Zonal distribution of fatty acid synthase in liver parenchyma of male and female rats

1. A sensitive radiochemical assay was established to determine the activity of fatty acid synthase in microdissected liver tissue of less than 1 microgram dry mass. 2. In female rats, the enzyme activity in perivenous tissue was twice that in periportal liver tissue while it was homogeneously distributed in livers of male animals. The overall activity was higher in female than in male animals. 3. The absolute activity, as well as the perivenous/periportal ratio, was reduced during starvation and in diabetes. They were greatly increased after refeeding to values above those observed in animals during normal feeding. 4. Ovariectomy or administration of testosterone to female rats resulted in a significant reduction of the zonal heterogeneity. 5. Castration or administration of estradiol to male animals was followed by an increase in the enzyme activity exclusively in the perivenous tissue, resulting in a zonal heterogeneity as observed in female rats.     

Hepatocyte heterogeneity in the metabolism of carbohydrates.

Periportal and perivenous hepatocytes possess different amounts and activities of the rate-generating enzymes of carbohydrate and oxidative energy metabolism and thus different metabolic capacities. This is the basis of the model of metabolic zonation, according to which periportal cells catalyze predominantly the oxidative catabolism of fatty and amino acids as well as glucose release and glycogen formation via gluconeogenesis, and perivenous cells carry out preferentially glucose uptake for glycogen synthesis and glycolysis coupled to liponeogenesis. The input of humoral and nervous signals into the periportal and perivenous zones is different; gradients of oxygen, substrates and products, hormones and mediators and nerve densities exist which are important not only for the short-term regulation of carbohydrate metabolism but also for the long-term regulation of zonal gene expression. The specialization of periportal and perivenous hepatocytes in carbohydrate metabolism has been well characterized. In vivo evidence is provided by the complex metabolic situation termed the 'glucose paradox' and by zonal flux differences calculated on the basis of the distribution of enzymes and metabolites. In vitro evidence is given by the different flux rates determined with classical invasive techniques, e.g. in periportal-like and perivenous-like hepatocytes in cell culture, in periportal- and perivenous-enriched hepatocyte populations and in perfused livers during orthograde and retrograde flow, as well as with noninvasive techniques using miniature oxygen electrodes, e.g. in livers perfused in either direction. Differences of opinion in the interpretation of studies with invasive and noninvasive techniques by the authors are discussed. The declining gradient in oxygen concentrations, the decreasing glucagon/insulin ratio and the different innervation could be important factors in the zonal expression of the genes of carbohydrate-metabolizing enzymes. While it is clear that the hepatocytes sense the glucagon/insulin gradients via the respective hormone receptors, it is not known how they sense different oxygen tensions; the O2 sensor may be an oxygen-binding heme protein. The zonal separation of glucose release and uptake appears to be important for the liver to operate as a 'glucostat'. Thus, zonation of carbohydrate metabolism develops gradually during the first weeks of life, in part before and in part with weaning, when (in rat and mouse) the fat- and protein-rich but carbohydrate-poor nutrition via milk is replaced by carbohydrate-rich food. Similarly, zonation of carbohydrate metabolism adapts to longer lasting alterations in the need of a 'glucostat', such as starvation, diabetes, portocaval anastomoses or partial hepatectomy.     

Hepatocyte heterogeneity in bile formation and hepatobiliary transport of drugs.

In the past two decades many studies have been devoted to the involvement of the periportal (zone-1) and perivenous (zone-3) hepatocytes in bile formation and hepatobiliary transport of endogenous and exogenous compounds. It became clear that such a heterogeneity in transport function can, in principle, be due to the different localization of the cells in the acinus with respect to the incoming blood, to intrinsic differences between the cells or to both. In this review we first discuss the techniques used to study hepatocyte heterogeneity in hepatobiliary transport function. Combinations of such techniques can be used to discriminate between cellular heterogeneity due to acinar localization as opposed to intrinsic differences. These techniques include: normal and retrograde perfusions of isolated perfused livers; autoradiographic, fluorimetric and histochemical localization of injected substrates; separation of isolated hepatocytes into fractions enriched in periportal and perivenous cells; measurements of fluorescent surface signals with microlight guides; selective zonal toxicity, and pharmacokinetic modelling and analysis. Subsequently, for each of the rate-limiting steps in the hepatobiliary transport of organic compounds, the basic mechanisms are summarized and the available knowledge on the involvement of the cells from the various zones in these transport steps is discussed. The available literature data indicate that heterogeneity in transport function is often due to the localization of the cells in the acinus: the periportal cells are the first to come into contact with the portal blood and are thus exposed to the highest substrate concentration. Consequently they obtain the most prominent task in further disposition of the particular compound. It follows that the extent of involvement of the perivenous cells in drug disposition is implicitly determined by the activity of the periportal cells. Because of the potential saturation of elimination processes in the periportal cells, the involvement of perivenous cells may vary with the input concentration. In addition, real intrinsic differences have been established in the hepatobiliary transport of some substrates. These are probably based on differences in the cellular content of carrier- and receptor-binding and/or metabolizing proteins. In some cases these intrinsic differences may be secondary to existing sinusoidal gradients of endogenous compounds, such as O2, amino acids, bile acids or monosaccharides. Yet, data on the heterogeneity of hepatocytes in the various transport steps are far from complete or are even totally lacking, especially for human liver. A multi-experimental approach and advanced technology will be needed in the future to gain more insight into the acinar organization of bile formation and hepatobiliary transport of drugs in the human.     

Alteration in the capacities as well as in the zonal and cellular distributions of pyruvate kinase L and M2 in regenerating rat liver

Pyruvate kinase L (PKL), the glucoregulatory isoenzyme of adult parenchymal cells, and M2 (PKM2), the isoenzyme of proliferating and non-parenchymal cells, were measured, using a specific anti-PKL antibody for differentiation, in total liver homogenates, in isolated parenchymal and non-parenchymal cells as well as in microdissected periportal and perivenous liver tissue from regenerating rat liver after two-thirds partial hepatectomy. Moreover, the zonal distribution of PKL was studied using immunohistochemical techniques. In total liver homogenates PKL activity per g liver decreased after partial hepatectomy, while PKM2 increased. Total PKL activity per 100 g body weight was restored to preoperational levels much more slowly than liver weight. During liver regeneration parenchymal cells acquired high PKM2 besides PKL activity. The isoenzyme outfit of non-parenchymal cells remained unchanged. Microdissection studies showed that PKL lost its normal perivenous to periportal gradient after partial hepatectomy and became evenly distributed within the liver acinus. PKM2 did not retain its even distribution, it became predominant in the periportal zone. Immunohistochemical staining revealed that after partial hepatectomy PKL was present in all parenchymal cells in an atypical non-zonal heterogeneous distribution. Normal specific activities as well as zonal and cellular distributions of both pyruvate kinase isoenzymes were restored 14-21 d after partial hepatectomy. During regeneration after 2/3 partial hepatectomy the liver loses its glucostat function as corroborated in this study by the decrease of the glycolytic capacity via the glucoregulatory PKL; this change of function is accompanied by a loss of PKL-zonation. This finding corroborates the view that zonation of carbohydrate-metabolizing enzymes is required only when the liver functions as a glucostat. The increase of PKM2 and the appearance of a zonal PKM2 heterogeneity are in line with the pattern of hepatocyte proliferation after partial hepatectomy.     

Distribution of cyclic AMP phosphodiesterase in microdissected periportal and perivenous rat liver tissue with different dietary states.

Cyclic AMP phosphodiesterase was measured in liver homogenates and microdissected periportal and perivenous liver tissue from rats in different dietary states under different conditions of substrate saturation and effector stimulation. A radiochemical microtest, more sensitive by 2-3 orders of magnitude than the usual assay, was established for the determination of the activity in liver samples corresponding to 200-800 ng dry weight. At saturating cyclic AMP concentrations (46 microM) phosphodiesterase was homogeneously distributed within the liver acinus of fed rats. Starvation for 48 h led to a decrease in the overall activity and to a heterogenous distribution with slightly higher activities in the perivenous zone. At physiological cyclic AMP concentrations (1.8 microM) phosphodiesterase showed a flat zonal gradient in livers of fed rats with higher levels in the periportal zone; after 48 h starvation it was homogeneously distributed. In the presence of cyclic GMP (2 microM) the basal activity at physiological substrate concentrations was stimulated to a greater extent in the perivenous zone. This led to a homogeneous activity distribution in the fed state and to a heterogenous pattern with a slight perivenous maximum in the fasted state. Thus there was no or only a small zonal heterogeneity of signal transmitting enzymes such as cyclic AMP phosphodiesterase and glucagon-stimulated adenylate cyclase (Zierz and Jungermann 1984). This similar signal transducing capacity in the periportal and the perivenous area will contribute to maintain the zonation of signal input due to the hormone concentration gradients across the liver acinus.     

Functional heterogeneity of periportal and perivenous hepatocytes

Periportal and perivenous hepatocytes differ in their content of many key enzymes and subcellular structures. The cells also receive different regulatory signals due to the gradients established during liver passage of oxygen, substrates and hormones. The signal heterogeneity is important not only for short-term regulation of metabolism but also for long-term control, i.e. the induction of liver cell heterogeneity. The zonal heterogeneity changes upon longer lasting physiological and pathological alterations of the metabolic situation such as starvation, diabetes or regeneration after partial hepatectomy; it develops only gradually during the first weeks of postnatal life. The model of 'metabolic zonation' proposes a functional specialization for the two zones: in the periportal zone oxidative energy metabolism with beta-oxidation and amino acid metabolism, ureagenesis, gluconeogenesis, cholesterol synthesis, bile formation and oxidation protection are the predominant activities, and in the perivenous zone glycolysis, liponeogenesis, ketogenesis, glutamine formation and biotransformation are the prevalent processes.     

Zonation of the metabolic action of vasopressin in the bivascularly perfused rat liver

Predominance of the vasopressin binding capacity in the hepatic perivenous area leads to the hypothesis that the metabolic effects of the hormone should also be more pronounced in this area. Until now this question has been approached solely by experiments with isolated hepatocytes where an apparent absence of metabolic zonation was found. We have reexamined this question using the bivascularly perfused liver. In this system periportal cells can be reached in a selective manner with substrates and effectors via the hepatic artery when retrograde perfusion (hepatic vein --> portal vein) is done. The action of vasopressin (1-10 nM) on glycogenolysis, initial calcium efflux, glycolysis and oxygen uptake were measured. The results revealed that the action of vasopressin in the liver is heterogeneously distributed. Glycogenolysis stimulation and initial calcium efflux were predominant in the perivenous area, irrespective of the vasopressin concentration. Oxygen uptake was stimulated in the perivenous area; in the periportal area it ranged from inhibition at low vasopressin concentrations to stimulation at high ones. Lactate production was generally greater in the perivenous zone, whereas the opposite occurred with pyruvate production. Analysis of these and other results suggests that at least three factors are contributing to the heterogenic response of the liver parenchyma to vasopressin: a) receptor density, which tends to favour the perivenous zone; b) cell-to-cell interactions, which tend to favour situations where the perivenous zone is amply supplied with vasopressin; and c) the different response capacities of perivenous and periportal cells.     

Hepatocyte heterogeneity in ammonia metabolism: impairment of glutamine synthesis in CCl4 induced liver cell necrosis with no effect on urea synthesis

After induction of a perivenous liver cell necrosis by CCl4 pretreatment of the rat, ammonia uptake by perfused liver is decreased. This was due to an inhibition of glutamine synthesis from added ammonia, whereas urea synthesis was not affected by CCl4 pretreatment. The data confirm recent findings on hepatocyte heterogeneity in ammonia metabolism and are explained by an impairment of perivenous glutamine synthetase, but not of periportal urea synthesis, by the perivenous liver cell necrosis induced by CCl4. Regarding the pathogenesis of hyperammonemia in acute severe liver disease like CCl4 poisoning, the data point to a role of an impaired glutamine synthesis, but not to an impairment of urea synthesis.     

Distribution of pyruvate kinase type L and M2 in microdissected periportal and perivenous rat liver tissue with different dietary states

Pyruvate kinase type L and M2 activities were measured in microdissected periportal and perivenous liver tissue from rats in different dietary states. A specific antibody against pyruvate kinase type L was used to distinguish the two isoenzymes. Using separated cells it was found that the L-isoenzyme was essentially restricted to the parenchymal and the M2-isoenzyme to the non-parenchymal cells. Pyruvate kinase type L activity in the perivenous zone was about twice as high as in the periportal zone in both male and female fed rats. Starvation for 48 h led to a decrease of the overall activity and to a lower perivenous-periportal gradient. After refeeding for 48 h the overall activity and the gradient were increased to above the normal level. Pyruvate kinase type M2 was homogenously distributed within the liver acinus. After 48 h starvation no change in the overall activity nor in the zonal distribution was observed. Refed rats exhibited a slightly reduced overall activity. Since the hepatocytes contain the total regulatory L- but no M2-pyruvate kinase the heterogeneous distribution of the L-isoenzyme under different dietary states supports the model of metabolic zonation of liver parenchyma with glycolysis predominantly in the perivenous zone.     

Periportal zonation of the cytosolic acetyl-CoA synthetase of male rat liver.

Several important metabolic functions of the mammalian liver have been shown to be located in zones with respect to the complex microcirculation of the organ. The zonal distribution of the cytosolic component of the acetyl-CoA synthetase activity has been investigated using the dual-digitonin-pulse-perfusion technique, which allows highly zone-selective sampling of cytosol from the periportal and perivenous zone of rat liver. Approximately 80% of the cytosolic enzymes are eluted from the hepatocytes in the periportal and perivenous sub-zones affected by digitonin, while less than 1% of the glutamate dehydrogenase activity (a marker enzyme of the mitochondrial compartment) is eluted. A twofold higher activity of the cytosolic form of acetyl-CoA synthetase is found in the periportal zone compared to the perivenous zone in fed male rats. Following a fasting/refeeding transition, this activity gradient is abolished in a manner similar to that observed for the enzyme acetyl-CoA carboxylase. Since the latter enzyme is utilizing the product of acetyl-CoA synthetase, acetyl-CoA, the similarity in the observed regulation suggests a functional coupling between cytosolic acetate activation and fatty-acid synthesis.     

Hypophysectomy does not alter the acinar zonation of gluconeogenesis or the mitochondrial redox state in rat liver.

The biochemical and functional heterogeneity of hepatocytes in different zones of the liver acinus may be related to the concentrations of hormones within the liver acinus. We examined the effects of hypophysectomy, which causes marked changes in plasma hormone levels and in activities of hepatic enzymes that are normally heterogeneously distributed, on the degree of metabolic zonation within the liver acinus. In hypophysectomized rats the activity of alanine aminotransferase was increased, but its normal zonation (predominance in the periportal zone) was preserved. The activity in cultured periportal and perivenous hepatocytes was increased by dexamethasone, but not by glucagon. Periportal hepatocytes from hypophysectomized rats expressed higher rates of gluconeogenesis in culture than did perivenous hepatocytes, irrespective of the absence or presence of dexamethasone, glucagon or insulin. Similar differences in rates of ketogenesis and in the mitochondrial redox state in response to glucagon were observed between periportal and perivenous hepatocytes from hypophysectomized rats as between cell populations from normal rats. Although hypophysectomy causes marked changes in hepatic enzyme activities, it does not alter the degree of zonation of alanine aminotransferase, gluconeogenesis or the mitochondrial redox state within the liver acinus.     

Bivascular liver perfusion in the anterograde and retrograde modes: Zonation of the response to inhibitors of oxidative phosphorylation

The action of cyanide (500 μM ), 2,4-dinitrophenol (50 μM ) and atractyloside (100 μM ) on glycogen catabolism and oxygen uptake was investigated in the bivascularly perfused liver of fed rats. Cyanide, 2,4-dinitrophenol and atractyloside were infused at identical rates into the hepatic artery in either the anterograde or retrograde perfusion. The accessible aqueous cell spaces were determined by means of the multiple-indicator dilution technique. Glucose release, oxygen uptake and glycolysis were measured as metabolic parameters. Oxygen uptake changes per unit cell space caused by atractyloside (inhibition) and 2,4-dinitrophenol (stimulation) were equal in the retrograde perfusion (periportal cells) and the anterograde perfusion (space enriched in perivenous cells); the decreases caused by cyanide were higher in the retrograde perfusion. Glucose release from periportal cells was not increased upon inhibition of oxidative phosphorylation, a phenomenon which was independent of the mechanism of action of the inhibitor. There were nearly identical changes in glycolysis in the periportal and perivenous cells. It was concluded that: (1) oxygen concentration in the perfused rat liver, if maintained above 100 μM , had little influence on the zonation of the respiratory activity; (2) in spite of the lower activities of the key enzymes of glycolysis in the periportal hepatocytes, as assayed under standard conditions, these cells were as effective as the perivenous ones in generating ATP in the cytosol when oxidative phosphorylation was impaired; (3) the key enzymes of glycogenolysis and glycolysis in periportal and perivenous cells responded differently to changes in the energy charge.     

Distribution of cyclic AMP phosphodiesterase in microdissected periportal and perivenous rat liver tissue with different dietary states

Summary Cyclic AMP phosphodiesterase was measured in liver homogenates and microdissected periportal and perivenous liver tissue from rats in different dietary states under different conditions of substrate saturation and effector stimulation. A radiochemical microtest, more sensitive by 2–3 orders of magnitude than the usual assay, was established for the determination of the activity in liver samples corresponding to 200–800 ng dry weight. At saturating cyclic AMP concentrations (46 M) phosphodiesterase was homogeneously distributed within the liver acinus of fed rats. Starvation for 48 h led to a decrease in the overall activity and to a heterogenous distribution with slightly higher activities in the perivenous zone. At physiological cyclic AMP concentrations (1.8 M) phosphodiesterase showed a flat zonal gradient in livers of fed rats with higher levels in the periportal zone; after 48 h starvation it was homogeneously distributed. In the presence of cyclic GMP (2 M) the basal activity at physiological substrate concentrations was stimulated to a greater extent in the perivenous zone. This led to a homogeneous activity distribution in the fed state and to a heterogenous pattern with a slight perivenous maximum in the fasted state. Thus there was no or only a small zonal heterogeneity of signal transmitting enzymes such as cyclic AMP phosphodiesterase and glucagon-stimulated adenylate cyclase (Zierz and Jungermann 1984). This similar signal transducing capacity in the periportal and the perivenous area will contribute to maintain the zonation of signal input due to the hormone concentration gradients across the liver acinus.     

Alteration with dietary state of the activity and zonal distribution of adenylate cyclase stimulated by glucagon, fluoride and forskolin in microdissected rat liver tissue

Adenylate cyclase activated by glucagon, fluoride and forskolin was measured in liver homogenates and microdissected periportal and perivenous tissue of fed and fasted rats. A radiochemical microtest, more sensitive by 2-3 orders of magnitude as compared with the usual assay, was established for the determination of the activity in liver samples corresponding to 200-600 ng dry weight. In liver homogenates from fasted as compared to fed animals the glucagon-stimulated and fluoride-stimulated activity was increased by 1.65-fold, while the basal and the forskolin-stimulated activity remained the same. In microdissected tissue of both fed and fasted animals the activity was stimulated in about 60% of the samples by glucagon, fluoride and forskolin (responsive samples). However, in about 40% of the microdissected tissue samples the activity could not be stimulated by any of the above activators (non-responsive samples). In responsive microdissected tissue of fasted as compared to fed animals, the glucagon-stimulated and fluoride stimulated activity but not the basal and the forskolin-activated activity was increased by 2-3-fold. In responsive microdissected samples of fed animals neither the basal nor the stimulated activities showed a significant periportal to perivenous gradient. In samples of fasted animals, however, a zonal gradient was observed for the glucagon-stimulated activity exhibiting a 1.5-fold higher rate in the perivenous zone.     

Heterogenous zonal distribution of lysosomal enzymes in rat liver

The activities of beta-N-acetylglucosaminidase, beta-glucuronidase, alpha-L-iduronidase and acid phosphatase were all significantly higher in the cell lysates from the periportal than from the perivenous region obtained by the regioselective digitonin treatment of the perfused liver. The activities of cathepsins B, H and L were only slightly higher in the periportal than in the perivenous cell lysates. These results support the view that there is little zonation of lysosomal degradation of proteins, whereas the enzymatic capacity for degradation of glycosaminoglycans may be more active in the periportal region.     

Glycogen synthesis via the indirect gluconeogenic pathway in the periportal and via the direct glucose utilizing pathway in the perivenous zone of perfused rat liver

Summary The isolated liver from 24 h fasted rats was perfused in a non-recirculating manner in the ortho-and retrograde direction with erythrocyte-containing (20% v/v) media to provide adequate oxygenation of the liver. Glucose and/or gluconeogenic precursors were added as substrates. Glycogen formation was determined biochemically and demonstrated histochemically. With glucose as the sole exogenous substrate glycogen was deposited in the perivenous area, with gluconeogenic precursors it was formed in the periportal zone during ortho-and retrograde flow. When glucose and gluconeogenic compounds were offered togethen, glycogen was deposited in both zones. The results cortoborate the model of metabolic zonation predicting that periportal glycogen is synthesized indirectly from gluconeogenic precursors while perivenous glycogen is formed directly from glucose.     

Flexibility of the hepatic zonation of carbon and nitrogen fluxes linked to lactate and pyruvate transformations in the presence of ammonia

It has been proposed that key enzymes of ureagenesis and the alanine aminotransferase activity predominate in periportal hepatocytes. However, ureagenesis from alanine, when measured in the perfused liver, did not show periportal predominance and even the release of the direct products of alanine transformation, lactate and pyruvate, was higher in perivenous cells. An alternative way of analyzing the functional distributions of alanine aminotransferase and the urea cycle along the hepatic acini would be to measure alanine and urea production from precursors such as lactate or pyruvate plus ammonia. In the present work these aspects were investigated in the bivascularly perfused rat liver. The results of the present study confirm that gluconeogenesis and the associated oxygen uptake tend to predominate in the periportal region. Alanine synthesis from lactate and pyruvate plus ammonia, however, predominated in the perivenous region. Furthermore, no predominance of ureagenesis in the periportal region was found, except for conditions of high ammonia concentrations plus oxidizing conditions induced by pyruvate. These observations corroborate the view that data on enzyme activity or expression alone cannot be extrapolated unconditionally to the living cell. The current view of the hepatic ammonia-detoxifying system proposes that the small perivenous fraction of glutamine synthesizing perivenous cells removes a minor fraction of ammonia that escapes from ureagenesis in periportal cells. However, since urea synthesis occurs at high rates in all hepatocytes with the possible exclusion of those cells not possessing carbamoyl-phosphate synthase, it is probable that ureagenesis is equally important as an ammonia-detoxifying mechanism in the perivenous region.     

Zonation of hepatic lipogenic enzymes identified by dual-digitonin-pulse perfusion.

The zonal distribution within rat liver of acetyl-CoA carboxylase, ATP citrate-lyase and fatty acid synthase, the principal enzymes of fatty acid synthesis, was investigated by using dual-digitonin-pulse perfusion. Analysis of enzyme mass by immunoblotting revealed that, in normally feeding male rats, the periportal/perivenous ratio of acetyl-CoA carboxylase mass was 1.9. The periportal/perivenous ratio of ATP citrate-lyase mass was 1.4, and fatty acid synthase exhibited the largest periportal/perivenous mass gradient, having a ratio of 3.1. This pattern of enzyme distribution was observed in male rats only; in females, the periportal/perivenous ratio of enzyme mass was nearly equal. The periportal/perivenous gradients for acetyl-CoA carboxylase, ATP citrate-lyase and fatty acid synthase observed in fed (and fasted) males were abolished when animals were fasted (48 h) and refed (30 h) with a high-carbohydrate/low-fat diet. As determined by enzyme assay of eluates obtained from the livers of normally feeding male rats, there is also periportal zonation of acetyl-CoA carboxylase activity, expressed either as units per mg of eluted protein or units per mg of acetyl-CoA carboxylase protein, suggesting the existence of gradients in both enzyme mass and specific activity. From these results, we conclude that the enzymes of fatty acid synthesis are zonated periportally in the liver of the normally feeding male rat.     

Intrahepatic distribution of human glutathione-dependent formaldehyde dehydrogenase

By means of a microelectrophoretic separation technique the different forms of alcohol dehydrogenase can be detected in microdissected liver tissue samples of the nanogram range. Alcohol dehydrogenase 3 (the glutathione-dependent formaldehyde dehydrogenase) was demonstrated to be zonally distributed in the human liver parenchyma. A periportal/perivenous gradient is evident in both sexes, however, the periportal/perivenous ratio is higher in males. The biological functions of this enzymatic form and the possible role of the periportal maximum are discussed.     

Clofibrate induces carnitine acyltransferases in periportal and perivenous zones of rat liver and does not disturb the acinar zonation of gluconeogenesis

Clofibrate induces hypertrophy and hyperplasia and marked changes in the activities of various enzymes in rat liver. We examined the effects of treatment of rats with clofibrate on enzyme induction and on rates of metabolic flux in hepatocytes isolated from the periportal and perivenous zones of the liver. Clofibrate induced the activities of carnitine acetyltransferase (90-fold), carnitine palmitoyltransferase (3-fold) and NADP-linked malic enzyme (3-fold) to the same level in periportal as in perivenous hepatocytes, suggesting that these enzymes were induced uniformly throughout the liver acinus. Increased rates of palmitate metabolism and ketogenesis after clofibrate treatment were associated with: a more oxidised mitochondrial redox state; diminished responsiveness to glucagon and loss of periportal/perivenous zonation. Despite the marked liver enlargement and hyperplasia caused by clofibrate, the normal periportal/perivenous zonation of alanine aminotransferase and gluconeogenesis was preserved in livers of clofibrate-treated rats, indicating that clofibrate-induced hyperplasia does not disrupt the normal acinar zonation of these metabolic functions.