Perivenous Zone Research Articles

Intravital microscopic investigation of xenogeneic microcirculation and impact of complement depletion by cobra venom factor.

Discordant xenografts are hyperacutely rejected within minutes. Disturbances in the microcirculation are considered to be the central mechanisms of hyperacute xenogeneic rejection (HXR). In this study intravital fluorescence microscopy was applied to investigate the dynamics of microcirculatory alterations in a setting in which HXR was inhibited by complement (C) depletion. Blood flow was measured as rat livers were perfused with isogeneic rat or xenogeneic human blood to assess the pattern of either physiological isogeneic hemoperfusion or in the course of HXR. Next, the complement system of the perfusate was inactivated by cobra venom factor (CVF) in order to inhibit HXR. Liver sinusoids of the isogeneic group were homogeneously perfused (sinusoidal perfusion rate 93.6+/-0.3%), whereas in the xenogeneic group the sinusoidal perfusion rate dropped to 67.1+/-3%. The perfusion in the periportal zone of an acinus was significantly lower ( 59.0+/-3.3%) than in the pericentral zone (76.2+/-3.1%). Treatment with CVF improved the sinusoidal perfusion to a value of 85.6+/-2.3%, physiological perfusion, however could not be reached. In contrast to the isogeneic group, massive white blood cell (WBC) and platelet accumulation was found in the xenogeneic group, especially in the terminal portal vessels and in the periportal zone of liver acini. WBC and platelet counts show that the adherence of these cells appears rapidly in the first 5 min after reperfusion as firm adherence. CVF was not able to inhibit WBC and platelet accumulation, indicating that WBC endothelial interactions do not require an intact complement system. Bile flow, a parameter of liver function, decreased only slightly during isogeneic perfusion. The addition of CVF to the rat blood reduced the bile flow to one half of the untreated isogeneic flow, indicating a hepatotoxic side-effect of CVF. In xenogeneic perfusion the bile flow dropped to 62.6% and with the addition of CVF to 37.5% in the first 15 min after reperfusion. The bile flow of the CVF treated groups recovered during the perfusion but could not reach isogeneic values.

Zonal location of compensatory hepatocyte proliferation following chemically induced hepatotoxicity in rats and humans.

Hepatocyte proliferation stimulated by partial hepatectomy occurs first in periportal cells, with midlobular and then perivenous cell division occurring later. We have previously shown that this pattern of compensatory cell proliferation also occurs following the hepatotoxicity of N-nitrosodimethylamine. We examined the generality of this pattern in livers of rats given a minimally toxic dose of an hepatotoxin and in liver biopsy samples from patients who had taken overdoses of acetaminophen. Proliferating hepatocytes were detected immunohistochemically (5'-bromodeoxyuridine or Ki-67 antigens). The perivenous necrogens N-nitrosodiethylamine, carbon tetrachloride (CCl4), bromobenzene, and acetaminophen all induced periportal hepatocyte proliferation. With CCl4, bromobenzene, and acetaminophen, the initial appearance of proliferating periportal hepatocytes was followed 12-24 hr later by division in the midlobular region, with a few cells dividing adjacent to the perivenous region of necrosis. The periportal necrogen allyl alcohol also induced periportal cell division. In the human studies, perivenous necrosis was accompanied by periportal and midlobular hepatocyte proliferation. These results suggest that regardless of its lobular location chemically induced hepatotoxicity stimulates cell proliferation that begins in the periportal zone and then moves in an orchestrated response into the midlobular and perivenous zones.

Zonal Induction of Mixed Lineage Kinase ZPK/DLK/MUK Gene Expression in Regenerating Mouse Liver

ZPK/DLK/MUK is a serine/threonine kinase believed to be involved in the regulation of cell growth and differentiation. To further explore the suggested participation of ZPK/DLK/MUK in this process, we examined the expression and cellular localization of ZPK/DLK/MUK mRNA in regenerating mouse liver following partial hepatectomy by ribonuclease protection assay andin situhybridization. The steady-state level of ZPK/DLK/MUK mRNA was very low in normal and sham-operated mouse livers, whereas a marked and transient increase was observed in the regenerating liver. While ZPK/DLK/MUK mRNAs were rarely detected in hepatocytes from all zones of the normal liver, hepatocytes of regenerating liver exhibit a gradient of expression ranging from low in the periportal zone, to intermediate in the mid-zone, to high in the pericentral zone. These findings demonstrate a transient stimulation of ZPK/DLK/MUK gene expression that correlates with the growth response of hepatocyte subpopulations in regenerating liver.

Differential expression of IGF-I and IGF-binding protein-1 and -2 in periportal and perivenous zones of rat liver.

IGF-I has important roles in regulating growth and metabolism. Circulating IGF-I is bound to specific binding proteins (IGFBP-1 to -6), with hepatocytes containing IGF-I, IGFBP-1 and -2 mRNA. Although many hepatic proteins are regionally expressed in the liver acinus, no studies have reported zonation of IGF protein expression. In this study we investigated the pattern of hepatic mRNA for the IGF proteins, vs the previously reported pepriportal gradient of phosphoenolpyruvate carboxykinase (PEPCK) expression. In situ hybridisation was used to analyse IGF-I, IGFBP-1, -2 and PEPCK mRNA in female Sprague-Dawley rats fed diets containing low (6%), normal (21%) or high (35%) protein. We report for the first time that IGFBP-1 and -2 and IGF-I are differentially expressed in the liver acinus. In the normal- and high-protein groups, levels of IGFBP-1 mRNA were higher in the perivenous region, i.e. the opposite gradient to PEPCK, with a higher gradient of IGFBP-1 expression in the high-protein group. In contrast, IGFBP-2 had a similar pattern to PEPCK, and a periportal gradient of IGF-I mRNA was also seen in the low-protein group. Using computerised image analysis, levels of IGFBP-1 and -2 mRNA were elevated 2- and 10-fold respectively, in the low- vs normal-protein groups. The level of IGF-I mRNA was reduced to 65% of normal, with circulating IGF-I levels at 30% and insulin levels 39% of normal. These results demonstrate that hepatocytes are a heterogeneous population with respect to regulation of IGF proteins, having specific expression patterns dependent on the position of the hepatocyte within the liver acinus.

In situ measurement of glutamate concentrations in the periportal, intermediate, and pericentral zones of rat liver.

We developed a quantitative histochemical assay for measurement of local glutamate concentrations in cryostat sections of rat liver. Deamination of glutamate by glutamate dehydrogenase (GDH) was coupled to the production of formazan and formazan precipitation was used for colorimetric visualization. The method was tested and validated with gelatin model sections with known glutamate concentrations. Calibration graphs showed linear relationships with high correlation coefficients (> 96%) between glutamate concentrations or section thickness and absorbance values. The method was reproducible, with a constant percentage of 60 +/- 5% of glutamate being converted in gelatin model sections containing glutamate concentrations of 2 mM and higher. Glutamate concentrations were estimated in periportal, intermediate, and pericentral zones of liver lobules that contain low, intermediate, and high GDH activity, respectively. In fed adult male rat livers, periportal zones contained the highest concentrations of glutamate (approximately 14 mM) and intermediate and pericentral zones approximately 13 and 9 mM, respectively. On starvation, glutamate concentrations increased only in the small rim of pericentral cells that express glutamine synthetase, to approximately 15 mM. In livers of fetal and newborn rats, glutamate was homogeneously distributed, with a concentration of approximately 5 mM. In suckling rat liver, distribution of glutamate was still homogeneous but the concentration was increased to approximately 8 mM. These glutamate distribution patterns were in agreement with those detected immunohistochemically.

Role of oxygen in the zonation of carbohydrate metabolism and gene expression in liver

Hepatocytes around the afferent (periportal) vessels differ from those around the efferent (perivenous) vessels in their contents of key enzymes, and therefore have different metabolic capacities. Thus, the model of "metabolic zonation" proposes that the periportal cells produce glucose via glycogenolysis and gluconeogenesis and that the perivenous cells utilize glucose via glycogen synthesis and glycolysis. The periportal and perivenous cells receive different signal patterns, because substrates including oxygen and hormones are degraded and products and mediators are formed during passage of blood through the liver. The different signal patterns should be important for both short-term regulation of metabolic rates and for long-term induction and maintenance of the enzyme equipments by control of gene expression. From the periportal to the perivenous zone, the concentration of the signal oxygen falls corresponding to a drop from about 13 (arterial) to 9 (mixed periportal) and then to 4 (hepatovenous) volume% gas atmosphere. For short-term regulation of metabolism, in perivenous-like cells net glucose production measured over a period of two hours was observed below 2%, net glycogen synthesis above 4%, and net lactate utilization above 6% oxygen. In periportal-like cells net glucose formation and net lactate utilization increased sharply from anoxia to 6% oxygen and then only moderately. For long-term regulation of gene expression, the glucagon (cAMP)-dependent activation of the PCK gene was modulated by oxygen. The transcriptional rate, the abundance of mRNA and the enzyme activity were increased to higher levels under arterial rather than under venous oxygen. Conversely, the insulin-dependent activation of the glucokinase gene was negatively modulated by oxygen. A heme protein appeared to be involved in oxygen sensing, since CO mimicked the effects of oxygen on the PCK gene. Hydrogen peroxide was produced by hepatocytes as a function of oxygen tension; exogenously added, it mimicked the effects of oxygen on PCK gene induction. Therefore, the heme protein containing an oxygen sensor could be a peroxide producing oxidase. It is not known at present whether the same oxygen sensor is also involved in the short-term regulation by oxygen of hepatic carbohydrate metabolism. Transfection of PCK promoter-CAT gene constructs into primary hepatocytes showed that oxygen modulated PCK gene activation in the region of -277/+73. This modulation was not mediated by isolated cAMP responsive elements.

Arterial oxygen partial pressures reduce the insulin-dependent induction of the perivenously located glucokinase in rat hepatocyte cultures: mimicry of arterial oxygen pressures by H2O2.

Liver glucokinase (GK) is localized predominantly in the perivenous zone. GK mRNA was induced by insulin maximally under venous O2 partial pressure (pO2) and only half-maximally under arterial pO2. CoCl2 and desferrioxamine mimicked venous pO2 and enhanced the insulin-dependent induction of GK mRNA under arterial pO2. H2O2 mimicked arterial pO2 and reduced insulin-induced GK mRNA under venous pO2 to the lower arterial levels. Thus the zonal O2 gradient in liver seems to have a key role in the heterogenous expression of the GK gene.

Functional localization of glucose transporter 2 in rat liver.

Heterogeneity of zonal hepatocytes is important to elicit specific liver function. We investigated the distribution of glucose transporter 2 (GLUT-2) in normal rat liver by immunostaining and Northern blot analysis. GLUT-2 stained by immunohistochemistry was distributed predominantly in the periportal hepatocytes and gradually thinned towards the perivenous zone. Ultrastructural immunostaining of GLUT-2 showed that it was localized on microvilli of the sinusoidal plasma membrane of hepatocytes but not on the basolateral plasma membrane. Consistent with the distribution of GLUT-2 protein, the level of GLUT-2 mRNA in periportal hepatocytes was 1.9-fold higher than in perivenous hepatocytes selectively isolated by the differential isolation technique. In addition, the mRNA level of phosphoenolpyruvate carboxykinase, one of the key enzymes of gluconeogenesis, was also twofold higher in the periportal hepatocytes. These results suggest that GLUT-2 contributes to the functional difference between periportal and perivenous hepatocytes in glucose metabolism of the liver.

Immunohistochemical Localization of Hepatic Nitric Oxide Synthase in Normal and Transgenic Sickle Cell Mice: The Effect of Hypoxia

Nitric oxide (NO) generated from L-arginine and molecular oxygen by nitric oxide synthase (NOS) has been shown to influence hepatocellular function and pathology in response to ischemia and certain hepatotoxins. In the present study, we examined the liver of a transgenic line of sickle cell mice for hepatocellular injury and localization of two isoforms of NOS, the endothelial constitutively expressed isoform (EcNOS) and the inducible isoform (iNOS) by immunohistochemistry. Diffuse expression of EcNOS was observed in hepatocytes of control and sickle cell animals maintained under room air conditions. In contrast, iNOS was observed only in the sickle cell mice, well-localized to hepatocytes surrounding the central veins of the lobules. When normal mice were exposed to hypoxic conditions for 4 to 5 days, iNOS immunostaining appeared de novo in a patchy distribution throughout the liver lobules. In the sickle cell mice, hypoxia appeared to increase the subjective intensity of pericentral staining of iNOS. Liver histology was normal in the sickle cell mice maintained under room air conditions, but showed multifocal areas of necrosis when sickling was exacerbated by chronic hypoxic conditions. However, a pericentral zone of preserved architecture was present, corresponding to the region of iNOS staining. We postulate that pericentral induction of iNOS under ambient conditions occurs in transgenic sickle cell mice in response to particularly intense hypoxic conditions near the central veins of the liver. Increases in NO synthesis may occur in this region, which would serve to protect these cells from ischemic damage either directly or by maintaining blood flow. These findings could be relevant to liver pathophysiology in patients with sickle cell disease.

The dynamics of local kinetic parameters of glutamate dehydrogenase in rat liver.

Kinetic parameters of glutamate dehydrogenase (GDH, EC 1.4.1.2) for glutamate were determined in periportal and pericentral zones of adult male and female rat liver lobules under normal fed conditions and after starvation for 24 h. GDH activity was measured as formazan production over time against a range of glutamate concentrations in serial cryostat sections using image analysis. Captured gray value images were transformed to absorbance images and local initial velocities (Vini) were calculated. A hyperbolic function was used to describe the relationship between substrate concentration and local Vini. Under fed conditions, Vmax values were similar in male and female rats (8 +/- 2 and 16 +/- 2 mumol min-1 cm-3 liver tissue in periportal and pericentral zones, respectively). Starvation increased Vmax, especially in pericentral zones of females (to 27 +/- 1 mumol min-1 cm-3 liver tissue). Under fed conditions, the affinity of GDH for glutamate was similar in male and female rats (2.5 +/- 0.5 mM and 3.5 +/- 0.8 mM in periportal and pericentral zones, respectively). Starvation had no effect on K(m) values in male rats, but in female rats affinity for glutamate decreased significantly in both zones (K(m) values of 4.0 +/- 0.1 mM and 8.6 +/- 0.8 mM, respectively). These local changes in the kinetic parameters of GDH indicate that conversion of glutamate to alpha-oxoglutarate cannot be predicted on the basis of GDH concentrations or zero-order activity in the different zones of liver lobules alone.

Quantitative assessment of primary cerium reaction product formed by glucose-6-phosphatase activity in a male rat liver: an image analysis study

Glucose-6-phosphatase (G6Pase) activity has been determined in periportal and pericentral areas of the liver of normal male rats. Measurements were performed on unfixed cryostat sections mounted on semipermeable membranes. In the present study, the oxidized primary reaction product of a cerium-based histochemical method [Ce(IV)perhydroxyphosphate] instead of the final reaction product after a second-step incubation was measured. For quantification of the amount of Ce(IV)perhydroxyphosphate formed the digital image analyzing system Quantimet 500+ was used. Estimated values of optical densities of Ce(IV)perhydroxyphosphate over test areas were employed for calculation of kinetic parameters of (G6Pase). Highest activities of G6Pase (higher Km and Vmax levels) were found in periportal areas of the rat liver, indicating a higher amount of active enzyme molecules and a lower affinity for the substrate. Differences in values for both Km and Vmax between periportal and pericentral zones were highly significant and closely comparable to those for male fed rats. Correlations between Km and Vmax were significant for periportal as well for pericentral liver areas. The results of the present study thus allow the same biological implications as histochemical methods employing a final reaction for quantification of enzyme activities. The present method avoids the drawbacks of enhancement reactions and demonstrates the feasibility of in situ analysis of enzyme kinetic parameters by quantification of oxidized primary cerium reaction products.

Zonation of parenchymal and nonparenchymal metabolism in liver.

The enormous number of different liver functions are carried out by parenchymal and four main types of nonparenchymal cells, either alone or in cooperation. Although the liver tissue is uniform on the level of histology, it is heterogenous on the level of morphometry and histochemistry. This heterogeneity is related to the blood supply; cells located in the upstream or periportal zone differ from those in the downstream or perivenous zone in their equipment with key enzymes, translocators, receptors, and subcellular structures and therefore have different functional capacities. This is the basis of the model of metabolic zonation, according to which glucose release from glycogen and via gluconeogenesis, amino acid utilization and ammonia detoxification, protective metabolism, bile formation, and the synthesis of certain plasma proteins such as albumin and fibrinogen occur mainly in the periportal area, whereas glucose utilization, xenobiotic metabolism, and the formation of other plasma proteins such as alpha 1-antitrypsin or alpha-fetoprotein occur predominantly in the perivenous zone. The mor- phologic and functional heterogeneity is the result of zonal differences in the activation of the cellular genome caused by gradients in oxygen, substrate, hormone, and mediator levels, in innervation, as well as in cell-to-cell and cell-to-biomatrix interactions.

Endotoxin- and cytokine-mediated effects on liver cell proliferation and lipid metabolism after partial hepatectomy: a study with recombinant N-terminal bactericidal/permeability-increasing protein and interleukin-1 receptor antagonist.

This study was performed to clarify the mechanisms underlying post-resection changes in liver cell proliferation and metabolism. To assess the role of gut-derived endotoxaemia and endogenous cytokines in these changes, the effects of peri-operative treatment with either the lipopolysaccharide-neutralizing bactericidal/permeability-increasing protein or interleukin-1 receptor antagonist were investigated at 24 h after two-thirds hepatectomy in rats. Peri-operative treatment with either agent caused enhanced expression of proliferating cell nuclear antigen (PCNA) and reduced lipid accumulation. Activity of the hexose monophosphate shunt was significantly decreased after partial hepatectomy and restored by interleukin-1 receptor antagonist only. After partial hepatectomy, bile canalicular alkaline phosphatase activity was significantly increased in pericentral zones and redistributed to both bile canalicular and sinusoidal membranes of hepatocytes. These effects were not significantly influenced by either treatment. It is concluded that endotoxin restricts liver cell proliferation and leads to lipid accumulation following partial hepatectomy, and that interleukin-1 is a principal mediator in these processes. Furthermore, interleukin-1 mediates a repression of the pentose phosphate pathway. These changes may be of significance with respect to liver function, at least in the early phase after partial hepatectomy.

Regenerative changes in hepatic morphology and enhanced expression of CYP2B10 and CYP3A during daily administration of cocaine.

The effects of daily cocaine administration for up to 14 days were studied in terms of hepatic morphology and the expression of cytochrome P450 (CYP) enzymes in the mouse liver. Daily intraperitoneal doses of 60 mg/kg of cocaine for 3 days induced severe hepatocellular necrosis in the pericentral zone and decreased activities of CYP1A2, CYP2A4/5, and CYP2Cx. The microsomal CYP2B10 protein content was increased by about 2.5-fold, but 2B10-dependent 7-pentoxyresorufin O-dealkylase (PROD) activity was barely detectable. Five or seven daily cocaine doses caused prominent pericentral inflammation and a significant (up to 14-fold) increase in the microsomal protein content and PROD activity. An increase in microsomal CYP3A was also evident, but CYP2A5 and CYP1A2 still remained at a low level. Immunohistochemical examination showed that the relative induction of CYP2B10 and CYP3A after treatment with cocaine was strongest in perivenous hepatocytes. Immunoinhibition experiments showed that CYP2B10 accounted for catalysis of only 15% to 20% of the enhanced microsomal cocaine N-demethylase (CNDM) activity, which correlated well with immunoreactive 3A protein, and could be blocked 70% to 90% by triacetyloleandomycin. After 10 or 14 daily doses of cocaine, regenerative changes with hepatocyte ballooning were observed, coinciding with increases in CYP1A2, CYP2A4/5, and CYP3A. These results suggest the following: (1) cocaine enhances its own cytochrome P450-dependent metabolism; (2) increased production of norcocaine in microsomes is catalyzed mainly by CYP3A enzyme(s), whereas 2B10, although markedly increased by cocaine treatment, has only a minor role in cocaine hepatotoxicity; and (3) despite increased microsomal CNDM activity, cocaine-induced liver injury is reversible in mice.

Regenerative changes in hepatic morphology and enhanced expression of CYP2B10 and CYP3A during daily administration of cocaine

The effects of daily cocaine administration for up to 14 days were studied in terms of hepatic morphology and the expression of cytochrome P450 (CYP) enzymes in the mouse liver. Daily intraperitoneal doses of 60 mg/kg of cocaine for 3 days induced severe hepatocellular necrosis in the pericentral zone and decreased activities of CYP1A2, CYP2A4/5, and CYP2Cx. The microsomal CYP2B10 protein content was increased by about 2.5-fold, but 2B10-dependent 7-pentoxyresorufin O-dealkylase (PROD) activity was barely detectable. Five or seven daily cocaine doses caused prominent pericentral inflammation and a significant (up to 14-fold) increase in the microsomal protein content and PROD activity. An increase in microsomal CYP3A was also evident, but CYP2A5 and CYP1A2 still remained at a low level. Immunohistochemical examination showed that the relative induction of CYP2B10 and CYP3A after treatment with cocaine was strongest in perivenous hepatocytes. Immunoinhibition experiments showed that CYP2B10 accounted for catalysis of only 15% to 20% of the enhanced microsomal cocaine N-demethylase (CNDM) activity, which correlated well with immunoreactive 3A protein, and could be blocked 70% to 90% by triacetyloleandomycin. After 10 or 14 daily doses of cocaine, regenerative changes with hepatocyte ballooning were observed, coinciding with increases in CYP1A2, CYP2A4/5, and CYP3A. These results suggest the following: (1) cocaine enhances its own cytochrome P450-dependent metabolism; (2) increased production of norcocaine in microsomes is catalyzed mainly by CYP3A enzyme(s), whereas 2B10, although markedly increased by cocaine treatment, has only a minor role in cocaine hepatotoxicity; and (3) despite increased microsomal CNDM activity, cocaine-induced liver injury is reversible in mice. (Hepatology 1996 Mar;23(3):515-23)

Regenerative changes in hepatic morphology and enhanced expression of CYP2B10 and CYP3A during daily administration of cocaine

The effects of daily cocaine administration for up to 14 days were studied in terms of hepatic morphology and the expression of cytochrome P450 (CYP) enzymes in the mouse liver. Daily intraperitoneal doses of 60 mg/kg of cocaine for 3 days induced severe hepatocellular necrosis in the pericentral zone and decreased activities of CYP1A2, CYP2A4/5, and CYP2Cx. The microsomal CYP2B10 protein content was increased by about 2.5-fold, but 2B10-dependent 7-pentoxyresorufin O-dealkylase (PROD) activity was barely detectable. Five or seven daily cocaine doses caused prominent pericentral inflammation and a significant (up to 14-fold) increase in the microsomal protein content and PROD activity. An increase in microsomal CYP3A was also evident, but CYP2A5 and CYP1A2 still remained at a low level. Immunohistochemical examination showed that the relative induction of CYP2B10 and CYP3A after treatment with cocaine was strongest in perivenous hepatocytes. Immunoinhibition experiments showed that CYP2B10 accounted for catalysis of only 15% to 20% of the enhanced microsomal cocaine N-demethylase (CNDM) activity, which correlated well with immunoreactive 3A protein, and could be blocked 70% to 90% by triacetyloleandomycin. After 10 or 14 daily doses of cocaine, regenerative changes with hepatocyte ballooning were observed, coinciding with increases in CYP1A2, CYP2A4/5, and CYP3A. These results suggest the following: (1) cocaine enhances its own cytochrome P450-dependent metabolism; (2) increased production of norcocaine in microsomes is catalyzed mainly by CYP3A enzyme(s), whereas 2B10, although markedly increased by cocaine treatment, has only a minor role in cocaine hepatotoxicity; and (3) despite increased microsomal CNDM activity, cocaine-induced liver injury is reversible in mice. (Hepatology 1996 Mar;23(3):515-23)

Glucagon stimulates phosphorylation of different peptides in isolated periportal and perivenous hepatocytes

The perivenous and periportal zones of the liver acinus differ in enzyme complements and capacities for gluconeogenesis, glycolysis and other metabolic processes. The biochemical factors governing this metabolic zonation are still poorly understood. Glucagon-mediated protein phosphorylation is an important factor in the regulation of hepatic metabolism. Here we show, by comparing the 32P-labelling pattern of isolated periportal and perivenous hepatocytes, that glucagon promotes the phosphorylation of zone-specific peptides as well as three common peptides (glycogen phosphorylase, glycogen synthase and pyruvate kinase) in the two cell types. We propose that the zone-specific phosphorylation of peptides is an important factor governing the shortterm zonation of metabolic processes in the liver.

Mass and Positional Isotopomer Analysis of Glucose Metabolism in Periportal and Pericentral Hepatocytes

To determine whether the source of carbon for the indirect pathway of hepatic glycogen synthesis differs between the periportal and pericentral zones, we studied seven 24-h-fasted conscious rats given a constant 2-h intraduodenal infusion of glucose, 40% labeled with [U-13C]glucose (99% 13C enriched), to raise and maintain plasma glucose concentration at approximately 10 mM. Glycogen, glutamate, aspartate, and alanine were selectively sampled from the periportal and pericentral zones of the liver by the dual-digitonin pulse technique and analyzed by 13C-NMR for positional isotopomer distribution and by gas chromatography-mass spectrometry for mass isotopomer distribution. Plasma glucose mass isotopomer distribution was determined from gas chromatography-mass spectrometry. The isotopomer distribution indicates that there was no significant difference between the zones with respect to 1) percent direct flux of glucose into the glycogen (periportal, 34 +/- 4; pericentral, 38 +/- 4), 2) extent of oxaloacetate/fumarate equilibration (periportal, 0.54 +/- .01;, pericentral, 0.53 +/- 0.01), 3) dilution of tracer in oxaloacetate (periportal, 0.64 +/- 0.07;, pericentral, 0.64 +/- 0.07), or 4) inflow of pyruvate versus tricarboxylic acid cycle flux (periportal, 0.70 +/- 0.20; pericentral, 0.68 +/- 0.16). Positional isotopomer populations, determined from the 13C-13C splitting in C3 and C4 of periportal and pericentral glycogen, were indistinguishable, indicating no significant differences in the source of the 3-carbon precursors for hepatic glycogen synthesis by the indirect pathway. In conclusion, glucose metabolism is the same in the periportal and pericentral zones with regard to 1) the relative flux of carbon via the direct/indirect pathways, 2) the source of the 3-carbon precursor used in the indirect pathway of glycogen synthesis, and 3) the flux of the 3-carbon precursors through the tricarboxylic acid cycle.

Variations in peroxisomal catalase of neonatal rat hepatocyte subpopulations. Effect of pre- and postnatal exposure to alcohol.

Alcohol consumption during pregnancy is teratogenic and induces severe alterations in hepatocytes. In the hepatocyte peroxisomal system, ethanol is converted in the presence of H2O2 to acetaldehyde and water. Therefore, peroxisomal catalase also acts as an antioxidant defence mechanism by removing H2O2 and preventing the formation of hydroxyl radicals in the cell. Alterations in peroxisomal catalase after pre- and pre+postnatal alcohol exposure were investigated in the rat. The effect of pre- and postnatal exposure to ethanol on hepatocyte subpopulations was analysed in isolated hepatocytes originating from periportal, intermediate and perivenous zones. Analysis of catalase revealed that the total activity and content of this enzyme were higher in 12-day-old cells than in cells from newborns and that this increment was more pronounced in treated cells. In controls, the amount of peroxisomal catalase increased mainly in periportal cells, whereas alcohol exposure induced a significant increase in the catalase of perivenous hepatocytes. We conclude that pre- and postnatal alcohol exposure mainly affects the perivenous hepatocyte peroxisomes and that the increase in peroxisomal catalase could constitute a defence mechanism against free radical generation induced by alcohol exposure during the perinatal period.

Image analysis and image processing as tools to measure initial rates of enzyme reactions in sections: distribution patterns of glutamate dehydrogenase activity in rat liver lobules.

To analyze regional differences in the activity of glutamate dehydrogenase in rat liver in situ, we developed an image recording and processing system for monitoring the formation of a colored final reaction product in time. All absorbance measurements of test and control reactions in time in consecutive sections were used to fit the data to a quadratic curve, with the derivative at t = 0 representing the initial velocity of formazan formation. The images of sections incubated for test and control reactions were topographically matched with an affine transformation using the positions of vessels as fiducials. Specific enzyme activity was calculated by subtracting the coefficients representing the initial velocity at corresponding locations in the test and control reactions and appeared to be 8 and 4 mumoles glutamate converted per min per cm3 of tissue at 20 degrees C in pericentral and periportal zones of fasted female rats, respectively. Those values are in agreement with biochemical data. The ability to construct two-dimensional images of cellular distribution patterns of enzyme activity in liver lobules is particularly useful for the study of metabolic zonation in this organ.