatocytes of zone 1 during the catabolism endogenous amino acids and proteins (18).This ammonia can be taken up by hepatocytes located downstream (those of zone 3) and utilized mainly in the synthesis of glutamine. Therefore, urea and glutamine synthesis can be considered as two sequential processes involved in ammonia detoxification. The proposal has been made that most of the incoming load of ammonia will be utilized in urea synthesis (low-affinity system) by hepatocytes in zone 1 (17). This would generate a profile of decreasing concentrations of ammonia in sinusoidal blood. Ammonia uptake by hepatocytes of zone 3 will be lower, and consequently the intracellular concentration of ammonia will also be lower. However, the interaction of ammonia with the glutamine synthetic pathway is still possible given the high affinity of this system for ammonia (17). Therefore, for the liver to accomplish the function of regulating the concentration of solutes in the terminal hepatic venules and in bile, it is important that there is zonal compartmentation of metabolic processes as well as interzonal interaction of hepatocytes performing these processes. In the case of ammonia metabolism, this interzonal interaction allows a fine adjustment of the concentrations of ammonia, urea, and glutamine in the terminal hepatic venules. Another interesting aspect studied by Haussinger et al. (21) is the possible role that the processes of

HEPATOCELLIJLAK

October 1~8

urea and glutamine synthesis play in regulating systemic pH. The role of the liver in the maintenance of systemic pH was the topic of a recent review (22) and has generated differing viewpoints (23). Hepatic urea synthesis provides a system for bicarbonate utilization as it is a very active process consuming stoichiometric amounts of HCO,and NH,+ (24). This may be crucial as a 100-g protein meal will yield -1 mol of HCO,,-, an amount that the kidney is unable to dispose of given the limited urinary volume (25). The conversion of bicarbonate and ammonia to urea by hepatocytes in zones 1 and 2 provides a mechanism for buffering the large load of base generated by protein catabolism. However, the conversion of glutamate to glutamine does not require bicarbonate and, thus, does not play a role in systemic buffering capacity. The shift of hepatic ammonia metabolism between urea and glutamine synthesis may help to correct systemic pH imbalances. For instance, during experimental acidosis there is a decreased rate of urea synthesis by hepatocytes of zone 1 accompanied by a concomitant increase in glutamine synthesis by hepatocytes of zone 3. This shift in metabolism will lead to a decrease in the rate of removal of base (bicarbonate) and, therefore, correct the systemic acidosis. In addition, the glutamine produced in the liver can be utilized by renal tubular cells to release ammonium ions into urine, which allows for excretion of the acid load. In systemic alkalosis, on the other hand, urea synthesis is increased to remove excess base (21). It has been proposed that during variations of systemic pH, the shift from ammonia removal via urea synthesis to net glutamine synthesis is accomplished by changes in the intercellular glutamine cycle (17,21). The relative metabolic contribution of the glutaminase pathway, located in zone 1. and of the glutamine synthetase pathway. located in zone 3, will be decreased by acidosis and alkalosis, respectively (17.26). Therefore, changes in systemic pH influence the relative rates of urea synthesis and the ac:tivity of glutaminase in zone 1, as well as the rate of glutamine synthesis in zone 3. Carbohydrate metabolic pathways and the removal of ammonia by the liver provide two examples of the compartmentation of functional processes within the acinus. The important point, however, is that there is coordination between processes predominantly performed in zones 1 or 3. This arrangement allows metabolic shifts to occur without reversing the metabolic machinery of individual hepatocytes. Moreover, this compartmentation is flexible, allowing zonal hepatocytes to vary their contribution to one or another process in response to microenvironmental changes.

HEX-XOGENEITY

Uptake From Sinusoidal Direct

Measurements

1133

Blood

of Zonal Uptake

Goresky (27) proposed that hepatocytes located in zone 1 should take up the greatest amount of substances from sinusoidal blood as the concentrations of incoming solutes should be highest at the acinar inlet. This uptake should result in a progressive decrease in the concentration of solutes in sinusoidal blood as the bolus traverses the acinus. Goresky then demonstrated that the hepatic uptake of [“Hlgalactose indeed followed the predicted pattern (28). Since then several others have studied the relative contribution of hepatocytes to solute uptake. Jones et al. (29) and St. Hilaire and Jones (30) used autoradiography to demonstrate decreasing cellular concentration within the acinus of ““I-cholylglycylhistamine (a bile acid analogue) and of epidermal growth factor, respectively. Similarly, perfusion of rat liver with fluorescent compounds of different characteristics such as fluorescein diacetate, fluorescein isothiocyanate, acridine orange, and rhodamine B (31) showed that profiles of decreasing cellular concentration developed between the hepatocytes of experiments. Morezones 1 and 3 during single-pass over, increasing the concentration of these substances resulted in the uptake of the compounds by hepatocytes more distally located (zones 2 and 3). Similar results were obtained in studies performed by Groothius et al. (32) using an endogenous bile acid, [“HIsodium taurocholate. The dependence of the uptake of substances on the direction of blood perfusion was demonstrated by experiments involving retrograde perfusion of the liver (perfusion from hepatic vein to portal vein). When either [“Hltaurocholate (32) or bromosulphthalein (BSP) (33) was perfused in a retrograde fashion, the result was the development of profiles of decreasing concentration in the direction of zones 3-1 with the same net uptake as obtained with anterograde perfusion.

Regulatory Role of Albumin: Reducing the Difference in Relative Concentration of Solutes in Hepatocytes When albumin is absent from the perfusate in an isolated liver preparation the liver avidly takes up organic anions such as BSP (34-36). The hepatocytes first exposed to this substance attain the highest relative concentration of BSP and a profile of decreasing cellular concentration is established in the direction in which the acinus is perfused. After the addition of albumin to the perfusate (36), the amount of BSP taken up is diminished and, consequently, the extracted fraction on a single pass is decreased. In addition, and most important to the regulation of

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TRABERETAL.

the distribution of solutes within hepatocytes, the relative concentration of BSP is similar in all hepatocytes. Thus, the cellular concentration gradients are abolished, BSP reaches every hepatocyte, and the concentration of BSP appearing in the terminal hepatic venules increases. Although the liver can efficiently extract bile acids and BSP from a perfusate without albumin, under physiologic conditions there are proteins in plasma to which these organic anions bind. Bromosulphthalein appears to bind to albumin (37), whereas bile acids bind to albumin and to high-density lipoproteins (38). Serum albumin provides a large number of high-affinity binding sites for bilirubin and, thus, plays a major role in the distribution of the pigment between plasma and the tissues; this protects the immature brain from the toxic effects of bilirubin and resultant kernicterus (39). The mechanism for the uptake of solutes predominantly bound to albumin is controversial. Forker and Luxon (40,41) and Weisiger and coworkers (42,43) were the first to propose that the rate of uptake of solutes such as bile acids, fatty acids, bilirubin, and BSP may depend on the concentration of the fraction bound to albumin and other plasma proteins rather than on the concentration of the free fraction as traditionally accepted. The hypothesized mechanism to explain this phenomenon was the presence of an albumin receptor on the hepatocyte sinusoidal membrane (43). However, one of the original proponents of this hypothesis has recently used mathematical modeling (44) and experiments investigating the uptake of fatty acids in the perfused rat liver (45) to argue that the uptake of solutes may be dependent on the free fraction of solute present at the hepatocyte membrane; thus, the rate of uptake would be dependent on the dissociation of ligand from albumin (45). Binding of solutes to plasma proteins, therefore, is one regulatory mechanism by which the concentrations of solutes reaching the systemic circulation are altered. In spite of these considerations, the full extent of the physiologic advantage of the binding of to albumin, which ultimately rethese substances by the liver, needs to be deduces their clearance fined.

Regulation of JntraceJJuJar Concentrations and Transport: Role of Intracellular Binding Proteins Perhaps the strongest case for the functional necessity of hepatocellular heterogeneity in the uptake process is found when one considers the role of intracellular binding proteins. Uptake at the cell membrane is only the initial event in a complex

GASTROENTEROLOGYVol.95,No. 4

process that will result in the processing and secretion of substances by the hepatic cell. After translocation at the sinusoidal membrane, a solute may interact with intracellular proteins and may be partially retained in the hepatocyte as a result of specific and nonspecific interactions. The specific interactions may help determine the eventual intracellular fate of the solutes and decrease the probability of solute movement back into the sinusoidal compartment. The concentration of proteins that act as intracellular binders in various hepatocytes may depend on the zonal location of the cell. Attempts have been made to assess the acinar distribution of some intracellular binding proteins. Redick et al. (46) showed that the glutathione S-transferases, which also act as intracellular binding proteins (47), were predominantly distributed in hepatocytes of zone 3. A similar acinar distribution for the activity of these enzymes was recently described in human liver by El Mouelhi and Kauffman (48).Other studies using either immunofluorescence (49) or immunoperoxidase (50) have shown that the binding of antibodies against glutathione transferase B or ligandin was uniformly distributed in all human hepatocytes. In contrast, the concentration of fatty acid-binding protein was found to be predominantly located in zones 1 and 2 of the acinus in male rats (51); however, this protein was uniformly distributed in the acini of female rats, suggesting that the relative intracellular concentrations of some binding proteins may be influenced by the hormonal milieu. It is conceivable that many of the solutes taken up by hepatocytes of zones 1 and 2 may reflux into the space of Disse. Although this reflux of solutes from zone 1 may be compensated by solute uptake into hepatocytes located in zones 2 and 3 (intracellular cycling), reflux of solutes from hepatocytes located at the end of the acinus, or zone 3, will enter the hepatic venule and, thus, reach the systemic circulation. Certain intracellular binding proteins may play a significant role in trapping transported solutes in hepatocytes. Intracellular binding proteins, such as the fatty acid-binding protein, may allow those hepatocytes in the acinus that are better suited to metabolize a particular substrate, such as fatty acids, to retain a higher concentration of that substrate. This trapping effect may be particularly important in preventing the reflux of substances from hepatocytes of zone 3, allowing a more selective regulation of what exits the acinus. This phenomenon of reflux from hepatocytes into the sinusoidal space has been well described for bilirubin (52).Wolkoff et al. (53) have shown that an increase in the concentration of ligand, an intracellular protein that binds bilirubin, increases the uptake of bilirubin from the sinusoidal

October

11188

blood by reducing the efflux of bilirubin from hepatocytes into the sinusoid. In studies using the isolated perfused liver, Baumgartner et al. (54) have demonstrated that taurodeoxycholate is retained longer and biotransformed to a greater extent in the hepatocytes of zone 3 than in those of zone 1. To this extent, it would be of interest to define the predominant zonal localization of the recently described cytosolic bile acid-binding protein (55). Thus, the heterogeneous distribution of intracellular binding proteins in the acinus may play an important role in the physiologic function of the liver and, by retaining solutes in hepatocytes that are best suited to process them, allow a more refined regulation of the concentration of solutes leaving the acinus via the terminal hepatic venule.

Bile Secretion Bile secretion is a major process by which solutes are removed from circulation or processed by hepatocytes, or both. and are excreted into the extracellular compartment of the intestinal lumen. Some modifications introduced by hepatocytes will render solutes incapable of being reabsorbed by the intestinal cells. Others, such as bile acids, will actively participate in the process of intestinal digestion and be reabsorbed. to a great extent, by the intestinal cells. Bile acids will be presented again to the hepatic acinus for uptake, completing the enterohepatic circulation of these substances. The site of uptake for taurocholate, a bile acid taken up via a secondary active transport mechanism, has been studied. The uptake of taurocholate measured in the perfused rat liver during anterograde and retrograde perfusion indicates that the distribution of carriers and Nat, K+-adenosine triphosphatase are similar throughout the sinusoidal membranes of the acinus (32). In addition, large doses of taurocholate are distributed similarly in all acinar hepatocytes (32). Therefore, the taurocholateuptake system appears to be equally distributed throughout the hepatocytes of the liver acinus. In animals without a gallbladder, in which the passage of bile into the intestinal lumen is continuous, it is expected that, under physiologic conditions, hepatocytes of zones 1 and 2 will contribute predominantly to the biliary secretion of taurocholate. IJnder conditions of an increased load of taurocholate. as may happen after the postprandial contractions of the gallbladder, it is conceivable that hepatocytes of zone 3 may also contribute to the biliary secretion of this bile acid. For those bile acids taken up by simple diffusion, the relative contribution of zonal bepatocytes to transport will depend not only on the direction of perfusion, but also, as

HEPA’TO(:ELLIJL.,~K

HE’l‘bXOC;ENEITY

1135

discussed, on the affinity of bile acids for plasma proteins. It is expected that most of these bile acids will be taken up in the proximal half of the acinus. It has been shown that the rate of biliary secretion of taurocholate and taurodeoxycholate is faster during anterograde than during retrograde infusions (32,54). As the rate of uptake of these bile acids is similar in all zones, and the rate of biliary secretion is slower in zone 3. it would appear that either the intracellular transit time, as described above, or the rate of secretion of taurocholate and taurodeoxycholate into the bile canaliculus is slower in hepatocytes of zone 3 (55). The secretion of bile acids into the bile canaliculus elicits the passage of water by what appears to be osmotic gradients (56,57). Bile formed in hepatocytes secondary to the secretion of bile salts has been called the “bile salt-dependent fraction.” From the analysis performed above. it would appear that the rat hepatocytes of zones 1 and 2 contribute predominantly to the secretion of this bile saltdependent fraction of canalicular bile. In this animal, hepatocytes of zone 3 also seem to (contribute to bile formation as damage to this zone by either bromobenzene or carbon tetrachloride is followed by a 300&400/o decrease in bile flow (58HiO). Therefore, under physiologic conditions in the rat, it is conceivable that hepatocytes of zone 3 contribute to bile formation predominantly through the secretion of the so-called bile salt-independent fraction of canalicular bile. This proposal (58) does not imply that bile secretion by this mechanism occurs only in the hepatocytes of zone 3. It only means that the main contribution of hepatocytes of zone 3 is to the secretion of this independent fraction rather than to

TPV

THV

ZI Figure 4. Hile formation

22

23

in the hepatic. ac:inus. It is proposed that predomiaantly to the hepatocytes of zone 1 mntributc: secretion of the bile salt-del)t,llclt,nt fraction of canalic:. ular bile (SSDF]. In contrast. hepatoc,ytes of 7.on6 3 most likely contribute to a lesser oxtr>nt to the sec:retion of the bile salt-dependent frac.tion of c.analicular bile. 1~Jnder Londitions in ivhich bile ac.itl load inczeases, i.e., after gallbladder contraction. hr~~atoc:ytns of zone 3 are probably involved to a greatrr clxtent in bile salt transport (recruitment phcinomctnc111). The c:ontribution of hepatocytes of zones 1 and 3 ttr the total secretion of the bile salt-independent frac:tion nt c:dnalic:ular bile (BSII;) is unknown. Ii is proposed. hr~wever. that hepatocytes of zone 3 actively c:ontributr to the formation of the bile salt-independent fraction ot c.Clnalic,ular bile. TPV, terminal portal venules: THV. terminal hepatic venulas.

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GASTROENTEROLOGY

bile salt transport. Although the mechanisms responsible for the secretion of the bile saltindependent fraction have not been fully elucidated, it is believed that bicarbonate secretion plays an important role (61).Figure 4 summarizes the proposal for a zonal compartmentation of the relative contribution of hepatocytes to bile formation. Whereas the hepatocytes of the acinus are perfused from zone 1 to zone 3, bile moves from zone exists that the bile 3 to zone 1. The possibility secreted by hepatocytes of zone 3, apparently rich in inorganic ions and poor in bile acids, may serve the purpose of providing fluid that would aid in the bulk movement of bile and dilute the solutes secreted by zones 1 and Z. More direct methods are needed to evaluate the hypothesis that hepatocytes of zones 1 and 3 make different contributions to the secretion of the bile salt-dependent and -independent fractions.

Physiologic Operational

Role of the Liver: An Definition of Liver Function

The liver is strategically located between the splanchnic and systemic circulation. Substances absorbed by the intestine and gastrointestinal hormones secreted by the intestine and pancreas, as well as solutes delivered via the hepatic artery, will be exposed to the liver for possible uptake and transformation (metabolic transformation and de novo synthesis). The fundamental role of the liver is to regulate the concentration of substances that will reach the brain, muscle, heart, lungs, and intestine, among other parts of the body, via the systemic circulation and bile. The liver performs this task by modulating the uptake-efflux of incoming substances as well as the rate of secretion of biotransformed or synthesized substances into sinusoidal blood and bile. The result of these processes is an adequate concentration of solutes in hepatic venules and in bile in response to the demands of the moment. The physiologic advantage of the organization of the liver into microcirculatory units comprising hepatocytes performing different functions (heterogeneity) and perfused unidirectionally becomes apparent once this main objective, the regulation of the concentration of substances in hepatic venules and in bile, is defined. Uptake, intracellular binding, metabolic processing, de novo synthesis, and secretion of substances into the sinusoidal blood, as well as into bile, represent the mechanisms by which the concentration of solutes is adjusted. Each of these processes is the subject of complex regulation involving changes in blood flow, afferent neural input, availability of substrates and cofactors, distribution of transport systems, and activity of rate-limiting enzymes, among other factors.

Vol. 95. No. 4

The unidirectional perfusion of the liver acinus represents an advantage for the sequential modification of concentrations in sinusoidal blood as blood traverses the acinus. This unidirectional perfusion in conjunction with the heterogeneity of function of hepatocytes results in a system with a great capacity for uptake, processing, and secretion of substances in hepatocytes of zones 1 and 2 and for the final adjustment of concentrations (via uptake-efflux, secretion) by hepatocytes of zone 3. This final adjustment of concentration in zone 3 may occur as a result of (a] differences in the availability of substrates; (b) a high affinity of hepatocytes of zone 3 for certain substances (i.e., intracellular binding proteins); or (c) the predominant expression in these hepatocytes of certain proteins (i.e., glutamine synthetase). Examples illustrating the function of this organization are the large capacity for uptake and biliary secretion of bile salts and other organic ions by hepatocytes of zones 1 and 2, and the specific properties of hepatocytes of zone 3 to handle ammonia, i.e., a higher affinity of these hepatocytes for ammonia and the selective synthesis of glutamine. Another advantage of the sequential perfusion of hepatocytes is that it allows intercellular cycling, i.e., the secretion of substances by hepatocytes located upstream and the uptake of these substances by hepatocytes located downstream. It is conceivable that cycling of substances between hepatocytes of zones 1 and 3 may play an important role in the modulation of processes occurring in zone 3 by hepatocytes located upstream; this point needs to be studied. It may be proposed that the cycling of substances represents a disadvantage for solutes that need to reach the systemic circulation. Although this may be the case, the degree of intercellular cycling may be regulated by the capacity for uptake of downstream hepatocytes of products secreted by upstream cells as well as by the predominant location of a physiologic process within the acinus. For instance, albumin, an important molecule that has to reach the systemic circulation, is not taken up by downstream hepatocytes, and the capacity for synthesis, as shown by experiments using in situ hybridization (621, is distributed in all hepatocytes. Conversely, glutamine seems to be synthesized and secreted exclusively by the last two hepatocytes surrounding the hepatic venules. A different example of the physiologic significance of hepatocyte heterogeneity is the metabolic zonation of the acinus for carbohydrate metabolism. In this case, the heterogeneity of function allows the performance of antagonistic processes (gluconeogenesis, glycolysis) in different segments of the acinus. The result still is the delivery of an amount of glucose in the hepatic

October 1988

HEPATOCELLIJLAK

venules that will result in an adequate concentration of this carbohydrate in the systemic circulation. We therefore propose that hepatocellular heterogeneity, rather than representing a biological curiosessential for the liver to ity. is an organization accomplish the regulation of the concentration of substances that are going to reach the systemic circulation and small intestine.

Regulation Influence

of Hepatocyte

sf Acinar

FUNCTIONAL

The regulation of hepatocyte heterogeneity in the adult liver seems to result, at least in part, from the unidirectional perfusion of the liver acinus. The importance of the microcirculation of the acinus is highlighted by the fact that heterogeneity is mainly established during the newborn period, when there is a transition from fetal to adult liver circulation (63,64). ‘I’he smooth endoplasmic reticulum (65,66) and numerous enzymes (67-69) equally distributed in the fetal liver develop a heterogeneous pattern in the newborn animal. In the adult animal there appear to be processes that are governed by both short-term regulation, without a change in the level of enzymes, and long-term regulation that require protein synthesis. Thurman and Kauffman (70) have summarized the evidence indicating that the direction of perfusion regulates the zonal compartmentation of the proc:esses of gluconeogenesis, glycolysis, and ketogenesis, as well as that of oxygen consumption. Changes in the direction of liver perfusion are rapidly followed by changes in the predominant contribution of hepatocytes of zones 1 and 3 to these processes. In contrast. other pathways. such as the predominant localization of monooxygenation and glucuronidation in zone 3, do not change with a switch from anterograde to retrograde perfusion. Figure 5 illustrates the data on the role of the direction of liver perfusion in the regulation of the zonal compartmentation of metabolic: exrents. as discussed by Thurman and Kauffman [7(l). Therefore, there seem to be processes that rapidly adapt to a change in the direction of blood tlow presumably secondary to changes in the availabilitv of substrates or cofactors. III contrast. other process& are either not influenced by changes in the direction of flow, or alternatively, due tc) the mechanisms involved in the adaptation [changes in gene transcription, in messenger ribonucleic acid (mRNA) translation, or in the half-life of the proteins involved], the modulating response is not apparent until hours after these changes in perfusion occur. Consequently, changes in the zonal location of monooxidation, described below, or glur:uronidation map have been missed during short

1137

COMPARTMENTATION

>02 UPTAKE GLUCONEOGENESIS

GLYCOLYSIS GLUCURONIDATION MONOOXYGENATION

THV

ZI

22

GLYCOLYSIS

23 .Oz UPTAKE GLUCONEOGENESIS GLUCURONIDATION MONOOXYGENATION

Heterogeneity

Microcirculation

HE:TEKCX;ENEITY

RETROGRADE PERFUSION THV

TPV

ZI

22

23

l*‘igure 5. Metabolic zonation and direc:tion ot liver perfusion. The predominant zonal loc:atiou of some metabolic. pathways depends on the direcAtion ot liver perfusion. This has been shown for gluconeogenesis. glycolysis, zand ketogenesis. Monooxygrnalion and glucuronidation were not apparently affected by the direction of liver perfusion in animals treated with phenobarbital [7(l). This figure has been motlitird from an illustration published by Thurman and Kaulim~~~~ [7(I). TPV, terminal portal venules: THV. trrmillciI hepatic: venules.

perfusions. We propose a working hypothesis that hepatocyte heterogeneity is established and later modulated mainly by the sequential perfusion of hepatocytes with portal and arterial blood, leading to a differential expression of metabolic processes in each zone. What factors determine the regulatory adaptation in response to changes in the direction of liver perfusion? It has been proposed that the zonal concentration of oxygen, as well as that of hormones and other cofactors (resulting from the unidirectional perfusion of the liver acinus), may be involved in the modulation of the metabolic compartmentation within zones (71). Several investigators (72-75) have attempted to measure the oxygen gradients that should develop in the liver acinus as the sequential removal of oxygen by hepatocytes proceeds. Although there is agreement that an oxygen gradient should exist, the slope of the concentration differences in the sinusoidal blood has not been defined. On the other hand. certain hormones have a high fractional clearance on first pass through the liver (76,77). and it has been proposed that there must be gradients of decreasing concentration of these hormones between hepatocytes of zone 1 and zone 3 (71). In addition, the availability of carbon substrates and liver innervation may also play a regulatory role in the development of hepatocyte heterogeneity (71). Further data are needed to define the role of each of these factors in the establishmnnt of hepatocyte

1138

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ET AL.

heterogeneity during liver development and in the modulation of heterogeneity in the adult liver. However, there may be factors other than sinusoidal gradients of solutes that modulate heterogeneity. This may be particularly important in regulating the expression of genes within different zones of the liver acinus. As previously mentioned, the enzyme glutamine synthetase is found only in those hepatocytes located in zone 3; moreover the histochemical localization of the enzyme appears to be limited to a l-Z-cell-thick area surrouding terminal hepatic venules (78). The developmental appearance of glutamine synthetase is also atypical in that the zonal location of this enzyme is evident several days before birth in the fetal rat liver, before the change in circulatory patterns occurs (79). It is possible that hepatocytes immediately in contact with the terminal hepatic venule and terminal portal venule may be regulated differently from other acinar hepatocytes. In this context, the interaction of hepatocytes with endothelial cells of the terminal hepatic venule may be an important factor modulating the expression of the glutamine synthetase gene within the liver acinus. Investigations in this area could lead to important insights into the intraacinar regulation of tissue-specific gene expression.

Cytochrome Intraacinar

PdfiO System: An Example of Regulation of Gene Expression

The hepatic mixed-function oxidase system is a major site for the metabolism of drugs. It is involved in the synthesis of hormones and prostaglandins as well as in the activation of substances to mutagenic, carcinogenic, and toxic compounds (80). Studies attempting to reconstruct the mixed-function oxidase system in vitro have established that the main components of this system are phospholipids, the enzyme nicotinamide adenine dinucleotide phosphate, reduced form (NADPH)-cytochrome PJsO reductase, and a series of hemoproteins known as cytochrome PdsO (81). There are many forms of indicates that cytochrome Pd5”, but present evidence there is one form of the NADPH-cytochrome Pas0 reductase (82). Taira et al. (83), using both an unlabeled antibody peroxidase-antiperoxidase method as well as an indirect fluorescence antibody technique, have observed that, in rat liver, the NADPH-cytochrome P4.50 reductase antibody was more capable of binding to hepatocytes of zones 2 and 3 than to hepatocytes of zone 1. In contrast to the reductase, the cytochrome P4so hemoproteins are members of a multigene family (84,85) or superfamily. Eight families of mammalian cytochrome PJsO genes, each with numerous members, have already been described on the basis of

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amino acid and nucleotide sequences as well as the response to chemical inducers (84). The presence of multiple cytochrome P 450 isoenzymes that are encoded by these genes is most likely responsible for the recognition and metabolism of the wide variety of endogenous and foreign substances with which this system interacts. The acinar localization of some of the forms of cytochrome P450 has been studied. Baron et al. (86,871, using an indirect fluorescence staining technique, showed that, in the livers of untreated rats, phenobarbital-inducible cytochrome P450 forms were predominantly located in hepatocytes of zone 3 with a profile of decreasing concentration from zone 3 to zone 1.Similarly, these authors showed that the distribution of the methylcholanthrene-inducible rats was also cytochrome P450 (PK,o MC) in control predominant in hepatocytes of zone 3 (87). These two isoenzymes appear to be present in small amounts (~5% of total cytochrome Paso) in the liver of uninduced rats (87). The localization of constitutive forms of cytochrome PQ5,,, the predominant forms in uninduced animals, has also been evaluated to some extent. Moody et al. (88) raised antibodies against cytochrome P450 isolated from the hepatic microsomes of untreated rats. The immunoglobulin G fraction precipitated 80% of the total microsomal cytochrome Pg5” and inhibited some P,,,-dependent enzyme activities. The distribution of the antibody, as assessed in frozen sections by direct immunofluorescence, was uniform. However, in paraffinembedded sections, the antibody was predominantly distributed in zones 2 and 3 (88); this disparity is unexplained. Other studies measuring total cytosuch as those of Gooding et al. chrome P450 activity, (89) using microspectrophotometry, have shown that in untreated rats there is a profile of increasing total cytochrome P450 activity from zone 1 to zone 3. These data suggest that the cytochrome P,,, enzymes are distributed throughout the acinus with a relatively higher concentration in the hepatocytes of zone 3. Although the physiologic advantage afforded by this arrangement is unclear at the present time, further information on the metabolism of hormones, prostaglandins, and other endogenous compounds may reveal the importance of this distribution of cytochrome P450. The study of the regulation of the expression of the genes that code for several of these hemoproteins has provided important information on the mechanisms responsible for the development of hepatocellular heterogeneity. We have studied the expression of cytochrome P,,,I, and P45,1Bgenes, the major phenobarbital-inducible forms, in the rat liver acinus. Studies by Harwick et al. (go), using nuclei isolated from whole liver, have demonstrated that the induc-

October

l!J88

tion of the cytochrome P450h and PJSOe genes in response to phenobarbital is mediated by an increase in the rate of transcription of the genes. However, the zonal differences in the amounts of PJSOb and P4joe protein within the hepatic acinus could still be regulated at either the level of transcription or translation. To investigate this question, it was first necessary to measure the levels of cytochrome P4501,and P 450e mRNA in the bepatocytes from different acinar zones. As an initial approach, hepatocytes from animals treated with phenobarbital were isolated from either the periportal or pericentral area using anterograde or retrograde perfusion of collagenase, respectively (91). The level of cytochrome PJSC,t,and P 4soo mRNA. measured using a complementary deoxyribonucleic acid probe that recognizes both mRNAs. was found to be higher in those hepatocytes isolated from the pericentral area of the acinus (92). The problems encountered in isolating hepatocytes from different zones required that these results be confirmed using an independent method. Hence, the localization of mRNA mjas determined in tissue sections using in situ hybridization. These studies revealed that, after a single injection of phenobarbital, cytochrome P4i,I,, and P450r mRNA increased in hepatocytes located in zones 2 and 3 of the liver acinus with no, or very little, increase in the 5-8 cells surrounding terminal portal venules (93). The distribution of mRNA induction was not dependent on the chemical inducer used, as a similar distribution M’as found after treatment with other inducers of P 4501,and P4.i,,?> such as polychlorinated biphenyls, organochlorine pesticides. and chlorpromazine (unpublished observations). The distribution of protein, determined by immunofluorescence using a monoclonal antibody, paralleled that of the mRNA with a delay in the attainment of the peak levels of -17 h (93). Hence. it appears that the heterogeneous expression of cytochromc P4jOb and Pa5,,, proteins results from the differential regulation of the transcription of the genes within the liver acinus. The molecular mechanisms and the factors, sinusoidal or intracellular. that modulate the differcntial transcription of cytochrome P45,,,, and P4Sof2 genes in the hepatic acinus are unknown. However, recent studies have indicated that inhibition of protein synthesis with cgcloheximide completely blocks the increased rate of transcription in isolated liver nuclei after treatment with phenobarbital as well as blocking the accumulation of Pas,,,, and P45,,,: mRNA (94). These experiments indicated that either actiI,e protein synthesis or the presence of a preexisting protein with a short half-life may be necessary for transcription of the P450kI and P45,,tS genes to increase in response to treatment with phenobarbital. The molecular mechanisms may involve the

HEPATOCELLlJl.AR

HE’I‘~ROGENEITY

1139

presence or induction of transacting activating proteins in the hepatocytes of zones 2 and 3, the presence or induction of repressors in zone 1, or both. Regardless, the induction of cytochrome P45,11, and P450p genes by phenobarbital represents a model for the elucidation of the role of differences in gene expression in the attainment of hepatocellular heterogeneity. A summary of the hypothesis on the regulation of cytochrome P4,,,1, and P450e gene expression within the hepatic acinus is depicted in Figure 6.

Summary

and Future Directions

Although morphologic, biochemical, and functional differences among hepatocytes of the liver acinus have been observed, the physiologic significance of hepatocyte heterogeneity has not been un-

ZONE

1

ZONE

INDUCTION OF mRNAs PROTEIN OCCURS IN HEPATOCYTES

AND SAME

2

ZONE

CYCLOHEXIMIDE BLOCKS INDUCTION

1 TRANSCRIPTIONAL

1 CONTROL

e

PROTEINS PERFORMING AS TRANS.ACTING FACTORS (?)

/” SINUSOIDAL MICROENVIRONMENT

Figure

3

z CELL TO CELL INTERACTIONS

6. Regulation of the expression of cytc~chrome P,->,,,, and P,?,,,, genes within the hepatir acinus. After treatment with phenobarbital, the cytochrome Pq5,,,, and P,.,,,,. apoproteins were induced predominantly in zones 2 and 3 of the hepatic acinus Studies using isolated hepatocytes from different hepatic: zones as well as in situ hybridization studies have demolkstrdted that the induction of cytochrome I’, ,,11, and P,?,,,, mRNA is distributed in the same hepatoqtes as the proteins. Pretreatment with the protein synthesis inhibitor cycloheximide prevents the increase in transcription of the cytochrome P,?,,,, and P.,,,,,. genes afler treatment with phenobarbital. This suggests that eithclr ac.tive protein synthesis or the presenc.e of ri prrexlsting protein with a short half-life may he net.essCrry for transcription 01 the I-‘,?,,,, and P,?,,,, genes to il)Lrease in response to treatment with phenobarbital. The molec.ular mech+ nisms may involve the presr:nr:e or induction of transaLting activating proteins in tile brp~~ttrcytes of zones 2 and 3. and the presence or induction of repressors in zonk 1. or both. The factors within the tonal hepatocytes or in the sinusoidal mi~.roenvironrn~~~~t that mt+ diate the differences in these fClctors art’ unknown. TPV. terminal portal venuln: TH\‘. tc-rminal hepatic: venult:: PB. phenobarbital.

1140

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ET AL.

derstood, partly because of the lack of an integrated view of liver function. We propose that many of the functions of the liver can be described as the capacity of the liver to regulate the concentration of solutes in terminal hepatic venules and in bile [and consequently in hepatic cells) and that hepatocyte heterogeneity represents a necessary feature to carry out these liver functions. Metabolic compartmentation, uptake, and biliary secretion are distributed in such a manner that transport and processing of large loads of substances are accomplished while fine adjustment of concentrations at the acinar exit is attained. Ultimately, hepatocyte heterogeneity provides the flexibility necessary for the hepatic units to perform complementary functional processes in a simultaneous manner. This functional flexibility allows adaptative responses of hepatocytes of the liver acini to various physiologic requirements. The past 30 yr have witnessed the description of the hepatic acinus (5,6), followed by numerous studies characterizing hepatocyte heterogeneity (l-4). Morphologic techniques involving light microscopy, electron microscopy, and immunohistochemical methods played major roles in this characterization. The notion that hepatocytes appeared different and that enzymes were distributed heterogeneously within hepatocytes of the liver acinus emanated from these studies. Attempts were made to assign functional significance to these observations. However, there are limitations to what the tissue distribution of a rate-limiting enzyme can tell us (2). The need to assess zonal function in a more direct manner became apparent and is the focus of several studies at present. The use of hepatocytes isolated from different acinar zones is appealing as conditions of the media can be clearly defined and it is possible to probe specific systems with a direct, simple experimental design. The disadvantages are the difficulties in clearly defining the origin in tissue of the isolated hepatocytes, the possible loss of heterogeneous characteristics by isolation, cell damage, and the inability to measure meaningful metabolic fluxes. Cells in culture have been used, and exposed to conditions presumably present in hepavaluable tocytes of zone 1 or zone 3 (8). Although information has been gained concerning factors that may modulate zonal metabolism using this system, this approach has several limitations including (a) rapid loss of some heterogeneous characteristics of the in vivo liver, (b) apparent changes in half-life of proteins and of mRNAs, and, most important, (c) decreased rate of transcription of some genes in hepatocytes in culture (95). The capability of assessing metabolic processes in the perfused liver has the advantage of probing the liver while maintaining cell-to-cell communication, interaction of cells with

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the biomatrix, and sinusoidal integrity. To this extent, the evaluation of metabolic fluxes using surface fluorescence represents an exciting approach to the assessment of the metabolic compartmentation of the acinus (96). However, there are limitations to the type of compounds that can be studied by this approach. In addition, there are difficulties in dissecting out mechanisms not only because of the complexities of the system but also because of the possibility that hepatocytes and circulatory patterns on the surface of the perfused liver may not be representative of the majority of the acini within the liver. Another potential approach to study the molecular mechanisms involved in the heterogeneous expression of genes in the liver may be the study of expression of the same genes in other tissues that demonstrate intratissue differences in gene expression. In this regard, we have recently demonstrated that the expression of cytochrome P4jOb mRNA in the small intestine is heterogeneous along the length of the small intestine as well as along the crypt-villus axis (97). Therefore, all systems that have been used have certain advantages and disadvantages. The type of approach to be used will have to be selected according to the question to be answered. The techniques of molecular biology will play an important role, especially in attempting to elucidate the molecular mechanisms responsible for hepatocyte heterogeneity.

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Keceived May 29, 1987. Accepted April 25. 1988. Address requests for reprints to: Jorge 1. Gumucio. M.D., Department of Internal Medicine (lllD), Veterans Administration Medical Center. 2215 Fuller Koad, Ann Arbor, Michigan 48105. This work was supported by grant AM32842 from the National Institutes of Health, a Veterans Administratioll Merit Review, a Veterans Administration Associate Investigatorship (P.G.T.]. and an American Liver Foundation Fellowship (I.(:.).