Zonation of the action of ethanol on gluconeogenesis and ketogenesis studied in the bivascularly perfused rat liver

Zonation of the actions of ethanol on gluconeogenesis and ketogenesis from lactate were investigated in the bivascularly perfused rat liver. Livers from fasted rats were perfused bivascularly in the antegrade and retrograde modes. Ethanol and lactate were infused into the hepatic artery (antegrade and retrograde) and portal vein. A previously described quantitative analysis that takes into account the microcirculatory characteristics of the rat liver was extended to the analysis of zone-specific effects of inhibitors. Confirming previous reports, gluconeogenesis and the corresponding oxygen uptake increment due to saturable lactate infusions were more pronounced in the periportal region. Arterially infused ethanol inhibited gluconeogenesis more strongly in the periportal region (inhibition constant = 3.99 ± 0.22 mM) when compared to downstream localized regions (inhibition constant = 8.64 ± 2.73 mM). The decrease in oxygen uptake caused by ethanol was also more pronounced in the periportal zone. Lactate decreased ketogenesis dependent on endogenous substrates in both regions, periportal and perivenous, but more strongly in the former. Ethanol further inhibited ketogenesis, but only in the periportal zone. Stimulation was found for the perivenous zone. The predominance of most ethanol effects in the periportal region of the liver is probably related to the fact that its transformation is also clearly predominant in this region, as demonstrated in a previous study. The differential effect on ketogenesis, on the other hand, suggest that the net effects of ethanol are the consequence of a summation of several partial effects with different intensities along the hepatic acini.     

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.     

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.     

Quantitative analysis of the pathways of glycogen repletion in periportal and perivenous hepatocytes in vivo.

In order to examine the pathways of hepatic glycogen repletion in the periportal and perivenous zones of the liver, [1-13C]glucose (99% enriched) was infused intraduodenally into conscious, 24-h fasted rats for 3 h. The liver was then quickly perfused in situ, and the cytoplasmic contents of the periportal and perivenous hepatocytes were selectively sampled by modification of the dual-digitonin-pulse technique (Quistorff, B., and Grunnet, N. (1987) Biochem. J. 243, 87-95). The 13C isotopic enrichment at each carbon position of the glucosyl units of hepatic glycogen was determined by 13C NMR and that of the C-1 position by gas chromatography-mass spectroscopy. From comparison of hepatic glycogen repleted by direct incorporation of plasma glucose (glucose----glucose-6-P----glucose-1-P----UDP-glucose----glycogen) was calculated to be 29% in the periportal zone and 35% in the perivenous zone, assuming equal glycogen synthetic rates within the two zones. Thus, the majority of glycogen is derived by an indirect route (glucose--------3-carbon unit--------glucose --------UDP-glucose--------glycogen) in both the periportal zone and in the perivenous zone. In conclusion, in a 24-h fasted rat there does not appear to be a major difference between the periportal and perivenous hepatocytes in the percent of glycogen synthesized by the direct pathway following a glucose load.     

The metabolism of fructose in the bivascularly perfused rat liver.

The metabolism of fructose was investigated in the bivascularly and hemoglobin-free perfused rat liver. Anterograde and retrograde perfusions were performed. In anterograde perfusion, fructose was infused at identical rates (19 mumols min-1 g-1) via the portal vein (all liver cells) or the hepatic artery (predominantly perivenous cells); in retrograde perfusion fructose was infused via the hepatic vein (all liver cells) or the hepatic artery (only periportal cells). The cellular water spaces accessible via the hepatic artery were measured by means of the multiple-indicator dilution technique. The following results were obtained. (i) Fructose was metabolized to glucose, lactate and pyruvate even when this substrate was infused via the hepatic artery in retrograde perfusion; oxygen consumption was also increased. (ii) When referred to the water spaces accessible to fructose via the hepatic artery in each perfusion mode, the rate of glycolysis was 0.99 +/- 0.14 mumols min-1 ml-1 in the retrograde mode; and, 2.05 +/- 0.19 mumols min-1 ml-1 in the anterograde mode (P = 0.002). (iii) The extra oxygen uptake due to fructose infusion via the hepatic artery was 1.09 +/- 0.16 mumols min-1 ml-1 in the retrograde mode; and, 0.51 +/- 0.08 mumols min-1 ml-1 in the anterograde mode (P = 0.005). (iv) Glucose production from fructose via the hepatic artery was 2.18 +/- 0.18 mumols min-1 ml-1 in the retrograde mode; and, 1.83 +/- 0.16 mumols min-1 ml-1 in the anterograde mode (P = 0.18). (v) Glucose production and extra oxygen uptake due to fructose infusion did not correlate by a single factor in all perfusion modes. It was concluded that: (a) rates of glycolysis are lower in the periportal area, confirming previous views; (b) extra oxygen uptake due to fructose infusion is higher in the periportal area; (c) a predominance of glucose production in the periportal area could not be demonstrated; and (d) extra oxygen uptake due to fructose infusion is not a precise indicator for glucose synthesis.     

Receptor-oriented intercellular calcium waves evoked by vasopressin in rat hepatocytes.

Agonist-induced intracellular calcium signals may propagate as intercellular Ca2+ waves in multicellular systems as well as in intact organs. The mechanisms initiating intercellular Ca2+ waves in one cell and determining their direction are unknown. We investigated these mechanisms directly on fura2-loaded multicellular systems of rat hepatocytes and on cell populations issued from peripheral (periportal) and central (perivenous) parts of the hepatic lobule. There was a gradient in vasopressin sensitivity along connected cells as demonstrated by low vasopressin concentration challenge. Interestingly, the intercellular sensitivity gradient was abolished either when D-myo-inositol 1,4, 5-trisphosphate (InsP3) receptor was directly stimulated after flash photolysis of caged InsP3 or when G proteins were directly stimulated with AlF4-. The gradient in vasopressin sensitivity in multiplets was correlated with a heterogeneity of vasopressin sensitivity in the hepatic lobule. There were more vasopressin-binding sites, vasopressin-induced InsP3 production and V1a vasopressin receptor mRNAs in perivenous than in periportal cells. Therefore, we propose that hormone receptor density determines the cellular sensitivity gradient from the peripheral to the central zones of the liver cell plate, thus the starting cell and the direction of intercellular Ca2+ waves, leading to directional activation of Ca2+-dependent processes.     

The zonation of liver and the distribution of fructose 2,6-bisphosphate in rat liver

Livers of starved rats refed for 2 h were perfused in situ by a modification of the dual digitonin pulse technique of Quistorff and Grunnet (Quistorff, B., and Grunnet, N. (1987) Biochem. J. 243, 87-95). A pulse of digitonin (2 mg/ml) was infused first antegrade through the portal vein followed retrograde through the vena cava, or in reverse order, 13 mg of digitonin per zone. Microscopic examination showed that this procedure permeabilized the periportal and perivenous zones of the liver without overlap, with a narrow unaffected band of hepatocytes between the zones. The distribution pattern between periportal and perivenous zones ratio for alanine transaminase, lactate hydrogenase, fructose-1,6-bisphosphatase, and phosphoenolpyruvate carboxykinase ranged from 1.5 to 3. Glucokinase activity was higher in the perivenous zone (periportal/perivenous ratio of 0.7) and glutamine synthetase was exclusively present in that zone. Fructose 2,6-bisphosphate concentration was nearly equal in the two zones.     

Hepatocyte heterogeneity in glutamate metabolism and bidirectional transport in perfused rat liver

1. The metabolic fate of infused [1-14C]glutamate was studied in perfused rat liver. The 14C label taken up by the liver was recovered to 85 +/- 2% as 14CO2 and [14C]glutamine. Whereas 14CO2 production accounted for about 70% of the [1-14C]glutamate taken up under conditions of low endogenous rates of glutamine synthesis, stepwise stimulation of glutamine synthesis by NH4Cl increased 14C incorporation into glutamine at the expense of 14CO2 production. Extrapolation to maximal rates of hepatic glutamine synthesis yielded an about 100% utilization of vascular glutamate taken up by the liver for glutamine synthesis. This was observed in both, antegrade and retrograde perfusions and suggests an almost exclusive uptake of glutamate into perivenous glutamine-synthetase-containing hepatocytes. 2. Glutamate was simultaneously taken up and released from perfused rat liver. At a near-physiological influent glutamate concentration (0.1 mM), the rates of unidirectional glutamate influx and efflux were similar (about 100 and 120 nmol g-1 min-1, respectively). 3. During infusion of [1-14C]oxoglutarate (50 microM), addition of glutamate (2 mM) did not affect hepatic uptake of [1-14C]oxoglutarate. However, it increased labeled glutamate release from the liver about 10-fold (from 9 +/- 2 to 86 +/- 20 nmol g-1 min-1; n = 4), whereas 14CO2 production from labeled oxoglutarate decreased by about 40%. This suggests not only different mechanisms of oxoglutarate and glutamate transport across the plasma membrane, but also points to a glutamate/glutamate exchange. 4. Oxoglutarate was recently shown to be taken up almost exclusively by perivenous glutamine-synthetase-containing hepatocytes [Stoll, B & H?ussinger, D. (1989) Eur. J. Biochem. 181, 709-716] and [1-14C]oxoglutarate (9 microM) was used to label selectively the intracellular glutamate pool in this perivenous cell population. The specific radioactivity of this intracellular (perivenous) glutamate pool was assessed by measuring the specific radioactivity of newly synthesized glutamine which is continuously released from these cells into the perfusate. Comparison of the specific radioactivities of glutamine and glutamate released from perivenous cells indicates that about 60% of total glutamate release from the liver is derived from the perivenous glutamine-synthetase-containing cell population. Following addition of unlabeled glutamate (0.1 mM), unidirectional glutamate efflux from perivenous cells increased from about 30 to 80 nmol g-1 min-1, whereas glutamate efflux from non-perivenous (presumably periportal) hepatocytes remained largely unaltered (i.e. 20-30 nmol g-1 min-1). 5. It is concluded that, in the intact liver, vascular glutamate is almost exclusively taken up by the small perivenous hepatocyte population containing glutamine synthetase.     

Postnatal differentiation of sex-specific distribution patterns of G6Pase, G6PDH and ME in the rat liver

The activity of the liver enzymes G6Pase, G6PDH and ME was studied in rats of 2-9 weeks old by histochemical means. In addition, G6PDH and ME activity was quantitatively determined in homogenates. In the 2nd and 3rd week G6Pase is similarly distributed in both sexes: while in the periportal zone high activity is demonstrable, the perivenous zone shows only low activity. After this period a nearly homogeneous distribution pattern becomes evident in all animals. Sex difference occurs after the 6th week: in the livers of male rats the periportal "maximum" is sometimes combined with a second peak in the perivenous area, in females a steep gradient emerges with high activity in the periportal zone and a low one in the perivenous zone. In the first postnatal weeks G6PDH activity is very low in parenchymal cells, but very prominent in Kupffer cells. Around the 5th week there is an increase, predominantly in the perivenous zone of both sexes. While there is again a further decrease demonstrable in male rats, the G6PDH activity of female rats rises to high adult values. This increase seems to be restricted to the perivenous zone. ME can be demonstrated at first in leucocytes. In the course of the 3rd week there is an increase of activity in both sexes: ME is demonstrable in parenchymal cells of the perivenous area and in scattered hepatocytes of the periportal area. In male rats, the perivenous activity is diminished towards the end of the investigation period, in females, however, a high activity remains in the perivenous zone. The data show that in females the activity of NADP dependent enzymes is high in the perivenous zone, so it may be assumed that a lipogenic area is situated around the terminal efferent vessels. Because of the sex difference this area may be hormone-dependent. The lipogenic area is situated opposite to the gluco(neo)genic area which corresponds to the periportal zone.     

Liver parenchyma heterogeneity in the response to extracellular NAD+

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

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.     

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.     

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.     

Hepatic heterogeneity in the response to ATP studied in the bivascularly perfused rat liver

The zonation of the purinergic action of ATP in the hepatic parenchyma was investigated in the bivascularly perfused rat liver by means of anterograde and retrograde perfusion. Livers from fed rats were used, and ATP was infused according to four different experimental protocols: (A) anterograde perfusion and ATP infusion via the portal vein; (B) anterograde perfusion and ATP via the hepatic artery; (C) retrograde perfusion and ATP via the hepatic vein; (D) retrograde perfusion and ATP via the hepatic artery. The following metabolic parameters were measured: glucose release, lactate production and oxygen consumption. The hemodynamic effects were evaluated by measuring the sinusoidal mean transit times by means of the indicator-dilution technique. ATP was infused during 20 min at four different rates (between 0.06-0.77 µmol min_-1 g liver_-1; 20-200 µM) in each of the four experimental protocols.The results that were obtained allow several conclusions with respect to the localization of the effects of ATP along the hepatic acini: (1) In retrograde perfusion the sinusoidal mean transit times were approximately twice those observed in anterograde perfusion. ATP increased the sinusoidal mean transit times only in retrograde perfusion (protocols C and D). The effect was more pronounced with protocol D. These results allow the conclusion that the responsive vasoconstrictive elements are localized in a pre-sinusoidal region; (2) All hepatic cells, periportal as well as perivenous, were able to metabolize ATP, so that concentration gradients were generated with all experimental protocols. Extraction of ATP was more pronounced in retrograde perfusion, an observation that can be attributed, partly at least, to the longer sinusoidal transit times. In anterograde perfusion, the extraction of ATP was time-dependent, a phenomenon that cannot be satisfactorily explained with the available data; (3) ATP produced a transient initial inhibition of oxygen uptake when protocols A and B were employed. These protocols are the only ones in which the cells situated shortly after the intrasinusoidal confluence of the portal vein and the hepatic artery were effectively supplied with ATP. The decrease in oxygen consumption was more pronounced at low ATP infusions when protocol B was employed. These observations allow the conclusion that the former phenomenon is localized mainly in cells situated shortly after the intrasinusoidal confluence of the portal vein and hepatic artery. Oxygen consumption in all other cells, especially the proximal periportal ones, is increased by ATP; (4) In agreement with previous data found in the literature, glycogenolysis stimulation by ATP was more pronounced in the periportal region. The cells that respond more intensively are not the proximal periportal ones, but those situated in the region of the intrasinusoidal confluence of the portal vein and the hepatic artery.     

Postnatal differentiation of sex-specific distribution patterns of G6Pase,G6PDH and ME in the rat liver

Summary The activity of the liver enzymes G6Pase, G6PDH and ME was studied in rats of 2–9 weeks old by histochemical means. In addition, G6PDH and ME activity was quantitatively determined in homogenates. In the 2nd and 3rd week G6Pase is similarly distributed in both sexes: while in the periportal zone high activity is demonstrable, the perivenous zone shows only low activity. After this period a nearly homogeneous distribution pattern becomes evident in all animals. Sex difference occurs after the 6th week: in the livers of male rats the periportal maximum is sometimes combined with a second peak in the perivenous area, in females a steep gradient emerges with high activity in the periportal zone and a low one in the perivenous zone. In the first postnatal weeks G6PDH activity is very low in parenchymal cells, but very prominent in Kupffer cells. Around the 5th week there is an increase, predominantly in the perivenous zone of both sexes. While there is again a further decrease demonstrable in male rats, the G6PDH activity of female rats rises to high adult values. This increase seems to be restricted to the perivenous zone. ME can be demonstrated at first in leucocytes. In the course of the 3rd week there is an increase of activity in both sexes: ME is demonstrable in parenchymal cells of the perivenous area and in scattered hepatocytes of the periportal area. In male rats, the perivenous activity is diminished towards the end of the investigation period, in females, however, a high activity remains in the perivenous zone. The data show that in females the activity of NADP dependent enzymes is high in the perivenous zone, so it may be assumed that a lipogenic area is situated around the terminal efferent vessels. Because of the sex difference this area may be hormone-dependent. The lipogenic area is situated opposite to the gluco(neo)genic area which corresponds to the periportal zone.Parts of this study were presented as an Inaugural Dissertation to the Medical Faculty of the University of Freiburg by H.H.Supported by grants from the Deutsche Forschungsgemeinschaft (Sa 127/7 and the SFB 46 (Molgrudent)     

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.     

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

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 together, glycogen was deposited in both zones. The results corroborate the model of metabolic zonation predicting that periportal glycogen is synthesized indirectly from gluconeogenic precursors while perivenous glycogen is formed directly from glucose.     

肝细胞的异质性：葡萄糖和酮体在大鼠肝脏的代谢

用大鼠肝脏门静脉或肝静脉周围的肝细胞来研究葡萄糖和酮体生成的区域分布。肝细胞通过毛地黄皂苷－胶原酶灌流技术分离。门静脉周围肝细胞的γ谷氨酰转肽酶的活性比肝静脉周围肝细胞高２．４倍;而谷氨酰胺合成酶的活性则相反,肝静脉周围肝细胞高出５６倍。门静脉周围肝细胞的内源性葡萄糖合成比肝静脉周围肝细胞高１．５７倍。给予刺激葡萄糖异生的底物,门静脉周围肝细胞的葡萄糖合成则增加１．７－２．１倍。肝静脉周围肝细胞的内源性酮体生成比门静脉周围肝细胞高１．３倍。给予能明显刺激酮体生成的辛酸盐,肝静脉周围肝细胞的酮体生成仅略为增加。我们的结果证实,在基础和刺激的条件下,葡萄糖的异生在门静脉周围肝细胞中优先,而酮体生成仅在肝静脉周围肝细胞占微弱的优势。     

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.     

Methods for the study of liver cell heterogeneity

A large number of histological, histochemical and biochemical techniques are available for studying liver cell heterogeneity. Structural differences are recognized by morphometric analyses of electron micrographs. The zonal heterogeneity of enzyme activities can be demonstrated by histochemistry and more precisely by ultramicrobiochemical assays in microdissected periportal and perivenous tissue. Immunohistochemistry is useful for quantifying and localizing proteins, especially isoenzymes, without depending on their biological activity. The zonal quantification of specific mRNA can be achieved by in situ hybridization. The different structural and enzymic equipment of periportal and perivenous tissue found by these techniques has led to the concept of metabolic zonation. This hypothesis can be confirmed by determination of metabolic rates in perfused liver after selective zonal damage, in separated periportal and perivenous hepatocytes as well as in periportal and perivenous tissue of perfused liver by non-invasive techniques.